dear editor-in-chief of journal of current biomedical reports i am pleased to submit a case report article entitled “pathophysiological evaluation of venous catheter infections in hemodialysis patients” to considerate for publication in journal of current biomedical reports. i declare that all the writers have contributed substantially to the manuscript and they have approved for the final submission. writers of this article have no claim regarding conflict of interest. for contact purposes, i have provided all the necessary information including my postal and email address and telephone in the above part of this letter. furthermore, i would like to disclose that the work described has not been submitted elsewhere for publication, in whole or in part and i, on behalf of all writers of this manuscript, completely accept the journal’s terms. in addition, the data/results of the manuscript are not plagiarized and the authors declare that are agree, if the manuscript is accepted for publication, to the transfer of all copyrights to journal of current biomedical reports *corresponding authors: dr. ayatollah nasrollahi omran faculty member and associate professor of islamic azad university of tonekabon e-mail: ayat51@ yahoo.com dear editor in chief of journal of current biomedical report s i am pleased to submit a case report article entitled “ pathophysiological evaluation of venous catheter infections in hemodialysis patients ” to considerate for publication in journal of current biomedical report s . i declare that all the writers have contributed substantially to the manuscript and they have approved for the final submission. writers of this article have no claim regarding conflict of interest. for contact purposes, i have provided all the necessary information including my postal and email address and telephone in the above part of this letter. furthermore, i would like to disclose that the work described has not been submitted elsewhere for publication, in whole or in part and i, on behalf of all writers of th is manuscript, completely accept the journal’s terms. in addition, the data/results of the manuscript are not plagiarized and the authors declare that are agree, if the manuscript is accepted for publication, to the transfer of all copyrights to journal of current biomedical report s *corresponding authors: dr. ayatollah nasrollahi omran faculty member and associate professor of islamic azad university of tonekabon e mail: ayat51@ yahoo.com /docprops/thumbnail.emf dear editor-in-chief of journal of current biomedical reports i am pleased to submit a case report article entitled “pathophysiological evaluation of venous catheter infections in hemodialysis patients” to considerate for publication in journal of current biomedical reports. i declare that all the writers have contributed substantially to the manuscript and they have approved for the final submission. writers of this article have no claim regarding conflict of interest. for contact purposes, i have provided all the necessary information including my postal and email address and telephone in the above part of this letter. furthermore, i would like to disclose that the work described has not been submitted elsewhere for publication, in whole or in part and i, on behalf of all writers of this manuscript, completely accept the journal’s terms. in addition, the data/results of the manuscript are not plagiarized and the authors declare that are agree, if the manuscript is accepted for publication, to the transfer of all copyrights to journal of current biomedical reports *corresponding authors: dr. ayatollah nasrollahi omran faculty member and associate professor of islamic azad university of tonekabon e-mail: ayat51@ yahoo.com journal of current biomedical reports jcbior.com volume 1, number 1, 2020 1 editorial letter a light shining through darkness: probiotic against covid-19 meysam hasannejad-bibalan1,∗, hamed hekmatnezhad2 1department of microbiology, school of medicine, guilan university of medical sciences, rasht, iran 2department of basic sciences, sari agricultural sciences and natural resources university, sari, iran keywords: probiotic, lactic acid bacteria, covid-19, sars-cov-2 the colonic microbiota play a key role in human gastrointestinal tract physiology include maintaining homeostasis of the large bowel and modulating the host immune response. lactic acid bacteria (lab) especially lactobacillus and bifidobacterium are the main part of gut microbiota, which are considered as health beneficial probiotic bacteria with valuable effects in humans [1, 2]. probiotic bacteria are currently used in industrial food production and therapeutically procedures [3]. the lactic acid produced by probiotic bacteria in the fermentation process causes decrease ph to inhibit the growth of putrefactive and pathogenic bacteria. besides, lab could have improvement effects on the nutritional value of fermented products through increase the production of the main food components such as vitamins, essential minerals, and amino acids [4]. probiotics have been shown to be effective in preventing and treating different disorders such as atopic dermatitis, urinary tract ∗corresponding author: dr. meysam hasannejad-bibalan, ph.d department of microbiology, school of medicine, guilan university of medical sciences, rasht, iran tel/fax: +981333690006 email: meysam_hasannejad@yahoo.com, hasannejad@gums.ac.ir https://orcid.org/ 0000-0002-9402-098x received: may 27, 2020 accepted: may 28, 2020 infections, colorectal cancer, and rheumatoid arthritis [5]. competition with disease-causing microbes, producing antimicrobial peptides (amps), modulate the microenvironment and regulate the host immune system makes probiotics a very interesting field for infectious disease research [6]. since december 2019, coronavirus disease 2019 (covid-19) caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) affected many people globally, and finding new treatment strategies is crucial [7]. to date, there is not strong practical evidence that showed the use of probiotics has promising effects in patients with covid-19. however, previous studies suggested that the use of probiotics may reduce or prevent some of the major complications of covid-19. the most significant cases are reducing antibiotic associated-diarrhea and ventilator-associated pneumonia, and prevention of acute respiratory distress syndrome (ards) and upper respiratory tract infections (rtis) [8, 9]. regarding the prevention of rtis, probiotics maybe not directly affect sars-cov-2 but can be efficient for reducing the risk of co-infections [9]. previous indirect evidence gives us hope that probiotics research might eventually deliver therapeutic options for fighting against covid19, but until reaching that goal further in vitro and in vivo researches will be highly needed. author contributions all authors contributed equally to this manuscript, and approved the final version of https://jcbior.com/ 2 manuscripts. conflict of interests the authors declare that they have no conflicts of interest. ethical declarations not applicable. financial support none to be declared. references 1. hasannejad bibalan m, eshaghi m, rohani m, esghaei m, darban-sarokhalil d, pourshafie mr, et al. isolates of lactobacillus plantarum and l. reuteri display greater antiproliferative and antipathogenic activity than other lactobacillus isolates. j med microbiol. 2017; 66(10):1416-20. 2. hasannejad bibalan m, eshaghi m, rohani m, pourshafie mr, talebi m. determination of bacteriocin genes and antibacterial activity of lactobacillus strains isolated from fecal of healthy individuals. int j mol cell med. 2017; 6(1):505. 3. el hage r, hernandez-sanabria e, van de wiele t. emerging trends in "smart probiotics": functional consideration for the development of novel health and industrial applications. front microbiol. 2017; 8:1889. 4. eshaghi m, bibalan mh, rohani m, esghaei m, douraghi m, talebi m, et al. bifidobacterium obtained from mother's milk and their infant stool; a comparative genotyping and antibacterial analysis. microb pathog. 2017; 111:94-8. 5. bustamante m, oomah bd, oliveira wp, burgos-díaz c, rubilar m, shene c. probiotics and prebiotics potential for the care of skin, female urogenital tract, and respiratory tract. folia microbiol (praha). 2020; 65(2):245-64. 6. vieco-saiz n, belguesmia y, raspoet r, auclair e, gancel f, kempf i, et al. benefits and inputs from lactic acid bacteria and their bacteriocins as alternatives to antibiotic growth promoters during food-animal production. front microbiol. 2019; 10:57. 7. li h, liu sm, yu xh, tang sl, tang ck. coronavirus disease 2019 (covid-19): current status and future perspectives. int j antimicrob agents. 2020; 55(5):105951. 8. mak jwy, chan fkl, ng sc. probiotics and covid-19: one size does not fit all. lancet gastroenterol hepatol. 2020; s2468-1253(20):30122-9. 9. baud d, dimopoulou agri v, gibson gr, reid g, giannoni e. using probiotics to flatten the curve of coronavirus disease covid-2019 pandemic. front public health. 2020; 8(186):15. https://pubmed.ncbi.nlm.nih.gov/28901907/ https://pubmed.ncbi.nlm.nih.gov/28901907/ https://pubmed.ncbi.nlm.nih.gov/28901907/ https://pubmed.ncbi.nlm.nih.gov/28901907/ https://pubmed.ncbi.nlm.nih.gov/28901907/ https://pubmed.ncbi.nlm.nih.gov/28868269/ https://pubmed.ncbi.nlm.nih.gov/28868269/ https://pubmed.ncbi.nlm.nih.gov/28868269/ https://pubmed.ncbi.nlm.nih.gov/28868269/ https://pubmed.ncbi.nlm.nih.gov/28868269/ https://pubmed.ncbi.nlm.nih.gov/29033923/ https://pubmed.ncbi.nlm.nih.gov/29033923/ https://pubmed.ncbi.nlm.nih.gov/29033923/ https://pubmed.ncbi.nlm.nih.gov/29033923/ https://pubmed.ncbi.nlm.nih.gov/28826763/ https://pubmed.ncbi.nlm.nih.gov/28826763/ https://pubmed.ncbi.nlm.nih.gov/28826763/ https://pubmed.ncbi.nlm.nih.gov/28826763/ https://pubmed.ncbi.nlm.nih.gov/31773556/ https://pubmed.ncbi.nlm.nih.gov/31773556/ https://pubmed.ncbi.nlm.nih.gov/31773556/ https://pubmed.ncbi.nlm.nih.gov/31773556/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc6378274/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc6378274/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc6378274/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc6378274/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc6378274/ https://pubmed.ncbi.nlm.nih.gov/32234466/ https://pubmed.ncbi.nlm.nih.gov/32234466/ https://pubmed.ncbi.nlm.nih.gov/32234466/ https://pubmed.ncbi.nlm.nih.gov/32339473/ https://pubmed.ncbi.nlm.nih.gov/32339473/ https://pubmed.ncbi.nlm.nih.gov/32339473/ https://pubmed.ncbi.nlm.nih.gov/32574290/ https://pubmed.ncbi.nlm.nih.gov/32574290/ https://pubmed.ncbi.nlm.nih.gov/32574290/ https://pubmed.ncbi.nlm.nih.gov/32574290/ journal of current biomedical reports jcbior.com volume 3, number 1, 2022 eissn: 2717-1906 1 case report metachronous occurrence of gastrointestinal stromal tumor and acute promyelocytic leukemia in a patient with ankylosing spondylitis: a case report fatemeh nejatifar1,*, habib zayeni2, marjan yaghmaie3, narges akrami4 1department of hematology and oncology, razi clinical research development unit, guilan university of medical sciences, rasht, iran 2department of rheumatology, guilan university of medical sciences, rasht, iran 3hematology, oncology and stem cell transplantation research center, tehran university of medical sciences, tehran, iran 4department of internal medicine, school of medicine, guilan university of medical sciences, rasht, iran abstract gastrointestinal stromal tumors are the most common mesenchymal tumor of the alimentary canal. acute promyelocytic leukemia (apl) is a unique subtype of acute myeloid leukemia. a 57-year-old male patient with a previous record with history of gastrointestinal stromal tumors (gist) and ankylosing spondylitis, was hospitalized with right ankle and foot swelling and pancytopenia. a diagnosis of apl was made by bone marrow aspiration and cytogenetic study. after a combined treatment with all-trans retinoic acid and arsenic trioxide, the patient achieved cytogenetic and molecular remission. according to past studies, the synchronous or metachronous coexistence of gists with other malignancies, such as lymphoma, liver cancer and pancreatic tumors has been widely reported. this study describes a patient with the metachronous occurrence of a cd34 positive gist and apl. we suggest, the minor alterations in hematological parameters must be taken into consideration for such cases. keywords: gist, acute promyelocytic leukemia, ankylosing spondylitis, hlab27 1. introduction acute promyelocytic leukemia (apl) is a distinct subtype of acute myeloid leukemia, characterized with promyelocytic proliferation and reveals chromosol translocation (15;17) and life-threatening coagulopathy. the presence of t (15;17) is considered a diagnostic marker of apl regarding to the blast count, such as our patient. gastrointestinal stromal tumors (gist) is the most common of soft tissue sarcoma of gastrointestinal tract and resulting by mutation of the receptor tyrosine kinase kit (cd 117) [1]. majority of gist express cd117 in immunohistochemistry. ihc demonstrates 60-80% *corresponding author: fatemeh nejatifar, md razi hospital, razi clinical research development unit, guilan university of medical sciences, sardar jangal street, rasht, iran tel/fax: +98 911 3433101 email: dr.f.nejatifar@gmail.com http://orcid.org/0000-0002-3858-8482 received: january, 17, 2021 accepted: february, 01, 2021 of gists express cd34 [1]. the development of apl during imatinib therapy is rare [1, 2]. kim et al. reported a case of 63-year-old woman, diagnosed with a hepatic gist and acute myeloid leukemia (aml) with kmt2a gene rearrangement [3]. gao et al. reported a case of 69-year-old man with synchronous occurrence cd117 positive aml and gist [1]. we report the case of the metachronous occurrence of a cd34 positive gist and apl as well as a history of ankylosing spondylitis (as). © the author(s) 2022 https://jcbior.com/ nejatifar et al. 2 2. case presentation a 57-year-old male patient suffering from right ankle and foot swelling was admitted in april 2018, to razi hospital which is the most important referral hospital in rasht, iran. the patient on admission revealed right ankle arthritis while the other physical examination was normal. at the time of hospitalization, the patient was pancytopenia. twelve years ago, the patient was diagnosed with gist and underwent surgery and treated with imatinib 400 mg/day for a period of 36 months. gist was 8*6*4 cm in the greater curvature of the stomach and its immunohistochemistry (ihc) staining was reported positive for cd34 and negative for cd117. he has been suffering from arthritis and low back pain for 5 years, and a diagnosis of positive hla-b27 as was made based on modified new york criteria [4]. as was refractory to nonsteroidal antiinflammatory drugs (nsaid); therefore, he has been under treatment with infliximab for three years. gist relapsed after 10 years and imatinib started again because the tumor was unresectable. at the recent hospitalization test revealed that white blood cell counts 1000 cells/μl (normal range: 4,00010,000 cells/μl) with 18% for diff neutrophil count,79% for lymphocyte count hemoglobin level of 7.2 g/ dl (normal range :13-15 g/dl), and platelet count of 61,000 (normal range: 150,000‑450,000 platelets/ μl). the patient’s other lab tests reported as follows: esr: 119 mm/hour, crp: +1 mg/l, pt: 12.5 sec, inr: 1, ptt: 32 sec, fibrinogen 346 mg/dl, fe: 39 mg/dl, tibc: 225 mg/dl, ferritin > 800 ng/ml, cr: 1 mg/dl, ldh: 278 u/l. the liver function test was normal. in peripheral blood smear, rbcs had mild anisocytosis and rouleaux formation. in addition, wbcs and platelets severely decreased. also, giant platelet was observed. however, no immature cells and tear drop was not observed (figure 1). pancytopenia persisted even after discontinuation of imatinib, and infliximab, and bone marrow aspiration (bma), as well as bone marrow biopsy (bmb) revealed a severe hypocellular marrow. differential counts of bma were as follows: blast = 2%, promyelocyte = 17%, myelocyte = 15%, metamyelocyte = 10%, band = 13%, segment = 12%, lymph = 7%, erythroid series = 30%, eosinophilic series = 2%, plasma cell = 5 %. ihc staining was performed but it was not helpful to confirm the diagnosis of apl. the cytogenetic result revealed a translocation between chromosomes 15 and 17 and gain of chromosome 21. (figure 2). pml/rara fusion transcript was also positive in bone marrow aspiration by rt-pcr at diagnosis. the treatment started with all-trans retinoic acid (atra) and arsenic trioxide (ato) because according to italian group for adult hematologic disease (ginema) and spanish pethema group [5], the patient is categorized as low risk. twenty-eight days later, blood cells became normal and bone marrow study revealed remission. three months after treatment, pml/rara was not detected in peripheral blood by quantitative rt pcr. the evaluation of patient after three years revealed hematological and molecular remission. 3. discussion although there is no known mechanism for imatinib and acute leukemia, sporadic acute leukemia cases and gist were reported during imatinib therapy for gist [1, 2, 6-8]. the development of apl during imatinib therapy is rare [1, 2]. it is to our knowledge this is the first case suffering from gist, apl and hla-27 positive ankylosing spondylitis simultaneously. our patient was cd117 negative gist. cd34 is a hematopoietic stem cell antigen that commonly present in gists but is less specific than kit (cd 117) [9]. our patient also presented cd34 on gist tumor cells. cd34, a hematopoietic stem cell antigen, is commonly present in gists but is less specific than kit (cd 117) [10]. the cd34 is also expressed in vascular figure 1. bone marrow aspiration. wright giemsa stain at magnification x100. nejatifar et al. 3 endothelial cells and has been shown leading to signal transduction and enhanced adhesiveness of cd34' hematopoietic cells. normal promyelocyte do not have cd34 while leukemic promyelocyte have overexpression this marker. our patient also presented cd34 on gist tumor and leukemic cells. we assume the concomitant of the cd34 expression on apl and gist cells may be a reason of diagnosis of these two cancers in our patient. joo et al., reported a case of 63-year-old woman of synchronous occurrence of kit-positive aml and gist [6]. miettinen et al’s reported 9 (0.48%) myeloid leukemia in 1892 gist patients, suggesting an apparent nonrandom association between gist and myeloid leukemia [7]. there were six patients with aml including one case of promyelocytic and three patients with chronic myeloid leukemia (cml). all patients, in that study, developed leukemia after gist, with the time interval varying from 1.7 to 21 years [7]. yu et al reported a 49-year-old woman with cd117 and cd34 positive gist given which was treated with imatinib. after one year the patient developed apl. the pml/rara fusion gene was positive and kit mutation was negative [8]. a genetic linkage to hla b27 has been demonstrated to increase the risk of developing hematological malignancies in patients with as [11]. despite of the presence of autoimmune disease prior immunosuppression therapy, and infliximab, they suggest prompt evaluation for patients who develop hematological abnormalities after being treated with infliximab. it is probably hard to determine which plays a more important role in developing leukemia. however, we suggest, the small changes in hematological parameters must be taken into consideration for such cases. a fundamental study is needed to investigate the possible mechanism of this relation. this study reports the case of a patient with the metachronous occurrence of a cd34 positive gist and apl. we suggest, the small changes in hematological parameters must be taken into consideration for such cases. acknowledgments we would like to thank the patient for allowing us to share his details, and thank to oncology ward staff. authors’ contributions fn: haematologist and medical oncologist managed the patient and participated in the drafted manuscript. hz: rheumatologist managed the patient. my: medical genetics and carried out cytogenetic study. na: collected data and figure 2. 47, xy, t (15;17) (q24; q21), +21[12]/46, xy [8], twelve metaphase cells analyzed displayed a translocation between the long arms of chromosomes 15 and 17 and gain of chromosome21.these results consistent with the diagnosis of apl. t (15;17) (q24; q21) is associated with a good prognosis. t (15;17) is seen often (40%) with additional karyotypic changes. nejatifar et al. 4 participated in the drafted manuscript. all the authors read and approved the final manuscript. conflict of interests the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. ethical declarations ethical approval to report this case was obtained from the ethical committee of guilan university of medical sciences with this code: ir.gums.rec.1400.159. consent for publication written informed consent was obtained from the patient for publication of this case report and any accompanying image. a copy of the written consent is available for review by the editor-in-chief of this journal. financial support self-funded. references 1. gao na, guo nj, yu wz, wang xx, sun jr, yu n, et al. synchronous occurrence of gastrointestinal stromal tumor and acute myeloid leukemia: a case report and review of the literature. oncol lett. 2016; 11(5):2977-80. 2. hechtman jf, dematteo r, nafa k, chi p, arcila me, dogan s, et al. additional primary malignancies in patients with gastrointestinal stromal tumor (gist): a clinicopathologic study of 260 patients with molecular analysis and review of the literature. ann surg oncol. 2015; 22(8):2633-9. 3. kim hj, baek sk, maeng ch, kim sy, park ts, han jj. acute myeloid leukemia with t(11;19)(q23;p13.1) in a patient with a gastrointestinal stromal tumor undergoing imatinib therapy: a case report. world j clin cases. 2020; 8(7):1251-6. 4. ranatunga s, miller av. active axial spondyloarthritis: potential role of certolizumab pegol. ther clin risk manag. 2014; 10:87-94. 5. sanz ma, lo coco f, martín g, avvisati g, rayón c, barbui t, et al. definition of relapse risk and role of nonanthracycline drugs for consolidation in patients with acute promyelocytic leukemia: a joint study of the pethema and gimema cooperative groups. blood. 2000; 96(4):1247-53. 6. joo yb, choi sh, kim sk, shim b, kim ms, kim yj. synchronous development of kit positive acute myeloid leukemia in a patient with gastrointestinal stromal tumor. korean j hematol. 2010; 45(1):66-9. 7. miettinen m, kraszewska e, sobin lh, lasota j. a nonrandom association between gastrointestinal stromal tumors and myeloid leukemia. cancer. 2008; 112(3):645-9. 8. yu yp, song p, mei jg, an zm, zhou xg, li f, et al. [acute promyelocytic leukemia developed during imatinib therapy for gastrointestinal stromal tumors]. zhongguo shi yan xue ye xue za zhi. 2017; 25(2):358-64. 9. miettinen m, lasota j. histopathology of gastrointestinal stromal tumor. j surg oncol. 2011; 104(8):865-73. 10. muroi k, nakamura m, amemiya y, suda t, miura y. expression of c-kit receptor (cd117) and cd34 in leukemic cells. leuk lymphoma. 1995; 16(3-4):297-305. 11. wick rr, nekolla ea, gaubitz m, schulte tl. increased risk of myeloid leukaemia in patients with ankylosing spondylitis following treatment with radium-224. rheumatology (oxford). 2008; 47(6):855-9. https://pubmed.ncbi.nlm.nih.gov/27123049/ https://pubmed.ncbi.nlm.nih.gov/27123049/ https://pubmed.ncbi.nlm.nih.gov/27123049/ https://pubmed.ncbi.nlm.nih.gov/27123049/ https://pubmed.ncbi.nlm.nih.gov/25564173/ https://pubmed.ncbi.nlm.nih.gov/25564173/ https://pubmed.ncbi.nlm.nih.gov/25564173/ https://pubmed.ncbi.nlm.nih.gov/25564173/ 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https://pubmed.ncbi.nlm.nih.gov/28446275/ https://pubmed.ncbi.nlm.nih.gov/22069171/ https://pubmed.ncbi.nlm.nih.gov/22069171/ https://pubmed.ncbi.nlm.nih.gov/7536510/ https://pubmed.ncbi.nlm.nih.gov/7536510/ https://pubmed.ncbi.nlm.nih.gov/7536510/ https://pubmed.ncbi.nlm.nih.gov/18390588/ https://pubmed.ncbi.nlm.nih.gov/18390588/ https://pubmed.ncbi.nlm.nih.gov/18390588/ https://pubmed.ncbi.nlm.nih.gov/18390588/ journal of current biomedical reports jcbior.com volume 3, number 3, 2022 eissn: 2717-1906 1 brief report prevalence and antibiotic susceptibility pattern of quinolones and cephalosporins resistant uropathogenic escherichia coli in the north iran saman shalibeik1, hossein ghafori2, kourush delpasand3,*, nafiseh zamani3, zaman darvish3, hassan pourmoshtagh4,* 1department of microbiology, faculty of biological sciences, falavarjan branch, islamic azad university, isfahan, iran 2department of microbiology, tonekabon branch, islamic azad university, tonekabon, iran 3poursina clinical research development unit, poursina hospital, guilan university of medical science, rasht, iran 4department of pediatrics, loghman-hakim hospital, shahid beheshti university of medical sciences, tehran, iran abstract escherichia coli is a gram-negative bacillus of the enterobacterales order that is considered an opportunistic pathogen. one of the places they can enter is the urinary tract, which is called the uropathogenic e. coli (upec) strains and the infections that result from it are called urinary tract infection (uti). the aims of this study was to obtain new information about the antibiotic susceptibility pattern of quinolones and cephalosporins resistant e. coli. in this retrospective cross-sectional study, all cases of urinary tract infections caused by e. coli isolates referred to razi hospital in rasht, the north of iran over a period of three years were evaluated. antimicrobial susceptibility patterns were evaluated using disk diffusion method. the total number of e. coli bacteria isolated from urine samples of patients with utis from 2016 to 2018 was 1224 cases. totally, 382 out of 612 tested e. coli isolates were ciprofloxacin resistant (62.4%). while, 251 out of 446 tested e. coli isolates were cephalosporins (56.3%). among the tested isolated, 206 cases were resistant to both antibiotics (cephalosporin and ciprofloxacin), of which 71 (34.5%) were recovered from male and 135 (65.5%) from female samples. the highest antibiotic susceptibility to e. coli was related to amikacin (74.8%) and followed by nitrofurantoin (67.5%). the results showed an alarming rate of cephalosporin and ciprofloxacin resistance among e. coli causing uti in our region. these findings suggest optimizing local stewardship programs and infection control policy. keywords: escherichia coli, urinary tract infection, quinolone, cephalosporin, antibiotic resistance 1. introduction currently, one of the most important problems of public health in the world are nosocomial infections. nosocomial infections are caused by organisms that *corresponding author: hasan pourmoshtagh, md department of pediatric, mosavi hospital, zanjan university of medical sciences, zanjan, iran tel/fax: +98 912 6402128 email: hpourmoshtagh@gmail.com http://orcid.org/0000-0001-8478-8226 kourush delpasand, md kd388@yahoo.com http://orcid.org/0000-0001-6842-3374 received: april, 02, 2022 accepted: august, 06, 2022 are resistant to or eventually become resistant to antibiotics [1, 2]. nosocomial infections generally refer to infections that occur 48 to 72 hours after admission, or three days after discharge, or 30 days after surgery © the author(s) 2022 https://jcbior.com/ https://jcbior.com/ shalibeik et al. 2 [3]. the world health organization (who) has conducted an epidemiological study of nosocomial infections in 14 countries and reported an overall prevalence of 8.7%. according to this report, the prevalence of nosocomial infections in iran is 4.5%, and the most common infections are related to urinary tract, and respiratory tract infections [4]. nosocomial infections increase the use of antibiotics and thus increase the resistance of bacteria to several drugs [5]. escherichia coli is the most common of the nosocomial infections. urinary tract infections (utis) is one of the most common bacterial infections in humans. although several different microorganisms, such as fungi, viruses, and bacteria, cause utis, but the uropathogenic e. coli (upec) strains are responsible for 80% of these infections. they may be symptomatic or asymptomatic and in most cases will not cause death [6-8]. antibiotic treatment is the most common way to treat infectious diseases, but overuse over the years can cause organisms to become resistant to any known antibiotic. antibiotic resistance is one of the health problems and its increase in developing countries such as iran is worrying [9, 10]. beta-lactams are the most widely used antibiotics in the treatment of bacterial infections. the widespread use of these antibiotics in pathogenic bacteria leads to the production and dynamic and continuous mutation of beta-lactamases in these bacteria, which increases their range of activity against antibiotics [11]. due to the effect of upec strain on a wide range of populations, it can be considered as a major target for antimicrobial therapy. quinolones and fluoroquinolones are first-line antibiotics in the treatment of utis, however, treatment of infections became more complicated with the advent of quinolones resistant strains [12, 13]. due to the importance of evidence based therapy, the present study aimed to evaluate prevalence and antibiotic susceptibility pattern of quinolones and cephalosporins resistant e. coli in the north of iran. 2. materials and methods 2.1 study area and subjects this is a retrospective cross-sectional study of all cases of utis caused by e. coli isolates that were admitted to razi hospital in rasht between 2016 and 2018. razi hospital is one of the largest hospital in guilan province, the north of iran consists of 281 approved hospital beds and 240 active beds. in this study, the sampling method was census and from the results of the hospital archives, information about age and sex was extracted from the patients' admissions in the hospital. the results of inpatients who had a positive culture of e. coli isolated from urine samples were studied. patients with incomplete file information (information about the main variables) and samples with patients with mixed culture report (simultaneous growth of two or more bacteria) and culture results of urine samples containing grampositive bacteria were excluded from the study. 2.2 antibiotic susceptibility testing antimicrobial susceptibility patterns were evaluated using disk diffusion method based on clsi guidelines [14]. the results of antibiogram tests, which include antibiotics of the quinolones (nalidixic acid and ciprofloxacin) and cephalosporins of the 1st to 4th generation, were extracted and the frequency of e. coli isolates resistant to this family was determined. the results of antibiotic susceptibility pattern of isolates causing utis to routine antibiotics used in the region were extracted from laboratory results and reported. 2.3 statistical analysis the collected and recorded results were analyzed by spss software version 22 (ibm corp., usa). the results are presented as descriptive statistics in terms of relative frequency. values were expressed as the mean ± standard deviation (continuous variables) or percentages of the group (categorical variables). 3. results the total number of e. coli isolated from urine samples of patients with utis from 2016 to 2018 were 1224 cases. the mean age of patients was 46 years and the age range of patients ranged from 16 to 97 years. totally, 382 out of 612 tested e. coli isolates were ciprofloxacin resistant (62.4%). while, 251 out of 446 tested e. coli isolates were cephalosporins (56.3%). among the tested isolated, 206 cases were resistant to both antibiotics (cephalosporin and ciprofloxacin), of which 71 (34.5%) were recovered from male and 135 (65.5%) from female samples. the antibiotic susceptibility pattern of e. coli isolates resistant to cephalosporin and ciprofloxacin were listed in table 1. shalibeik et al. 3 the highest antibiotic susceptibility to e. coli was related to the antibiotic amikacin (74.8%). the highest antibiotic susceptibility to e. coli was related to amikacin (74.8%) and followed by nitrofurantoin (67.5%). 4. discussion the wide spread of antibiotic-resistant bacterial species is one of the most serious challenges for physicians and health centers, which will lead to high costs in this regard. research shows that the prevalence of antibiotic resistant e. coli is increasing in iran; however, the patterns are varied geographically [15]. in the present study, the rate of cephalosporin resistance in utis caused by e. coli was 56.3%. similar resistance rates are reported in some iranian studies which does not recommend the use of cephalosporins and penicillins in the treatment of utis caused by e. coli without the laboratory report [16, 17]. in accordance to our findings, there are several reports that indicate to a high prevalence of fluoroquinolones resistant e. coli in iran. studies in the united states show that e. coli antibiotic resistance increased from 3 percent to 17.1 percent between 2002 and 2010 [18]. but the global data suggest that the rate of fluoroquinolones resistance is on the rise, due to multifactorial causes including the antibiotic over abuse, and emergence of multidrug-resistant clone [19-21]. due to the retrospective nature of this study, the main limitations of the present study are the existence of a defect in the records and the lack of some clinical. also, due to the limited study area, the results can not be generalized to the whole region and thus require more extensive studies to determine the overall status of medical and educational centers in the future. the results of recent study determined a regional pattern of e. coli antibiotic resistance that causes utis. the results showed an alarming rate of cephalosporin and ciprofloxacin resistance among e. coli causing uti in our region. these studies could lead to improved infection control programs in regional health centers as well as optimization of laboratory reports to help treat patients. prevention of resistant isolates requires targeted optimization of treatment protocols based on laboratory results or the pattern of antibiotic resistance in the area. due to changes in the susceptibility pattern of e. coli, as well as the time and place of increasing the rate of antibiotic resistance of this bacterium, continuous antibiogram tests are recommended. also, designing prospective studies to investigate the effect of targeted use of antibiotics based on laboratory results in the process of reporting multidrug-resistant 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https://pubmed.ncbi.nlm.nih.gov/32747356/ https://pubmed.ncbi.nlm.nih.gov/32747356/ https://pubmed.ncbi.nlm.nih.gov/32380906/ https://pubmed.ncbi.nlm.nih.gov/32380906/ https://pubmed.ncbi.nlm.nih.gov/32380906/ https://pubmed.ncbi.nlm.nih.gov/32380906/ https://pubmed.ncbi.nlm.nih.gov/32380906/ journal of current biomedical reports jcbior.com volume 3, number 1, 2022 eissn: 2717-1906 1 original research first report of simultaneous assessment of hepatitis a virus and hepatitis e virus seroprevalence among soldiers in iran khodayar ghorban1, maryam dadmanesh2, hasan mohammadnezhad3, hamed naziri4,* 1department of immunology, school of medicine, aja university of medical sciences, tehran, iran 2department of infectious diseases, school of medicine, aja university of medical sciences, tehran, iran 3department of microbiology, shahr-e-qods branch, islamic azad university, tehran, iran 4department of microbiology, school of medicine, guilan university of medical sciences, rasht, iran abstract hepatitis e virus (hev) and hepatitis a virus (hav) have been considered as common causes of viral hepatitis to military service members since their discovery. although iran has been recognized as an endemic country for hav and hev there is no study about hav and hev simultaneous prevalence among iranian soldiers. the purpose of this study was to assess anti-hav and anti-hev antibodies in iranian soldiers concurrently. in this cross-sectional study, a total of 400 serum samples were collected by simple random sampling for detection of anti-hav and anti-hev igg by commercial hev enzymelinked immunosorbent assay (elisa) kit. out of 400 individuals, all participants were male with ages ranging from 18 to 26 years. totally, 12% and 0.5% of the tested specimens were positive for anti-hav and anti-hev respectively. there were no significant differences between the subjects grouped according to age groups. our finding presented a dramatic decrease in seropositivity against hav. therefore, vaccination for hav should be reevaluated for military services in iran. keywords: hav, hev, soldiers, iran 1. introduction despite significant decline in the prevalence of hepatitis a virus (hav) and hepatitis e virus (hev) infections in industrial countries recently, there remain a serious health problem in third world [1, 2]. all type of acute and chronic viral hepatitis are one of the most important public health concern worldwide [3, 4], and both hav and hev infections are the most important cause of oral-fecal hepatitis and are transmitted mainly through the fecal-oral route; either via person-to-person direct contact or consumption of contaminated food or water [5, 6]. while hev infection frequently occur in young adults, hav is more common in early childhood and immune *corresponding author: hamed naziri, ph.d department of microbiology,school of medicine, guilan university of medical sciences, rasht, iran tel/fax: +98 13 33326061/+98 13 33326061 email: hamed.naziri65@gmail.com http://orcid.org/0000-0001-6290-8944 received: october, 13, 2021 accepted: december, 02, 2021 response against it usually leading to long-lasting protection [7]. furthermore the prevalence of hav in developing countries (27 % versus 81% in high class countries) is significantly higher than that of hev in endemic areas (7.8% versus 45%) [8]. access to safe water, basic sanitation and hygiene play an important role in disease transmission, especially in low-income regions [9]. most sporadic cases and outbreaks of acute hepatitis are caused exclusively by hev as an important public health concern in primarily developing countries in asia and africa, frequently related to contaminated water [7, 10]. the iranian soldiers who developed hepatitis e was found to have anti-hev antibodies were primarily estimated to be © the author(s) 2022 https://jcbior.com/ ghorban et al. 2 2% and almost 98% of them were at risk for hev infection [11]. in many parts of the world, the proportion of antihav seroprevalence has been declining in recent years. probably, it is related to the improvements in public health programs, availability of clean water and sanitary conditions [12, 13]. in addition, vaccinations against hav have provided a safe and effective means of preventing of hepatitis a [14]. like hav, hev is usually an acute self-limiting disease and with no progression to a chronic disease state. since, there is no a suitable cell culture system for efficient hev replication and susceptible small laboratory animal, construction a live/attenuated vaccines is not possible [15]. the purpose of this study was to evaluate the seroprevalence characteristics of hav and hev and their co-infection among iranian soldiers to determine whether vaccination should be administered routinely in iranian army draftees. 2. materials and methods 2.1 patients and methods this cross-sectional study was approved by the ethical committee of aja university of medical science. it was carried out among 400 iranian soldiers in a military unit from may to december 2019 (tehran, iran). the average age of the study population was 21.6±1.9 years. all soldiers completed a questionnaire to assess other descriptive characteristics such as ethnicity, career before recruitment, education level, history of vaccination, and time of in-service. five ml blood samples were collected from all soldiers after obtaining an informed consent. serum stored in -20 ˚c until anti-hav igg and anti-hev igg antibodies determination. detection of anti-hav igg and anti-hev igg was performed by commercial hev enzyme-linked immunosorbent assay (elisa) kit (diapro, diagnostic bioprobes, milano, italy) according to manufacturer’s instruction. samples with anti-hev igg or anti-hav igg ratio <0.90 were recognised as negative, those with ratio ≥0.90 but <1.10 were considered as borderline, and samples with ratio ≥1.10 were defined as positive. 2.2 statistical analysis statistical analysis was done through spss (version 17) statistical software (scientific package for social sciences, chicago, il). data were subjected to the analysis of variance (anova) to compare the proportions between groups. the level of p-value less than 0.05 were considered significant. 3. results totally 400 soldiers were tested for detection of anti-hav igg and anti-hev igg antibodies. the mean age was 21.63 ± 1.92 (range from 18 to 24). out of 400 individuals, all of them were male. the duration of regular full-time service was 11.13±5.49 (range from 3 to 24 months). in 322 (80.5%) of them had high school diploma and higher degree and 78 (19.5%) had education less than a high school diploma. the majority of soldiers (96%, n=384 were student before coming to compulsory military service. the overall anti-hav igg and anti-hev igg was found in 48 (12%) and only two (0.5%) of 400 participants respectively (table 1). none of seropositive participants revealed any clinical symptoms similar to acute or chronic hepatitis at the time of sampling. previous history of jaundice, receiving blood transfusion and history of hepatitis were negative in all soldiers. there were no statistically significant between the age, ethnicity, career, education level, and time of in-service with anti-hav igg and anti-hev igg antibodies. the seropositive of hav is in rural and urban areas were 60.6 % and 39.4% respectively. there was no significant difference between these groups (p >0.5). 4. discussion this is the first cross-sectional study that compares prevalence of anti-hav and anti-hev antibodies among iranian soldiers simultaneously. the results of this study suggest that prevalence of anti-hav antibodies have declined dramatically during recent years. both hav and hev infection can lead to severe and long-term disease including liver table 1. age-specific seroprevalence of anti-hav antibody among soldiers age group (year) negative positive 18-21 188 (91.2%) 18 (8.8%) 21-26 164 (84.5%) 30 (15.5%) total 352 (88%) 48 (12%) ghorban et al. 3 damage which may last for six months or longer [16]. hav and hev infections are more common in overcrowding regions with inappropriate hygiene, lower income countries and directly related to sanitation. safety and clean water were available among iranian forces. health care such as the using of bottled water or healthy food is strongly recommended during missions in endemic areas. in one previous study, prevalence of hev and hav infection have been reported about 9.3% and 90% respectively [17, 18]. our results did not confirm the hypothesis that vaccination for hepatitis a is not necessary for iranian military conscripts and some previous reports about prevalence of hav among them. also the seroprevalence of hav was less than estimated in other studies [19, 20]. the best protection way against hav is vaccination among high risk groups. one of the noticeable health problems in military service is hav which deployed to countries with low socioeconomic status. an objective of this study was to assess the seroprevalence of anti-hav among iranian soldiers. our results represented low anti-hav seroprevalence which was unexpected. this proportion is completely different with previous reports of anti-hev prevalence in the iran, which were 80.3%–97% in izadi et al. and ghorbani et al. studies respectively, our proportion was significantly lower than those stated in more recent studies [11, 19]. one earlier study among iranian blood donors represented seroprevalence rates of 70.3% [21]. nevertheless, these results are also in discordance with seroprevalence was observed in previous studies, which may have been caused by hygiene promotion in iran. the current study exhibited that 0.5% of tehran soldiers were positive for anti-hev. previous studies reported similar hev seroprevalence among iranian soldiers in non-endemic regions [11]. while hepatitis e is endemic in developing countries such as iran [7], our data showed less prevalence of hev compared to 30% in previous data. also, our finding revealed an unprecedented reduction in seropositivity against hav which shows individuals are at risk for hav infection. therefore, vaccination for hav should be reconsidered for military services in iran. we identify the limitations of our study such as the overall sample size is small and lack of molecular tools. in addition to these limitations, clinical data collected during the soldiering have not contained some risk factors, such as contact with animals, food regime, and travel to endemic regions. in summary, there was a dramatic decline in seropositivity against hav in our study. as a consequence, iran should reevaluate the need for vaccination against hav for its military services. authors’ contributions hn: designed and performed experiments, analyzed data and co-wrote the paper. kg: designed and performed experiments, analyzed data. md, hm: analyzed data and co-wrote the paper. hn: designed experiments, supervised the research. all authors 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21. hesamizadeh k, sharafi h, keyvani h, alavian sm, najafitireh shabankareh a, sharifi olyaie r, et al. hepatitis a virus and hepatitis e virus seroprevalence among blood donors in tehran, iran. hepat mon. 2016; 16(1):e32215. https://pubmed.ncbi.nlm.nih.gov/27298557/ https://pubmed.ncbi.nlm.nih.gov/27298557/ https://pubmed.ncbi.nlm.nih.gov/26421130/ https://pubmed.ncbi.nlm.nih.gov/26421130/ https://pubmed.ncbi.nlm.nih.gov/26421130/ https://pubmed.ncbi.nlm.nih.gov/26421130/ https://pubmed.ncbi.nlm.nih.gov/25149658/ https://pubmed.ncbi.nlm.nih.gov/25149658/ https://pubmed.ncbi.nlm.nih.gov/25149658/ https://pubmed.ncbi.nlm.nih.gov/25149658/ https://pubmed.ncbi.nlm.nih.gov/31993027/ https://pubmed.ncbi.nlm.nih.gov/31993027/ https://pubmed.ncbi.nlm.nih.gov/31993027/ https://www.sid.ir/en/journal/viewpaper.aspx?id=100506 https://www.sid.ir/en/journal/viewpaper.aspx?id=100506 https://www.sid.ir/en/journal/viewpaper.aspx?id=100506 https://pubmed.ncbi.nlm.nih.gov/15831565/ 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https://pubmed.ncbi.nlm.nih.gov/27099669/ https://pubmed.ncbi.nlm.nih.gov/27099669/ https://pubmed.ncbi.nlm.nih.gov/27099669/ https://pubmed.ncbi.nlm.nih.gov/27099669/ https://www.sid.ir/en/journal/viewpaper.aspx?id=94597 https://www.sid.ir/en/journal/viewpaper.aspx?id=94597 https://www.sid.ir/en/journal/viewpaper.aspx?id=94597 https://pubmed.ncbi.nlm.nih.gov/27110256/ https://pubmed.ncbi.nlm.nih.gov/27110256/ https://pubmed.ncbi.nlm.nih.gov/27110256/ https://pubmed.ncbi.nlm.nih.gov/27110256/ journal of current biomedical reports jcbior.com volume 2, number 4, 2021 eissn: 2717-1906 1 case report pneumomediastinum, pneumopericardium pneumothorax and subcutaneous emphysema in iranian covid-19 patients manouchehr aghajanzadeh1, mohammad haghighi2,*, siamak rimaz2, ali alavi fomani3, azita tangestaninejad3, mohammad taghi ashoobi3, yousha pourahmadi3, mohammad mojtaba rohani4 1department of thoracic surgery, guilan university of medical sciences, rasht, iran 2anesthesiology research center, department of anesthesiology, alzahra hospital, guilan university of medical sciences, rasht, iran 3inflammatory lung diseases research center, department of internal medicine, razi hospital, school of medicine, guilan university of medical sciences, rasht, iran 4department of radiology, razi hospital, guilan university of medical science, rasht, iran abstract recently, spontaneous pneumomediastinum (pm), pneumopericardium (pp), pneumothorax (pt), and subcutaneous emphysema (se) were reported as infrequent complications in coronavirus disease 2019 (covid-19) patients in intensive care (icu). here we report these complications in nine iranian patients of covid-19. nine patients with reported pm, pp, pt, and se in covid-19 who were hospitalized in arya hospital, rasht, iran, for three months, were followed to record demographic data and clinical characteristics of these patients. in nine pm-developed patients, six cases represented pt, one patient with pp, and four patients with pt and se. four patients expired and only five patients survived. pm, pp, pt, and se are uncommon complications in covid-19 patients and were reported frequently in male patients. early diagnosis and treatment could save the patients since these complications are related to poor prognosis and prolonged hospitalization. patients with mild covid19 and mild pulmonary damage have a favorable outcome. keywords: covid-19, subcutaneous emphysema, spontaneous pneumomediastinum, pneumopericardium, pneumothorax 1. introduction severe acute respiratory syndrome coronavirus-2 (sars-cov-2) infection has developed as the greatest pandemic in this era with high morbidity and mortality and multiple complications. there are lots of review studies about coronavirus disease 2019 (covid-19) and its complications [1-4]. irregular reported numbers of spontaneous pneumomediastinum (pm), pneumopericardium (pp), pneumothorax (pt), and subcutaneous emphysema (se) cases in patients with covid -19, and also in the patients without mechanical *corresponding author: mohammad haghighi, md department of anesthesiology, alzahra hospital, guilan university of medical sciences, rasht, iran tel/fax: +98 911 3315256 email: manesthesist@gmail.com http://orcid.org/0000-0003-0331-1683 received: september, 15, 2021 accepted: december, 14, 2021 ventilation, which results in barotrauma [5]. the pathophysiologic mechanism of pm is an increased gradient pressure between the alveoli and parenchyma of lung that leads to severe alveolar injury and the air dissects the surrounding bronchovascular sheaths, and this air enters into the mediastinum and produces pm and disturbance to cervical se and pleural space pt [3, 5, 6]. alveolar wall is more prone to rupture due to inflammation in covid-19 patients and alveolar wall is exacerbated by severe cough or any problems which increase the intra-alveolar pressure [5]. invasive positive pressure ventilation can cause © the author(s) 2021 https://jcbior.com/ aghajanzadeh et al. 2 pt and/or pm spontaneously. barotrauma is the cause of pm, pt, pp, and se [3]. in this care report, we focus only on hospitalized covid-19 patients who developed these complications. some of patients have primarily mild to moderate disease condition, but deteriorated progressively, and also represented the associated factors to prognosis these complications. 2. case presentation in this case report, we evaluated case series and described the record of nine patients in arya hospital, rasht, iran during three months in covid-19 fourth outbreak from april until july 2020. patients demographic data, medical histories, clinical and laboratory finding, chest computer tomography (ct) results, and patient’s outcome (death or discharged), were recorded. all subjects gave their informed consent to participate in the study. according to our evaluation, seven out of nine patients with covid-19 were male and two of them were female. the age range was about 32 to 68 years by mean of 48.27±10. diagnosis of covid-19 was confirmed by polymerase chain reaction (pcr). among nine patients, three of them reported the history of hypertension, four of them had type 2 diabetes mellitus, two patients had bronchial asthma, and also two of them had obesity. after two to six days of hospitalization, all of the nine patients represented one or more following complications: pm, pp, pt, and se. three patients with low oxygen saturation (between 50% and 86%), improved up to 90–93% on bipap mask at 12 l/min. chest ct was done to evaluate the severity of lung damaged due to sarscov-2 at the early stage (supplementary figures 1–8). the most common radiologic findings were groundglass pattern, integrated opacity, and thickening of septum. four patients suffering minor pulmonary damage (10–35% of lung), three of them had moderate injury (50% of lung) and four cases had severe forms of injury (over 70% of lung). none of patients represented pm, pp, pt, or se complications, while these complications happened after 1 to 8 days of hospitalization. laboratory findings have revealed following data: the count of neutrophil increased but lymphocyte decreased, c reactive protein (crp) was higher than 100 mg/l, also the levels of ferritin and lactate were increased. arterial blood gases evaluation was as follows: the mean of initially po2 was 78 mmhg (59– 83), but later decreased to 54 mmhg (48–85). the mean of initially pco2 was 60 mmhg (48–68), and the mean of and o2 saturation was 80.2% (60–98%). all patients were received one ceftriaxone course of treatment in combination with remdesivir, dexamethasone, and levofloxacin. cardiothoracic surgeon consulted all cases. throughout hospitalization, eight patients needed oxygen therapy, six patients were transferred to the intensive care unit (icu), and underwent invasive ventilation. six patients needed chest tube, and six patients with pt needed subclavicular incision for evacuation of se, while the rest of them were managed conventionally. four patients died after 8-20 days, with the median of 6-12 days of hospitalization. the remaining five patients who had moderate lung damage at admission time, did not need surgical drainage and were discharged in good clinical condition after hospitalization (8-12 days). clinical characteristics of all patients were explained in detail in table 1. 3. discussion pm is presented in two types, benign or malignant, which in some patient it can be fatal [7]. benign type of pm can turn to malignant forms, in the case of rapidly rise pressure in the mediastinum, which causes all spaces to closed with no way for air escape. malignant pm could consequence mechanical obstruction and presents as pericardial tampon, especially in the present of cough. this event can interfere with the cardiovascular function and causes reduction in circulation volume and tension pt form [5, 8, 9], which none of the nine reported patients represented this complication. in some patients with pm, air can escape into route to the neck, retroperitoneum or to the right and left pleural space, and causes bilateral pt, which can turn into tension pt form [10]. a retrospective study has reported that 13 patients with covid-19 have developed spm with or without pt and se [5, 8, 9]. in this case report, no patient had isolated pt and some of these patients needed invasive positive pressure ventilation. furthermore, none of reported patients have represent with retroperitoneal emphysema. moreover, this case report has showed high level serum of ldh, which might be due to cellular damage. also, severe diffuse alveolar damage causes of the alveolar damage, and air escape along the bronchoalveolar sheath and mediastinum space [8]. aghajanzadeh et al. 3 t a b le 1 . d e m o g ra p h ic a l a n d c li n ic a l c h a ra c te ri st ic s o f p a ti e n ts p a ti e n t b y n u m b e r g e n d e r a g e y e a r p u lm o n a r y i n ju r y b y c t p t p m p p s e h r o 2 sa t t y p e 2 d ia b e te s m e ll it u s c t s -c i s u r a st h m a o u tc o m e 1 m 6 4 7 0 % y e s y e s n o y e s 6 8 8 8 % y e s y e s y e s n o y e s d e a th 2 m 6 1 5 8 % n o y e s n o y e s 8 2 6 7 % n o y e s n o n o n o d e a th 3 m 3 4 7 0 % n o n o y e s y e s 1 0 0 8 0 % n o n o y e s y e s n o d e a th 4 m 5 7 6 4 % y e s y e s n o y e s 9 5 6 0 % y e s y e s y e s n o n o d e a th 5 f 6 8 2 8 % y e s y e s n o y e s 7 0 9 2 % y e s y e s n o n o n o d is c h a rg e 6 m 4 4 2 0 % y e s n o n o n o 7 0 9 8 % n o n o y e s y e s n o d is c h a rg e 7 m 6 2 5 5 % y e s y e s n o y e s 1 2 0 6 0 % n o y e s y e s n o y e s d is c h a rg e 8 m 5 3 5 0 % y e s n o n o n o 1 0 0 8 7 % y e s y e s y e s n o n o d is c h a rg e 9 f 3 8 2 5 % y e s n o n o n o 7 5 9 0 % n o n o n o n o n o d is c h a rg e c t : th o ra x c o m p u te r to m o g ra p h y , p t : p n e u m o th o ra x , p m : p n e u m o m e d ia st in u m , p p : p n e u m o p e ri c a rd iu m , s e : su b c u ta n e o u s e m p h y se m a , h r : h e a rt r a te , s a t o 2 : o x y g e n s a tu ra ti o n , c -t : c h e st t u b e , s -u -i : su b c la v ic u la r in c is io n , s u r : su rg e ry aghajanzadeh et al. 4 a similar study, reported coivd-19 patients that represented pneumothorax (ptx), pm, and se spontaneously or after mechanical ventilation [11]. we observed the occurrence of these complications in nine patients. in these reported patients, four of them represented spontaneously complications without invasive mechanical ventilation, while other represented complications in patients with invasive mechanical ventilation. as reported in other studies with the majority of male gender, seven of patients were male and two of them were female. the pathophysiological mechanism of complications was air leakage through damage of alveolar walls by inflammation during of cytokine storm [5, 12-14]. despite to some studies with low mortality rate, in this case report, patient’s mortality rate due to pm, pp, pt, and se was four out of nine patients. pm, pp, pt, and se are rare complications in severe covid-19 pneumonia.these complications are contributed to poor prognosis and prolonged hospitalization. patients with mild covid-19 and mild pulmonary damage have a favorable outcome. supplementary files supplementary file 1. acknowledgments we would like to thank all individuals who volunteered to participate in this study. furthermore, thank you to hospital staff and specialists for assistance with conforming and recording cases. authors’ contribution concept and study design: ma, mh, sr; methods and experimental work: aa, at, mta; results analysis and conclusions: ma, mh, yp; manuscript preparation: mmr, aa, at, mta. all authors read and approved the final version. conflict of interests no potential conflict of interest was reported by the authors. ethical declarations all subjects gave their informed consent to participate in the study. also, all methods were performed in accordance with the relevant guidelines and regulations. consent for publication written informed consent was obtained from the patients for publication of this case report. a copy of the written consent is available for review by the editor-in-chief of this journal. availability of data and material corresponding author (mohammad haghighi) is available in the case in requiring, and all data and tables, used in the manuscript are prepared originally by authors financial support self-funded. references 1. hamad am, elmahrouk af, abdulatty oa. alveolar air leakage in covid-19 patients: pneumomediastinum and/or pneumopericardium. heart lung. 2020; 49(6):881-2. 2. murillo brito d, villalva c, simón a, guisado-clavero m. covid-19 pneumonia associated with spontaneous pneumomediastinum and pneumopericardium. ctsnet. 2020:12485831. 3. martinelli aw, ingle t, newman j, nadeem i, jackson k, lane nd, et al. covid-19 and pneumothorax: a multicentre retrospective case series. eur respir j. 2020; 56(5) :2002697. 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covid-19 pneumonia: time-dependence after the onset of symptoms. eur radiol. 2020; 30(11):6161-9. 14. kolani s, houari n, haloua m, alaoui lamrani y, boubbou m, serraj m, et al. spontaneous pneumomediastinum occurring in the sars-cov-2 infection. idcases. 2020; 21:e00806. https://books.google.com/books?id=hux1bwaaqbaj&pg=pa156-ia1&dq=mason+rj,+slutsky+a,+murray+jf,+nadel+ja,+gotway+mb.+murray+%26+nadel%27s+textbook+of+respiratory+medicine+e-book:+elsevier+health+sciences;+2015&hl=en&sa=x&ved=2ahukewjz0l-bual1ahwqgbqkhz8hcycq6af6bageeai#v=onepage&q=mason%20rj%2c%20slutsky%20a%2c%20murray%20jf%2c%20nadel%20ja%2c%20gotway%20mb.%20murray%20%26%20nadel's%20textbook%20of%20respiratory%20medicine%20e-book%3a%20elsevier%20health%20sciences%3b%202015&f=false https://books.google.com/books?id=hux1bwaaqbaj&pg=pa156-ia1&dq=mason+rj,+slutsky+a,+murray+jf,+nadel+ja,+gotway+mb.+murray+%26+nadel%27s+textbook+of+respiratory+medicine+e-book:+elsevier+health+sciences;+2015&hl=en&sa=x&ved=2ahukewjz0l-bual1ahwqgbqkhz8hcycq6af6bageeai#v=onepage&q=mason%20rj%2c%20slutsky%20a%2c%20murray%20jf%2c%20nadel%20ja%2c%20gotway%20mb.%20murray%20%26%20nadel's%20textbook%20of%20respiratory%20medicine%20e-book%3a%20elsevier%20health%20sciences%3b%202015&f=false https://books.google.com/books?id=hux1bwaaqbaj&pg=pa156-ia1&dq=mason+rj,+slutsky+a,+murray+jf,+nadel+ja,+gotway+mb.+murray+%26+nadel%27s+textbook+of+respiratory+medicine+e-book:+elsevier+health+sciences;+2015&hl=en&sa=x&ved=2ahukewjz0l-bual1ahwqgbqkhz8hcycq6af6bageeai#v=onepage&q=mason%20rj%2c%20slutsky%20a%2c%20murray%20jf%2c%20nadel%20ja%2c%20gotway%20mb.%20murray%20%26%20nadel's%20textbook%20of%20respiratory%20medicine%20e-book%3a%20elsevier%20health%20sciences%3b%202015&f=false https://pubmed.ncbi.nlm.nih.gov/32474630/ https://pubmed.ncbi.nlm.nih.gov/32474630/ https://pubmed.ncbi.nlm.nih.gov/32474630/ https://pubmed.ncbi.nlm.nih.gov/32474630/ https://pubmed.ncbi.nlm.nih.gov/32395425/ https://pubmed.ncbi.nlm.nih.gov/32395425/ https://pubmed.ncbi.nlm.nih.gov/32395425/ journal of current biomedical reports jcbior.com volume 3, number 2, 2022 eissn: 2717-1906 1 case report symptomatic reinfection with covid-19: a case-report study in iran tofigh yaghubi kalurazi1, ehsan amini-salehi2, samira rahimi-shamami3, rahmat poursmail3, farahnaz joukar2, soheil hassanipour4, fariborz mansour-ghanaei2,* 1department of health, nutrition and infectious diseases, school of medicine, guilan university of medical sciences, rasht, iran 2gastrointestinal and liver diseases research center, guilan university of medical sciences, rasht, iran 3student research committee, school of medicine, guilan university of medical sciences, rasht, iran 4gi cancer screening and prevention research center, guilan university of medical sciences, rasht, iran abstract coronavirus disease 2019 (covid-19) resulted in a worldwide concern. the role of the immunity system and memory cells in this disease and their ability in preventing a secondary infection is a controversial issue. here we presented a case of reinfection with this virus eight months after the first episode. a twenty-one-year-old man was referred to our local hospital on 19 february 2020 with symptoms of viral infection. covid-19 infection was confirmed by rt-pcr. he got hospitalized for 5 days. eight months later on 4 october 2020, he was again referred with symptoms of viral infection and para-clinical tests confirmed covid-19 infection. he got hospitalized for 6 days in the second episode. although the immunity system plays important role in covid-19 infection through the presence of memory cells it doesn’t guarantee permanent immunity to this virus. reinfection with covid-19 is possible and has been reported in some other studies. keywords: covid-19, immunity system, sars-cov2, reinfection, secondary infection 1. introduction according to world health organization (who), by october 23, 2021 more than 243 million people got infected by severe acute respiratory syndrome coronavirus 2virus (sars-cov-2) in the world and more than 4.9 million people died since its emergence in wuhan, hubei province, china [1, 2]. an important issue in the course of the coronavirus disease 2019 (covid-19) pandemic which is caused by sars-cov-2 is the role of the immune system and how much the immune system is capable of preventing a secondary infection [3, 4]. infection with sars-cov-2 leads to immune system response with specific memory t cells and b cells and *corresponding author: fariborz mansour-ghanaei, md gastrointestinal and liver diseases research center, razi hospital, sardar-jangle ave., p.o. box: 41448-95655, rasht, iran tel/fax: +98 13 33325783/+98 13 15534951 email: fmansourghanaei@gmail.com http://orcid.org/0000-0002-6264-0025 received: october, 23, 2021 accepted: may, 01, 2022 sars-cov-2-specific immunoglobulin g antibody (igg) which appear in the plasma of infected individuals [5-7]. although the persistence of neutralizing antibodies (immunoglobulin m and immunoglobulin g) in sars-cov-2 infection lasts about 40 days, the persistence of these antibodies in other respiratory viruses last for several months [8]. though the presence of memory cells has been seen reinfection with sars-cov-2 is still a controversial issue [9]. some studies believe that reinfection with coronavirus is unlikely [10], while some other studies reported reinfection with coronavirus [5, 11, 12]. losing immunity to other types of coronavirus family © the author(s) 2022 https://jcbior.com/ https://jcbior.com/ yaghubi kalurazi et al. 2 within one to three years has been reported in some studies [13-17]. here we present a case who was reinfected with the coronavirus in the north of iran where the infection rate was one of the highest in the world. 2. case presentation a twenty-one-year-old man with a history of smoking cigarettes and hookah and without any other underlying diseases and resident of rostamabad (guilan province, north of iran) was referred to razi hospital (rasht, guilan province, north of iran) at 9.00 post meridiem (pm) on 19 february, 2020 with symptoms of viral infection. he was conscious with a glasgow coma scale (gcs) of 15/15 and his symptoms were started 5 days before his referral. his symptoms included: fever and chills, fatigue, headache, myalgia, and purulent cough. his sense of smell and sense of taste were normal but he reported that he had lost his appetite. he showed no signs of respiratory distress syndrome (including color changes, grunting, retractions, and wheezing), and his o2 saturation, respiratory rate, and pulse rate were normal (supplementary table1). because the place where he lived was one of the regions with a high prevalence of covid-19 infection at that time, for confirming the diagnosis, a nasopharyngeal swab test for detecting sars-cov-2 with real-time polymerase chain reaction (rt-pcr) was done for him and the test result was positive. also, a chest x-ray (cxr) (figure 1), and a computed tomography scan (ct scan) were done for him. he got hospitalized in the ward for 5 days and a blood examination was done for him based on the hospital protocols (supplementary table 2). during his hospitalization, he admired one ampule of apotel (500 milligrams/q 4 hours) for controlling his fever, tablets of azithromycin (500 milligrams/ twice daily), ceftriaxone (500 milligrams/one injected ampule), chloroquine (200 milligrams/once daily), and oseltamivir (75 milligrams/once daily). most of the symptoms lasted for 3 days or less since he got hospitalized except for the loss of appetite which lasted for 4 days (supplementary table 1). when he got discharged, another blood sample was done for him (supplementary table 2). eight months later on 4 october 2020, he was admitted again to the hospital at 2 pm with symptoms of lethargy, headache, myalgia, purulent cough, and the loss of appetite. the patient felt the symptoms less severe than the first time. he showed no signs of respiratory distress syndrome and his o2 saturation was normal. he was conscious with a gcs of 15/15 (supplementary table1). the second time, he was referred to our hospital 3 days after the onset of symptoms. he said he was in a hookah house for the past two weeks. a nasopharyngeal swab test for detecting sars-cov-2 rt-pcr was done for him and the test result was positive. a ct scan was done for him as well. he got hospitalized in the ward for 6 days and a blood examination was done for him based on the hospital protocols (supplementary table 2). during his hospitalization for the second time, he admired one ampule of apotel (500 milligrams/q 4 hours) for controlling his fever, tablets of azithromycin (500 milligrams/twice daily), ceftriaxone (500 milligrams/one injected ampule), and one ampule of dexamethasone (8 milligrams). most of the symptoms of the patient lasted for 2 days or less since he got hospitalized except for the loss of appetite which lasted for 3 days (supplementary table1). when he got discharged, another blood sample was done for him (supplementary table 2). figure1. chest x-rays (cxr) in the first time infection yaghubi kalurazi et al. 3 3. discussion iran is one of the countries with a high prevalence of covid-19 with more than 5 million confirmed cases and more than 112 thousand deaths [2]. reinfection with the sars-cov-2 virus is not well understood and only a few case reports of reinfection with the sars-cov-2 virus have been reported in belgium [18], the usa [19], ecuador [12], and some other countries [11]. although the number of reinfected case reports in comparison to the number of all covid-19 cases is so small but we cannot conclude whether reinfection with this virus is rare or not. it is important to know that we survey only symptomatic patients who are admitted to hospitals and because we don’t have a broad testing and general supervision of the society, we can't judge the frequency of asymptomatic reinfection in people who recovered from their first episode of the disease [20]. an interesting point of our patient is that he said he felt the symptoms more severe in the first episode whereas the cases reported from the usa [19] and ecuador [12] showed the symptoms much more severe in the second episode of infection. the studies from hong kong and belgium did not show a significant difference between the first and second episode symptoms [18]. moreover, a chest ct scan also showed less severe lungs involvement in the second episode. some mechanisms can explain reinfection with sars-cov-2. our patient was a smoker and smoking is an independent factor in the transmission of the virus [20]. it is also important that our patient told us about his attendance in a hookah house within two weeks before his second referral and because of his attendance in such a public place without strong hygiene protocols; he might have got reinfected in that place [21]. the duration of symptoms was longer the first time. one mechanism that can explain the less severity and duration of symptoms in the second event is the presence of immune memory cells which are made during the first exposure of the patient to the virus [5, 6]. as we know human body's immune system consists of innate and adaptive immunity. adaptive immunity includes cell-mediated (t cells) and humoral immunity (b cells and antibodies) [22]. in the first exposure to a pathogen, memory cells are made and these memory cells cause a faster response in the following exposures [23]. another mechanism may be a higher dose of infection with the virus during the first exposure [24]. it is also probable that the infection with the sars-cov-2 for the first time was with a stronger virulent type of virus [25]. unfortunately, we didn’t do genome sequencing for identifying the gene content of the virus in the first and second exposure to understand whether the reinfection was with the same type of virus or a different type. in the para clinic exam, we see a higher lymphocytosis the second time which confirms the less severity of the disease. previous studies showed the relationship between lymphopenia and the severity of covid-19 infection [26, 27]. tan et al. showed low lymphocyte percentage can be used as a prognostic factor in the disease progression and he explained how this virus can target lymphocytes [27]. we think that the severe lymphocytosis in the second exposure is due to some mechanism. first, there is a faster and stronger response in the second exposure to the virus due to memory cells generation [23], note that our patient was not immunocompromised and had not any underlying disease. second, genetics play an important role in the function of the immune system [28, 29]. the relationship between genetics and covid-19 infection has been studied before [30]. we think that the stronger response the second time can also be related to the genetic response of our patient. third environmental factors can also affect the immune system response [30, 31]. forth, the time of referral to the hospital can be an important issue. for the first time, our patient was referred to our hospital for 5 days and the second time 3 days after the onset of symptoms. we started medication and antibiotic therapy sooner the second time and this action could lead to better immune response the second time. in conclusion, covid-19 is a viral disease with high prevalence worldwide. identifying the pattern of virus infection can lead us to a better understanding and management of the disease. this case presentation showed us one episode of infection with covid-19 doesn’t guarantee the individuals to be protected against the virus. however, this doesn’t deny the necessity of vaccination but reflects that even vaccinated people and people with a previous infection should take care and obey the quarantine protocols. supplementary files supplementary file 1. https://jcbior.com/index.php/jcbior/article/view/133/docx yaghubi kalurazi et al. 4 authors’ contributions all authors contributed equally and approved the final version of manuscript. conflict of interests the authors declare no competing interests. ethical declarations the present study was approved by the ethics committee of guilan university of medical sciences, rasht, iran (ir.gums.rec.1400.065). consent for publication written informed consent was obtained from the patients for publication of this case report. a copy of the written consent is available for review by the editor-in-chief of this journal. financial support self-funded. references 1. zhu n, zhang d, wang w, li x, yang b, song j, et al. a novel coronavirus from patients with pneumonia in china, 2019. n engl j med. 2020; 382(8):727-33. 2. who 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poorsaravani2,3,*, sadroddin mahdipour4, sareh shafaei4, haleh masoudi5, anahita shabani5 1skin research center, department of dermatology, razi hospital, school of medicine, guilan university of medical sciences, rasht, iran 2school of medicine, ramsar campus, mazandaran university of medical sciences, ramsar, iran 3pediatric diseases research center, guilan university of medical sciences, rasht, iran 4department of dermatology, school of medicine, ramsar campus, mazandaran university of medical sciences, ramsar, iran 5department of pediatrics, school of medicine, ramsar campus, mazandaran university of medical sciences, ramsar, iran abstract skin manifestations, a common problem in infants, can be a serious concern for parents. most manifestations are benign and transient, but some of them need more evaluation regarding whether they can negatively affect infant health. in this study, it is aimed to evaluate the frequency of skin manifestation in newborns admitted to the department of newborns and nicu from 2019 to 2020. this cross-sectional was performed on infants hospitalized in the department of pediatrics and nicu of a pediatric hospital in guilan, iran, from 2019 to 2020. the sampling was performed using the census method. the information was gathered using a checklist of infant and mother characteristics. out of 323 newborns, 164 cases had skin lesions (50.8%). the lesions of erythema toxicum, cutis marmorata, diaper dermatitis, milia, salmon patch, and mongolian spots were presented at 14.9%, 9.9%, 8.1%, 5.6%, 4.3%, and 2.8%, respectively. only 5.38% of infants required treatment. there was no significant relationship between skin lesions and demographic factors of gestational age, type of delivery, or the family history of dermatological diseases. the rate of skin lesions was moderate to high in hospitalized newborns. in addition, erythema toxicum, cutis marmorata, diaper dermatitis, salmon patch, and mongolian spots were more prevalent in infants. these findings can help pediatric physicians effectively in their early diagnosis and therapeutic procedures. keywords: skin lesions, newborn, nicu, erythema toxicum 1. introduction cutaneous lesions are very common in the neonatal period, and they are mostly transient, physiological, reversible and self-limiting. unfortunately, some of these skin conditions are pathological and require intervention and collaboration between neonatologists and dermatologists [1, 2]. several maternal and environmental factors, including prematurity, *faranak poorsaravani, md pediatric diseases research center, guilan university of medical sciences, rasht, iran tel/fax: +911 2372858 email: faranakpoursaravani@yahoo.com http://orcid.org/0000-0003-2336-9153 received: april, 13, 2022 accepted: april, 01, 2023 congenital infections, ethnicity, and maternal drug use, can affect the onset, type, and evolution of skin lesions. the variety of manifestations geographically and ethnicity and their role in the prevalence of birthmarks are well documented [3, 4]. neonatal skin lesions are a serious concern for parents. many skin diseases in infancy and childhood recover spontaneously and do not have much importance, but some of them may be signs of major © the author(s) 2022 https://jcbior.com/ https://jcbior.com/ eftekhari et al. 2 and systemic diseases [5, 6]. therefore, the timely skin evaluation of neonates and infants is important and is a good indicator for the health of infants. despite numerous studies on skin lesions [7-10], there is no known procedure for treating them in infants, particularly in northern iran. on the other hand, in the neonatal intensive care unit (nicu), cutaneous lesions can expose newborn infants to infections. approximately 9% of all births require a nicu [11, 12]. according to a recent cohort study in 2020 in iran, about 34.2% of nicu admitted neonates had skin injuries, and in addition to neonatal conditions, equipment and neonatal care play a significant role in the incidence of skin injuries [13]. this study aimed to investigate the dermatological manifestations in a sample of neonates in their neonatal period in the north of iran and also identify factors related to the onset of neonatal cutaneous manifestations. 2. materials and methods 2.1 study design in a retrospective cross-sectional study, a total of 323 neonates admitted to the department of pediatrics and the neonatal intensive care unit (nicu) at the 17shahrivar hospital of rasht, guilan province, in the north of iran, were evaluated from 2019 to 2020. the sampling was performed using the census method. the study design was approved by regional ethic committee (ir.mazums.rib.rec.1400.038). after approval of the ethics committee and signing of the testimonial by the neonate’s parents, the skin surfaces, including soles, palms, genitalia, and scalp of all admitted neonates within the first 28 days of life were carefully examined by a dermatologist and a neonatologist. the manifestations were considered to be transient and benign lesions, vascular lesions, infectious lesions, pigmented lesions, and lack of development of the vasomotor system. a checklist was used to record fetal and maternal history (gender, birth weight, gestational age, type of delivery, maternal age, social history and comorbidities, parity, and family history of skin disease). in terms of gestational age, all subjects were classified as preterm (37 weeks), term (> 37 weeks), or post-term (> 42 weeks). 2.2 statistical analysis the data were analyzed in spss software version 18 using the chi-square test and fisher's exact test. variables were reported as average with standard deviation (sd), and qualitative variables as frequency and percent. the p value <0.05 was considered significant. 3. results in this study, of a total of 323 newborns evaluated (mean age 8.11±8.05), 173 (53.89%) were males and 148 (46.11%) were females. the mean birth weight was 2992.5±827.3 g, and the mean maternal age was 29.58±6.4 years. also, 24.8% of newborns were delivered vaginally, while 75.2% were delivered via cesarean section. about 29.19% of them were preterm, 61.49% term, and 9.32% post-term. at least 164 (50.8%) newborns had one skin lesion, and 114 (35.58%) of mothers reported at least one disease during pregnancy. neonatal parameters and their correlations with cutaneous lesions are reported in table 1. maternal parameters and their correlations with cutaneous lesions are reported in table 2. neonatal acne was significantly associated with neonatal age (p <0.05), so that it was more prevalent in older neonates. besides, a significant relationship was seen between gender and skin lesion total (p = 0.006). a higher prevalence of diaper dermatitis was observed in the female gender (13.5% (20 cases) versus 3.46% (6 cases), p = 0.01). a significantly higher incidence of congenital melanocytic nevus regarding birth weight is also found (p = 0.001), so that it was more prevalent in neonates with a higher birth weight. the prevalence of cutaneous lesions was significantly correlated with maternal diseases, and it was lower in those with maternal diseases (37 cases, 31.9%) (p = 0.021). besides, cutaneous lesions were positively table 1. neonatal demographic and clinical parameters variables number percent gender male 173 53.89 female 148 46.11 gestational age preterm 94 29.19 term 198 61.49 post-term 30 9.32 causes of hospitalization icter 188 60.45 infection 44 14.14 prematurity 34 10.93 others 45 14.48 eftekhari et al. 3 correlated with gestational diabetes mellitus, and they were lower in those with gestational diabetes (10 cases, 25%) (p = 0.036). there was no significant relationship between skin lesions and demographic factors of gestational age, type of delivery, or the family history of dermatological diseases. according to the main findings of the present study, out of 323 newborns, 164 cases had skin lesions (50.8%). the lesions of erythema toxicum, cutis marmorata, diaper dermatitis, milia, salmon patch, and mongolian spots were presented at 14.9%, 9.9%, 8.1%, 5.6%, 4.3%, and 2.8%, respectively. only 5.38% of infants required treatment. in table 3, the types of cutaneous lesions and their frequency in neonates are shown. 4. discussion skin manifestations, as a common problem in infants, can be a serious concern for parents. most manifestations are benign and transient, but some of them need more evaluation regarding whether they can negatively affect infant health. to our knowledge, this study was the first conducted in a fully neonatal period of 28 days of infant life at guilan, in the north of iran. similar studies in iran and in regard to the neonatal period of 28 days are few and far between. the results of the present study indicated that 50.5% of neonates had skin lesions. the most prevalent lesions were lesions of erythema toxicum, cutis marmorata, diaper dermatitis, milia, salmon patch, and mongolian, respectively. in a study by firouzi et al., skin lesions were present in 79.8% of the cases. the prevalence of milia, erythema toxicum, salmon patches, and mongolian spots were 45.2%, 43%, 37.3%, and 37%, respectively [14]. in our study, erythema toxicum was noted in 15.1% of the population, which was in accordance with non-iranian studies that reported a lower prevalence of erythema toxicum [1]. according to a study in 2017, erythema toxicum and mongolian spots were seen in 50% of infants at birth [15]. in our study, erythema toxicum was more prevalent in neonates (15.1%), whereas mongolian spots were less prevalent (2.8%). according to previous studies, the prevalence of mongolian spots was reported to be over 90% among americans and asians [17–19]. karegar maher et al. reported the rates of mongolian spots and salmon patches to be 32.3% and 14.5%, respectively, among 1000 newborns [20]. khoshnevisasl et al. evaluated the incidence of birthmarks in 500 neonates born, of which 95.6% of them had at least one lesion. the most common skin table 2. maternal parameters and diseases variables level number percent type of delivery nvd 80 24.8 c/s 242 75.2 smoking smoker 1 0.31 passive smoker 12 3.73 addiction 1 0.31 gestational diabetes mellitus 40 12.5 hypothyroidism 40 12.5 hypertension 11 3.43 iron deficiency anemia 5 1.56 preeclampsia 4 1.25 peptic ulcer 2 0.62 hyperthyroidism 2 0.62 kidney stone 2 0.62 food allergy 2 0.62 minor thalassemia 2 0.62 brain tumor 1 0.31 urinary tract infection 1 0.31 emboli 1 0.31 g6pd deficiency 1 0.31 table 3. the frequency of cutaneous lesions detected in 323 neonates in the 28 first days of their life cutaneous lesions number percent erythema toxicum neonatorum 48 14.9 cutis marmorata 32 9.9 diaper dermatitis 26 8.1 milia 18 5.6 salmon patch 14 4.3 mongolian spot 9 2.8 congenital melanocytic nevus 3 0.9 lanugo hair 2 0.6 capillary hemangioma 2 0.6 allergy to medical tape 2 0.6 neonatal acne 2 0.6 heatstroke 2 0.6 morbiliform rash 1 0.3 ictiosis 1 0.3 accessory nipple 1 0.3 epidermolysis bullosa 1 0.3 total 164 50.8 eftekhari et al. 4 lesions were epstein’s pearl (60.4%) and mongolian spots (56%), respectively [21]. the lower rate of skin manifestations in the present study could be due to evaluating the full neonatal period of 28 days of infancy and also the lower rate of maternal hormones in the period of 28 days as compared with the first 3 days of life in previous studies. according to the results of the present study, there was no significant relationship between skin lesions and demographic factors of gestational age, type of delivery, or the family history of dermatological diseases. in a study by firouzi et al., natural vaginal delivery, use of medication, term gestation, and maternal disease were associated with a higher incidence of cutaneous lesions in neonates [14]. based on a study by karegar maher et al., maternal age was the only factor that showed a statistically significant association with the salmon patch. in addition, the upper eyelid and sacral regions were found to be the most common sites of the salmon patch and mongolian spots, respectively. the inclusion of a larger sample of neonates in studies by firouzi et al., and karegar maher et al., resulted in differences in findings. khoshnevisasl et al. evaluated the incidence of birthmarks in neonates born. they reported erythema toxicum was significantly associated with the type of delivery. in addition, there was a significant relationship between milia and mongolian spots with increasing gestational age [21]. some studies mentioned the higher prevalence of toxic erythema in infants with a higher gestational age. the higher rate of preterm infants in our study could be one of the possible causes of the lower prevalence of toxic erythema as compared with previous studies [15, 22-24]. in the present study, 5.38% of neonates with skin manifestations required treatment. six newborns required treatment for diaper dermatitis and one newborn for intertrigo. besides, milia, toxic erythema, postural melanoma, mongolian spot, salmon patch, and cutis marmorata do not require treatment and are self-limiting. hemangiomas in small children are selflimiting. severe neonatal acne needs treatment. congenital melanocytic nevi also only need to be monitored and do not require emergency treatment. as a limitation of retrospective studies, the number of studied variables and cofactors was limited. however, results of present study provide valuable insight for future general studies. to the best of our knowledge, this was the first study to be conducted in our region, during a fully neonatal period of 28 days. the results of the present study indicated that the rate of skin lesions was moderate to high in hospitalized newborns. in addition, erythema toxicum, marmorata, diaper dermatitis, salmon patches, and mongolian spots were more prevalent in infants. according to other results of our study, infant gender (female) and gestational diabetes can be considered as risk factors that increase skin lesions in newborns. besides, there was a positive association between neonatal acne and infant age, and between maternal melanocytic nevus manifestation and higher birth weight. considering that infants without existing indications can be exposed to higher risks, our findings can help pediatric physicians effectively in the early diagnosis and therapeutic procedures. authors' contributions concept and design: he, ss, hm. data collection, and field works: he, fp, sm, as. results analysis and interpretation: fp, sm, as. drafting and revision: ss, hm. all authors read and approved the final version of the manuscript. conflict of interests none to be declared. ethical declarations the study design was approved by the ethical committee of the mazandaran university of medical sciences [ir.mazums.rib.rec.1400.038]. financial support self-funding. references 1. haveri ft, inamadar ac. a cross-sectional prospective study of cutaneous lesions in 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branch, islamic azad university, isfahan, iran abstract akkermansia muciniphila is an anaerobic species of gut microbiome that has been proposed as a new functional microbiota with probiotic properties. recent research has shown the amazing abilities of probiotic bacteria, a. muciniphila, which resides in most people's intestines. these bacteria affect the body if it increases or decreases abdominal fat. the presence of a. muciniphila has opened new ways for the use of this plentiful intestinal symbiont in next generation therapeutic products, as well as targeting microbiota dynamics. a. muciniphila is particularly effective in increasing mucosal thickness and enhancing bowel barrier function. as a result, host metabolic markers improve. the host functions that are disrupted in various diseases with a particular focus on metabolic disorders in animals and humans. a specific protein in the outer membrane of a. muciniphila called amuc-110 could in the future be a strong candidate for drug production. as a result, we suggest that microbes and our microbiology or gut microbiome knowledge could be a new source for future treatments. the objectives of this review are to summarize the data available on the distribution of a. muciniphila gut in health and disease, to provide insights into the environment and its role in the creation of microbial networks at the mucosal interface, as well as to discuss recent research on its role in regulation. keywords: akkermansia muciniphila; type 2 diabetes; obesity; prebiotics introduction akkermansia was introduced by dutch microbiologist anton akkermansia (born october 28, 1940: died august 21, 2006) who studies the biology of microorganisms. this genus contains a single species called akkermansia muciniphila [1]. the word mucio is derived from the latin root of mucin in the gastrointestinal tract and phila is derived from the latin word filos. muciniphila also calls mucin an inhibitor. a. muciniphila is a dominant human intestinal mucin-degrading bacterium in a genus * corresponding author: dr. bahram nasr esfahani, ph.d department of bacteriology and virology, school of medicine, isfahan university of medical sciences, isfahan, iran tel/fax: +98 313 7922478 email: nasr@hlth.mui.ac.ir https://orcid.org/0000-0003-3264-3361 received: june, 28, 2020 accepted: august, 09, 2020 proposed in 2004 by muriel derrien and colleagues. it’s a gram-negative, oval-shaped, strictly anaerobic, non-motile non-spore-former. odd that such an abundant organism had to wait so long to be discovered. probably this has to do with the conditions used to its isolation, which is an anaerobic medium containing gastric mucin as the only carbon and nitrogen source. the genus akkermansia belongs to the division verrucomicrobia [2,3]. microbial ecosystem of a. muciniphila most likely, there is a relationship between fecal https://jcbior.com/ mohammadzadeh rostami et al. 2 microbiota and health indicators. not just a pure microorganism but an ecosystem that restores the complex balance between host biology and the environment. according to the abundant study of a. muciniphila, it is related to extensive metagenomics frequency and abundance of bacterial genes [4]. role of a. muciniphila in obesity and type 2 diabetes obesity-related metabolic disorders and cardiac metabolism are widespread worldwide. among all environmental factors, the intestinal microbiota is now an important intervention in host energy and metabolism that is susceptible to several noncommunicable diseases [5]. among the nextgeneration bacteria that appear to be useful, a. muciniphila is the future candidate. without a doubt, a. muciniphila is inversely related to obesity, diabetes, cardiovascular disease and superficial inflammation [2]. gut microbiota and its changes in the composition and biodiversity, can play an appropriate role in the expansion of metabolic diseases. in humans, most of the papers show that a depauperated microbial diversity is associated with a higher insulin resistance, obesity and inflammation although oral administration of a. muciniphila to mice fed with high-fat diet (hfd) extremely improves glucose homeostasis, the antidiabetic properties of this bacterium have not been indicated in humans because of its growth properties and oxygen sensitivity that set the use of living a. muciniphila improper for putative therapeutic chances. [6]. in addition, numerous documents have shown that pasteurization of a. muciniphila increases both stability and efficiency of strains [7]. this gives you the powerful a. muciniphila as the next generation of candidates to produce new foods or medicinal supplements with beneficial properties. finally, a specific protein in the outer membrane of a. muciniphila, called amuc_1100, which is one of the most abundant proteins involved in pellet formation, can be a powerful candidate for the production of subsequent drugs [8-11]. we suggest that microbes and microbiology, or our gut microbiome knowledge, could be a new source for future treatments. obesity provides the basis for developing type 2 diabetes and cardiovascular disease. these two pathologies are part of the metabolic syndrome. it is also an important issue in public health [7]. intestinal microbes play a vital part in regulating host metabolism and surface inflammation. several researches have shown that our eating lifestyles greatly impact the composition and function of the gut microbiota and may ultimately play a role in initiating or protecting against metabolic disorders [12,13]. from peribiotics to next-generation beneficial microbes: focus on identifying a. muciniphila one possible way to influence the intestinal microbiota, which is well documented, is to use selected microbes that market the host health benefits to the host by defining "living microorganisms that are used properly." other methods, such as the use of probiotics over the past 20 years, have received much attention [14]. the concept of prebiotics, first observed by gibson and rutherford (1995), has led to a large number of dietary supplements, leading to significant market growth [15]. peribiotics are the selective stimulation of the growth or activity of one or a limited number of microbial genera (species) or species in the gut microbiota that provide health benefits to the host [16]. according to the methodological and fundamental research of microbiologists, there has been a great deal of progress recently in our understanding of gut microbiota. however, the subject of peribiotics has not fully understood. despite various research on the molecular mechanisms underlying how prebiotics and intestinal microbes interact with the host, the identification of the candidate bacterium elaborate in useful effects on energy, glucose, lipid metabolism and immunity is still difficult [17]. a. muciniphila is one of the most abundant single species in the human gut microbiota (0.5% 0.5% of total bacteria) and in 2004 by maurice drine in his doctoral research at the university of wageningen as a specialist in the use of the distinct and specific mucus [18, 19] the discovery began with the fact that the human body produces "peribiotics" or microbial layers called mucus (sputum), which is a glycoprotein and offspring especially in the large intestine that destroy it goes [20]. while experiments on non-microbial mice have shown that a. muciniphila shows immunity and metabolic signaling, especially in the colon, the exact function of these microbes is unclear [21, 22]. further evidence of the function of a. muciniphila has been repeatedly identified by other prebiotic studies using inulin-type fructans, initially known as bifidogenic compounds that can increase the quantity of bifidobacterium species. they thanked the development of new techniques independent of mohammadzadeh rostami et al. 3 culture. the study investigated the effect of this type of prebiotics on a microbial complex in mice [23]. the first surprise was that more than 100 different species were affected by prebiotics [14]. among these bacteria, they found that the relative abundance of a. muciniphila increased more than 100-fold after peribiotic intake and reached more than 4.5% in highfat diets, whereas this effect on a normal diet (2.5% 0.09) was less in his model [22]. notably, these findings are approved in different sets of studies [23, 24]. some studies show that a. muciniphila was lower in the gut microbiota of diabetic rats and mice that were genetically or affected by obese diets. however, few studies have reported the frequency of a. muciniphila in mice with increased glucose and fat intake [15]. insulin-rich fructans have also been widely shown to show obesity-related metabolic disorders, including fat mass depletion, insulin resistance, hepatic steatosis, and intestinal barrier strengthening [25]. importantly, in humans, the frequency of a. muciniphila has been reduced in several pathological conditions, including obesity, type 2 diabetes, gastrointestinal inflammation, hypertension, and intestinal disease [26, 27]. on the other hand, it has been found that antidiabetic therapies, such as the use of metformin and bariatric surgery, are associated with a significant increase in mucosal levels. thus, much evidence has been shown that a. muciniphila can protect against specific metabolic disorders and risk factors for cardiovascular metabolism associated with a low degree of inflammation [28]. the role of a. muciniphila on and around the intestine the gut microbiota is the largest human microbial reservoir that has a prominent role in human health and is involved in regulating many host physiological pathways such as regulating gastrointestinal stimulation, digestive barrier permeability, lipid distribution, and energy homeostasis [5]. a great deal of data suggesting the relative frequency of a. muciniphila in obesity and metabolic disorders in mice and humans, we decided to investigate the usual association between a. muciniphila and metabolic enhancers [2, 5, 29]. carlota dao et al found that the use of a. muciniphila, a live fillet with 2.108 bacterial cells per day, partially protected diet-induced obesity in mice [2]. certainly, when dietary intake was not altered or dietary fat was removed, rats had a 50% lower weight gain treated with the live drug a. muciniphila. this protection is reflected by less subcutaneous and internal fat mass as well as by increased markers of fatty acid oxidation (acox1, cpt1a, acacb) in adipose tissue [30, 31]. in addition, animals treated with the live a. muciniphila substrate no longer showed insulin resistance, infiltrating inflammatory cells (cd11c) in adipose tissue which is one of the most important features of obesityassociated with superficial inflammation [32]. interestingly, the metabolic improvements following the administration of live a. muciniphila were comparable with results obtained from the treatment with oligofructose or inulin [30, 32]. however, live a. muciniphila did not mimic prebiotics food intake behaviors. there is also a great interest in a. muciniphila because of its association with animal and human health. remarkably, decreased levels of a. muciniphila have been observed in patients with inflammatory bowel disease (mainly ulcerative colitis) and metabolic disorders, suggesting that they may have potential anti-inflammatory properties [5 ,33.] conclusion several studies have shown that a. muciniphila can reduce the serum level of inflammatory cytokines like il-2, ifn-γ, il-12p40, and mcp-1, and also the lipid overload process linked with the ldl receptor pathway by decreasing apob48 and apob100 on ldls. meanwhile, a. muciniphila can modulate the immune system by production of short-chain fatty acid which is elaborate in signaling to the host by inhibiting histone deacetylase (hdac) or by activating g-protein-coupled receptors, which triggers other metabolic pathways [34,36]. the main steps towards the growth of a. muciniphila as a new probiotic candidate have been completed. initially, the identification of a. muciniphila being grown on a defined medium according to performance in humans. second, it has been found that inactivation of the bacteria by pasteurization improves its stability and durability. third, amuc_1100 introduced as a key mechanism of interaction between a. muciniphila and its host. fourth, the evidence that a. muciniphila may be safely managed in the targeted people [9, 17, 35]. eventually, the pasteurized bacteria and the isolation of bacterial components like the fairly small 30-kda amuc_1100 cause to putative progress of drugs based on a. muciniphila related products that could target inflammatory bowel disease or diseases that compromise the function of the bowel barriers [33]. as mohammadzadeh rostami et al. 4 a result, comprehensive information on a. muciniphila has become progressively available, and many of the criteria set for evaluating novel food safety in europe can be fulfilled. however, no studies on the toxicological properties of a. muciniphila, including dose-response studies, long-term studies, and reproduction, have been published so far. such studies are likely to be important before authorities complete a comprehensive safety assessment. author contributions all authors contributed equally to this manuscript and approved the 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https://pubmed.ncbi.nlm.nih.gov/32153527/ https://pubmed.ncbi.nlm.nih.gov/32153527/ https://pubmed.ncbi.nlm.nih.gov/32153527/ https://pubmed.ncbi.nlm.nih.gov/32153527/ journal of current biomedical reports jcbior.com volume 2, number 2, 2021 eissn: 2717-1906 1 brief report hiv/aids awareness and attitude among factory workers in shiraz, iran behzad dehghani1, zeinab shahsavani2, abbas dehghani1,* 1department of bacteriology and virology, school of medicine, shiraz university of medical sciences, shiraz, iran 2student research committee, school of nutrition and food sciences, shiraz university of medical sciences, shiraz, iran abstract hiv has been introduced as a causative agent for aids which is still considered as a major health problem for the country and a burden on the health care system. raising awareness and knowledge plays a critical role in controlling hiv infection in all social groups. therefore, this study aimed to investigate the awareness and attitudes of factory workers regarding hiv/aids infection in shiraz, iran. this cross-sectional study was conducted on 92 factory workers in shiraz, selected via the random sampling technique. a standard researchermade questionnaire was used to collect data. around 84% of participants were male and 65.2% had 30 years of age or more. the majority of responders considered shared syringe as the main transmission route in iran. the awareness level of the general aspects of hiv infections and possible transmission modes were high. a large proportion of responders believed that patients should be supported by the governments and that they have the right to lead a normal life, meanwhile they were scared for their children having an infected classmate. it can be concluded that the public programs to increase awareness had a positive effect on factory workers' knowledge. however, some misconceptions toward hiv patients were identified that need to be corrected. in addition, further studies should be conducted in other cities to reach an accurate estimate of hiv/aids awareness among factory workers as a big part of our society. key words: hiv, aids, awareness, factory workers, iran 1. introduction acquired immune deficiency syndrome (aids) has started to spread since 1970s rapidly and became a mysterious pandemic in the 1980s; it was revealed that human immunodeficiency virus (hiv) caused aids. since then, it has emerged as the most difficult challenge to public health [1, 2]. the most recent who report estimated people living with hiv at the end of 2019 around 38 million [3]. although the four main hiv transmission routs includes unprotected sexual intercourse, contaminated blood transfusion, breast milk, transmission from an infected mother to her baby at * corresponding author: abbas dehghani, msc department of bacteriology and virology, school of medicine, shiraz university of medical sciences, shiraz, iran tel/fax: +98 713 2304356 email: abbasdeh@mail.ir https://orcid.org/0000-0002-2386-7454 received: april, 08, 2021 accepted: may, 07, 2021 birth, this report introduced sexual intercourse as the main transmission route especially among the age group 15-49 years [3, 4]. while in most regions of the world a decline of the epidemic has been reported, in the middle east and north africa regions a growing epidemic among key populations has been indicated recently [4]. based on the recent reports from iran, it was estimated that 59,000 [5] individuals are living with the hiv and based on previous studies, sexual intercourse, needle sharing, and mother to child transmission have been reported as the main routes of aids transmission [6]. © the author(s) 2021 https://jcbior.com/ dehghani et al. 2 prevention of hiv infections was suggested as the most cost-effective measure to control this disease which needs to raise awareness in all social groups of a country [7], and risky practices and insufficient knowledge are major burdens in preventing the spread of hiv. during the last decades, several public programs were conducted to increase hiv/adis awareness in iran and several studies tried to assess knowledge and attitude toward this disease in different parts of the society. a large proportion of studies have focused on some specific groups including students [8, 9], medical staff [10, 11], and hairdressers [12, 13], and it seemed factory workers who consist a large part of our society were ignored. with this background in mind, the aim of this study was to evaluate the knowledge and attitudes toward hiv infection and assess the effect of public programs among factory workers in shiraz, south iran. 2. materials and methods 2.1 study area in this cross-sectional study, 92 workers enrolled who were randomly selected from different firms in shiraz, south iran, during 2017-2018. a standardized questionnaire previously designed by our research group was used including a series of questions about hiv infection, different transmission routes, and treatment. different inclusion criteria were considered including, 1) iranian nationality, 2) being muslim, 3) working in a firm in shiraz, and 4) hiv-negative status. in addition, participants with relatives suffering from hiv/aids were excluded. to assess the reliability of the questionnaire, it was administered to 15 participants two times with a retest interval of a week, which showed moderate-to-high reproducibility. 2.2 measurements data were collected through a standardized questionnaire containing 26 questions. the questionnaire was considered to be flexible to responders and they were able to answer any question even though they were not informed in this regard. 2.3 statistical analysis in the present study, the sample size was calculated using cochran’s formula, and the estimated power was around 0.8, which was acceptable. to evaluate the construct and concurrent reliabilities the cronbach’s alpha coefficient was used. besides, the pearson’s product-moment correlation was employed to analyze the confirmatory and explanatory factors. all data were coded using microsoft excel (microsoft, mountain view, ca), and data analysis was performed using the spss, version 22 (ibm corp., somers, ny). chi-square test and cross tabulation were used to compare groups and a p-value of <0.05 was considered statistically significant. 3. results in all 92 workers enrolled, of which 83.7% were male, the majority of participants were more than 30 years old (65.2%), the education level of the majority of them was high school diploma (42.4%) and the majority of participants had diploma (42.4%). our results showed that the majority of the workers (96.1%) involved in the study had heard about hiv/aids. table 1 also shows the results of the participants’ knowledge and reveals that generally their awareness about hiv and aids was satisfied and the mean of correct answers for questions was 80.6%. around 96% believed that aids is a serious disease and adherence to moral principles can prevent the spread of aids. out of 89 participants who answered the question about the major hiv transmission route in iran, 73.9% selected shared syringes used by addicted people. besides, 16.3% and 6.5% of them believed sex and blood transfusion are the other transmission routes of aids in iran, respectively. the results of awareness about hiv transmission methods showed 95.7% of participants believed that contaminated dental and surgical instruments as well as sexual intercourse (marriage) could transmit this virus. furthermore, the percentage of responders believed that donated organs (blood or tissue) and mother to baby through womb can spread hiv were 94.6% and 92.4%, respectively. moreover, more than half of responders (52.2%) believed that hiv can be transmitted via breastfeeding. a large percentage of the responders did not consider sharing the items or places belong to aids/hiv patients including the swimming pool or bathrooms with the aids patients (73.9%), hiv patients’ personal items (55.4%) (i.e., clothes, comb, towel, etc.), their tear (excreta) (70.7%), touching the aids patients (88%), using the items and food of dehghani et al. 3 aids patients (69.6%), as the hiv transmission routes. it was noticeable that 85% feared using the aids patient’s razor (shaver). table 2 shows the workers' attitude toward hiv infected patients. comparison study based on sex, age, and education level revealed that in all cases there was no significant difference among participants groups. discussion compared to two previous studies conducted by this research group in shiraz which studied awareness among high school students [9] and college students [14]; it can be concluded factory workers and high school student share the same idea and, in both groups, more than 50% believe that hiv infections is table 1. the responders’ knowledge on hiv/aids general knowledge correct answer (%) dose not know (%) is aids caused by a virus? 84.8 13 is aids a contagious disease? 94.6 5.4 is aids a hereditary disease? 81.5 0 is aids a curable disease? 87 1.1 is aids mostly observed in developing or underdeveloped countries? 20.7 2.2 is aids a serious disease or as simple as catching a cold? 95.7 0 does a person infected with hiv have a less resistant body against other diseases? 92.4 0 is there any vaccine against hiv infection? 83.7 1.1 can patient have negative diagnosis results? 53.3 1.1 does a person transmit the hiv virus without showing the aids symptoms? 80.4 1.1 will morality prevent the spread of aids? 95.7 0 can controlling the blood of donators be effective to prevent the spread of hiv? 90.2 0 can the sterilization of dental instruments by dentists prevent aids infection? 95.7 0 will early diagnosis of hiv virus in people help the prevention of aids infection? 76.1 2.2 which part of body is affected by hiv virus? 83.7 5.4 which method is used to detect hiv virus in blood? 75.5 1.1 table 2. the responders’ attitudes towards hiv/aids patients general attitudes totally agree (%) agree (%) no comment (%) disagree (%) completely disagree (%) dose not answer (%) students infected with hiv virus should be educated in a separate school 39.1 17.4 12 16.3 15.2 0 if our children have a hiv infected classmate, we should change the school of our children 38.0 17.4 13.0 20.7 10.9 0 hiv infected patients should be supported 70.7 28.3 1.1 0 0 0 hiv infected patients must benefit from social rights such as studying, working, ... 63.0 30.4 3.3 1.1 2.2 0 we must allow the aids patient to use public bathrooms or swimming pools. 30.4 28.3 18.5 8.7 14.1 0 one of the main reasons of aids infection is disobeying religious and moral principles 46.7 39.1 8.7 5.4 0 0 people should be aware of aids disease since it's a health and public issue 83.7 14.1 2.2 0 0 0 dehghani et al. 4 mainly found in developing and underdeveloped countries. although, in contrary, around 70% of college students disagreed with this idea. interestingly in all three reports, responders considered the shared syringe among addicted people as the major transmission route in iran, along with, in second and third ranks, sex and blood transfusion were placed. knowledge about transmission methods in three studies showed similarity in some cases including contaminated medical instruments, sexual intercourse, donated organs (or blood), and mother to baby through the womb which were appropriately high in all studies. while, in extremely rare cases, hiv can be transmitted by sharing razors, using the aids patient’s razor was considered by 74.2% of college students, 76% high school students, and 85% factory workers as a mode of transmission. it is established that breastfeeding substantially increases the risk of hiv-1 transmission from mother to child [15], the awareness about this route amongst high school students was 62% which was higher than factory workers (52.2%) and significantly higher than college students (42.7%). comparison results of three studies in misconceptions about modes of transmission showed high school students had the highest rates of misconceptions. using hiv patients’ personal items showed varied results, while only 18.2% of college students feared of using shared personal items, this subject was significantly high among factory workers (44.6%) and high school students (67%). additionally, sharing the swimming pool or bathrooms was considered by 15% of college students, 32% of high school students, and 26.1% of factory workers as a route of transmission. besides, 8% of college students, 18% of high school students, and 12% of workers considered touching as a route. also urine, tears, and saliva was recognized as a spread way by 25.2%, 24%, and 29.3% of college students, high school students, and factory workers, receptively. college students showed the best attitude toward hiv patients in comparison with high school students and factory workers. while a majority of college students disagreed with using separate schools for hiv infected students and changing the school if there is an hiv positive classmate; the majority of high school students and workers agreed with these subjects. furthermore, contrary to the majority of high school students who believed that public bathrooms or swimming pools are not supposed to be available for aids patients, the majority of college students, and workers thought they must be allowed to use these facilities. to the best of our knowledge, there was no published study on hiv awareness in factory workers in iran as well as limited studies were done in other countries. mullany et al., 2003 researched to assess hiv awareness among 725 burmese migrant factory workers in tak province, thailand [16]. the average percentage of questions answered correctly about hiv transmission was 41.3% which was lower than our results which were around 77%. besides, only 12% of burmese workers believed that hiv was not transmitted through casual contact (kissing, coughing, sharing a toilet, etc.) which was significantly higher in the present study (88%). in general, it can be concluded that the level of awareness in the present study was significantly higher than in mullany’s study. in another study, abera et al., 2003 conducted a crosssectional study to investigated hiv/aids awareness among workers in the informal sector in ethiopia [17]. while, in the present study we could not define any different between male and female responders, in abera’s study females were less aware than males. furthermore, 88% ethiopian workers considered sex as a rout of transmission which was lower than our results (95.7%). only 13.9% of ethiopian workers found hiv patients’ personal items as the hiv transmission routes [17]; however, this was around 45% among iranian workers which showed a big misunderstanding about hiv transmission route. in addition, islam et al., 2010 collected data using a questionnaire from 123 participants (female migrant workers) who were ready to fly from bangladesh to other countries to take up an overseas job. similar to our result the majority of bangalees workers were familiar with potential modes of acquiring hiv infection. contrary to our study, only 46.3% of bengalese workers found unprotected sex as a potential mode of hiv infection, while it was 95.7% among iranian workers [18]. hasan et al., 2013 [19] investigated the level of awareness on hiv/aids amongst the garment workers in bangladesh. in this study, 303 workers in three selected garment factories in dhaka city were enrolled. similar to our study the vast majority of dehghani et al. 5 workers knew aids as a transmissible and preventable disease. contrary to our results, bangalees workers believed that sexual intercourse is the main route of hiv transmission; however, iranian workers considered shared syringe as the major route which may be related to different social disorders in iran and bangladesh. additionally, while, hasan et al., showed the level of awareness increased significantly with age; in the present study, we did not find any relationship. the main limitation of the present study was the inclusion of 92 factory workers, which was due to the short duration of the study. hence, the sample size was too small to find a comprehensive perspective on the level of awareness among workers. however, our results could show the level of knowledge about the transmission and treatment of hiv/aids which can be practical for further studies. in general, the hiv/aids awareness of factory workers was satisfactory and in many cases, the prevalence of correct answers was higher than 90%. it seems there is no need to raise awareness about general aspects of hiv/aids and their transmission routes. however, there are some misconceptions and misunderstandings toward hiv-positive patients, especially regarding infected students which showed the immediate need of raising awareness. acknowledgments the authors would like to acknowledge all the study participants and ali dehghani and yalda dehghani for their invaluable assistance in editing this manuscript. author contributions conception or design of the work: bd and zs; data collection: zs, ad; data analysis and interpretation: bd, zs; drafting the article: bd, ad; critical revision of the article: bd, ad. all authors read and approved the final version of manuscript. conflict of interests the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. ethical declarations the researchers obtained the approval of the human research ethics committee of the university. in addition, prior to the study, the written informed consent forms were read and signed by each participant in the study. financial support self-funded study. references 1. unaids. report on the global hiv/aids epidemic july 2002. available from: http://data.unaids.org/pub/report/2002/brglobal_aids_report_ en_pdf_red_en.pdf 2. jaiswal s, magar bs, thakali k, pradhan a, 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https://pubmed.ncbi.nlm.nih.gov/14722454/ https://pubmed.ncbi.nlm.nih.gov/14722454/ https://pubmed.ncbi.nlm.nih.gov/14722454/ https://pubmed.ncbi.nlm.nih.gov/12655834/ https://pubmed.ncbi.nlm.nih.gov/12655834/ https://pubmed.ncbi.nlm.nih.gov/12655834/ https://doi.org/10.4314/ejhd.v17i1.9781 https://doi.org/10.4314/ejhd.v17i1.9781 https://doi.org/10.4314/ejhd.v17i1.9781 https://doi.org/10.1007/s10903-010-9329-5 https://doi.org/10.1007/s10903-010-9329-5 https://doi.org/10.1007/s10903-010-9329-5 https://pubmed.ncbi.nlm.nih.gov/23710426/ https://pubmed.ncbi.nlm.nih.gov/23710426/ https://pubmed.ncbi.nlm.nih.gov/23710426/ journal of current biomedical reports jcbior.com volume 3, number 1, 2022 eissn: 2717-1906 1 original research anti-bacterial effect of white tea extract on streptococcus mutans and streptococcus salivarius nazanin bashardoust1, shima daryoush2,*, adel ashouri3, parisa rahimirad2 1department of oral and maxillofacial pathology, dental sciences research center, school of dentistry, guilan university of medical sciences, rasht, iran 2student research committee, school of dentistry, guilan university of medical sciences, rasht, iran 3private dentist, rasht, iran abstract due to the increasing frequency of drug-resistant strains among different types of microorganisms, the finding of antimicrobial and antifungal compounds of natural substances, which certainly has fewer side effects, has long been of interest to researchers. therefore, the aim of this investigation was to study the effect of white tea extract on reducing streptococcus mutans and streptococcus salivarius. white tea was prepared in powder form and then its hydroalcoholic extract of it was prepared. the antimicrobial effects of extracts on s. mutans and s. salivarius were performed using well diffusion and broth microdilution methods. all experiments were performed in triplet replications. antibacterial susceptibility testing revealed that white tea extract at concentration of 500 µg/ml had a remarkable antibacterial activity against s. mutans, and s. salivarius with a zone of inhibition, 25±1 mm and 30±1, respectively. the minimum inhibitory concentration (mic) values of the both tested strains were estimated 31.25 µg/ml. moreover, the minimum bactericidal concentration (mbc) values of white tea extract were estimated 62.5 µg/ml against both streptococci. considering the inhibitory power of the white tea extract on the gram-positive bacteria of s .salivarius and mutans, it can be concluded that this plant can be used in various industries, including the pharmaceutical and sanitary industry and it can also improve oral health as s. mutans and salvarius are the most important bacteria causing decays. keywords: camellia sinensis, streptococcus mutans, tea, microbial sensitivity tests 1. introduction after water, tea (camellia sinensis (l.) kuntze) is the most consumed beverage among the people of the world. tea contains green tea polyphenols ambe phytoextracts, and is one of the most traditional and widespread beverages in china and japan, derived from the young branches of camellia sinensis. cathechins and flavins in tea are also considered active ingredients in microbiology [1]. in 2005, iran's share of world tea consumption was 3.3 percent, while it accounted for only 1.4 percent of world production *corresponding author: shima daryoush, dds school of dentistry, guilan university of medical sciences, rasht, iran tel/fax: +98 911 3103121 email: shima.daryoush@gmail.com https://orcid.org/0000-0003-2945-5132 received: august, 26, 2021 accepted: december, 27, 2021 [2]. a class of polyphenols called catechins stands out among their bioactive ingredients, of which epigallocatechin gallate is the most common and has high antioxidant properties [3]. other phenolic compounds for instance caffeine, gallic, proanthocyanidols, chlorogenic or cinnamic acids, quercetin, and theophylline; and minerals as fluorine, manganese or chromium are also found in teas. studies mostly focused on their antioxidant potential and their effect on the prevention and treatment of degenerative diseases. various studies have © the author(s) 2022 https://jcbior.com/ bashardoust et al. 2 investigated on the effect of teas in controlling cardiovascular diseases, increasing bone density, protecting against neurodegenerative diseases and improvement of type 2 diabetes [3]. white tea is known to be the least processed form of tea and have a series of powerful bioactivities, such as antioxidant, anti-inflammatory, anti-mutagenic, and anti-cancer activities [4]. it is reported that ingesting white tea for a long time has a protective role for tissues from acute oxidative stress [5]. black tea is advisable as anticariogenic food because plaque ph values are not below essential value [6]. compared with black and green teas, just few studies have been worked on the antimicrobial activity of white tea. white tea is different from others regarding the processing methods. this kind of tea is extracted from young leaves, even before the buds are completely bloomed [7]. both white and green tea are well-known to contain catechins like epigallocatechin gallate (egcg), epicatechin gallate, epicatechin in large volumes, and epigallocatechin, isolated green tea catechins could be suppressor of elastase and collagenase. in comparison noticeably higher anti elastase and anti-collagenase activities are exhibited in white tea. early investigations have studied that egcg exhibited strong collagenases inhibitory activity that decrease the organic matrix during erosion. based on the study results, green and white tea consumption enhanced microhardness of dentine, which is showing functional remineralization. this could be a result of the tea action on the collagen network, which may result in collagen stabilized and left intrafibrillar spaces of collagen network open for remineralization [8]. white tea mouthrinse powerfully inhibits formation of plaque although not as comparable to mouthrinses which contain chlorhexidine. hence, for those who prefer herbal products, white tea mouthrinse is an appropriate option. as the processing temperature is low in comparison with processing temperature of other kind of teas, white tea retains its nutrients. the components of white tea are alanine, threonine, histidine, aspartic acid, glutamic acid, and amino acids. polyphenols, which is a natural antioxidant is found in the important ingredients in white tea [9]. some studies have shown that there is a relation between dental caries and salivary level of s. mutans, a gram-positive species [10]. while some studies have implied that lactobacillus salivarius (gram-positive anaerobic bacteria) wb21 can be helpful for controlling dental caries, and other oral diseases [11]. in this study we are trying to investigate the effect of white tea extract on the reduction of s. mutans and salivarius bacteria according to dental caries. tea is extremely popular and usually people use it daily, if we can prove that one type of it is useful to improve oral health, hygiene or immune system against cancer and to control caries that would be valuable information. it can also suggest a healthy lifestyle .this study directly measures the effectiveness of tea extracts on microorganisms that defects oral health and uses global standards and guidelines. 2. methods and materials 2.1 bacterial strains this present in vitro study was conducted on two strains of gram-positive cocci including s. mutans (atcc 35668), and s. salivarius (atcc 19258). bacterial strains were taken from pastor technolog institute (tehran, iran). the strain was obtained from the strain stock through being cultured overnight in aerobic conditions at 37 °c on a triptic soy agar (tsa) plate containing fibrous sheep blood 5% (merck, germany). 2.2 characterization of extract initially, 500 grams of white tea in the department of agriculture in the university of guilan was turned into powder by an electric mill and kept in a glass jar. first, the equipment, including glassware such as beaker, is sterilized in autoclave (omran, type h2a) for 15 minutes at 121 °c and under at least 15 psi of pressure. after sterilization, 250 g of white tea powder was weighed with a scale and poured into the beaker, and the beaker was transported to 1000 ml of distilled water. then, it was placed on a heater stirrer (ika, rh basic 2) and used. next, stirred it from the magnet (ika, rh basic 2) until the final volume reached 500 ml after a few hours. after the extract was cooled, it was filtered by filtration or sterile gas, then the solution was placed under the laminar flow hood (wieteg, wlc-v1500) by uv light for 24 hours to be sterilized and free of bacteria. finally, it was kept in a container in the refrigerator, away from light. 2.3 antimicrobial susceptibility testing antibacterial property of the white tea extract was assessed method on muller-hinton agar (mha) via bashardoust et al. 3 well diffusion (merck, germany), enriched with 5% sheep blood [12]. briefly, a saline suspension of bacterial colonies was made to prepare the bacterial suspension, and adjusted to the tube according to 0.5 mcfarland standard (1.5 × 108 colony forming units (cfu)/ml), then inoculated on the mha surface via sterile swab. for extract antimicrobial activity test, 5 mm wells were punched into the plates. lastly, 10 µl of extract (500 µg/ml) was added into the wells. prepared plates were incubated at 37 °c for 24 hours on a rotary shaker at 160 rpm, then the growth inhibition clear zone was evaluated. furthermore, susceptibility to antibiotic was determined toward gentamicin (10 µg), and tetracycline (30 µg) antibiotic discs (mast, uk) as the positive control, and distilled water as negative control. all tests were done in triplicate. the minimum inhibitory concentration (mic) of white tea extract was obtained via standard broth microdilution according to the guidelines of clinical and laboratory standards institute (clsi) (30th edition) [13]. mueller hinton broth (merck, germany) containing 5% sheep blood was used to determine the mics for all bacterial strains. strains were cultured in 96-well microplates. to evaluate the inhibitory effects of the antibacterial agents on bacterial growth, each well was supplemented with a range of concentrations (500 µg/ml to 31.25 µg/ml) of the active agents. the final inoculum concentration was approximately 5 x 105 cfu/ml in each well. following at 37 °c and 16-18 hours of incubation, the wells were checked for any microbial growth, and the mic was characterized as the minimum concentration that did not produce visual growth. the minimum bactericidal concentration (mbc) was illustrated as the minimum concentrations resulting microorganisms 99.9% mortality rate in the primary inoculums by seeding on mha contain 5% sheep blood [14]. 2.4 statistical analysis spsstm software version 21.0 (ibm crop., usa) was used for analysis. the outcomes are provided as relative frequency. the values arereported as mean ± standard deviation (continuous variable) or group percentage (categorical variable). 3. results antibacterial susceptibility testing revealed that white tea extract at concentration of 500 µg/ml had a remarkable antibacterial activity against s. mutans, and s. salivarius with a zone of inhibition, 25±1 mm and 30±1, respectively. the comprehensive results of antibacterial activity of white tea extract illustrated in table 1. the mic and mbc activity of white tea extract were determined by broth microdilution technique. the mic values of the both tested strains were estimated 31.25 µg/ml. moreover, the mbc values of white tea extract were estimated 62.5 µg/ml against both streptococci. 4. discussion complexity of mechanisms that create antibiotic resistance has led to the problem of bacterial resistance to antibiotics that have always plagued medical systems in recent decades. therefore, finding new antimicrobial compounds with minimal side effects is a topic that has always been on the minds of t a b le 1 . a n ti b a c te ri a l a c ti v it y o f w h it e t e a e x tr a c t b y w e ll d if fu si o n m e th o d s tr a in e x tr a c t (5 0 0 µ g /m l) p o si ti v e c o n tr o l n e g a ti v e c o n tr o l g e n ta m ic in t e tr a c y c li n e d is ti ll e d w a te r s . sa li v a ri u s 3 0 ± 1 m m 4 5 m m 3 0 m m 0 s . m u ta n s 2 5 ± 1 m m 3 5 m m 5 0 m m 0 bashardoust et al. 4 researchers. in this study we assessed white tea extract’s effect on s. mutans and s. salivarius which had an antibacterial result on them by forming zone of inhibition, more effectively on s. salivarius than s. mutans. in a study, the antibacterial effects of polyphenols on several species of pathogenic bacteria, including escherichia coli were evaluated. the mic for e. coli was several times more abundant than other bacteria, such as s. aureus (1519±949 and 192±91 µg/ml, respectively) and even more than salmonella (795 ± 590 g µg/ml) [15]. in contrast, there are conflicting reports about the antimicrobial activity of tea extract against pathogenic bacteria. according to hara and ishigami salmonella typhimurium and campylobacter jejuni are resistant to tea extract, while other researchers have reported s. typhimurium susceptibility to aqueous extract of tea. this conflict can be due to different method used in the study and species [16]. in a study of the effect of a kind of tea called oolong on s. mutans, by analyzing extract of the tea, researchers found a fraction of it (otf10) a polyphenolic compound with an inhibitory effect on the enzyme glucosyltransferase 1, bacterial enzyme necessary to form dextran plaque, and as a result, tooth decay. interestingly, catechins and other small tea polyphenols did not have such an effect, but black tea thioflavin did. this inhibitory effect, which prevents bacteria from adhering to dental plaque and tooth decay, was greater in otf10 than in oolong tea extract this different conclusion may be because of different type of tea that was used [17]. herbal polyphenols are thought to exert their inhibitory effects on bacterial growth by producing hydrogen peroxide [18]. at the same time, bactericidal cathechins have been shown to damage two layers of membrane fat [19]. although polyphenols are potent antioxidants, they may act as peroxidants under certain conditions [20]. it is possible that tea polyphenols exert their inhibitory effect on bacterial growth in this way. in another study, the effect of white tea aqueous extract on serum levels of antioxidant enzymes in arsenic-exposed rats was investigated. the results showed that consumption of white tea by reducing the activity of antioxidant enzymes and strengthening the antioxidant defense system reduces the oxidative stress caused by arsenic [20]. in one study, the antimicrobial effect of green tea extract on s. mutans and enterococcus faecalis was investigated. sodium hypochlorite and chlorhexidine were used to determine the susceptibility and resistance of bacteria to green tea. results showed that in s. mutans, the diameter of the green tea growth inhibition zone was not significantly different from chlorhexidine (p = 0.305). since the results of ranjbar et al.’s investigation were inconsistent with the results of the present study, it is possible that this may be due to different methods of studies [21]. by studying the results of research on the antimicrobial properties of white tea extract, it is understood that some factors such as; climatic conditions, plant habitat cultivation, plant cultivation and maintenance conditions, drying method, extraction method and concentration used in studies can affect the amount of antimicrobial effects and other effects related to plant extracts. also, increasing the concentration increases the lethality of the extracts. considering the results and inhibitory power of white tea extract on the gram-positive bacteria of s. salivarius and s. mutans, it can be concluded that this plant has the potential to be used in various industries, including pharmaceutical and health industries, due to its antibacterial properties. authors’ contributions all authors have cooperated in conception and design, or analysis and interpretation of the data for this study; drafting the article or revising it for important content and they approved the final version. conflict of interests the authors declare that there is no conflict of interest. ethical declarations study did not include any human participants and was ethically approved by national research ethics committee and the code given was ir.gums.rec.1396.1238. financial support self-funded. bashardoust et al. 5 references 1. granato d, prado-silva ld, alvarenga vo, zielinski aaf, bataglion ga, morais drd, et al. characterization of binary and ternary mixtures of green, white and black tea extracts by electrospray ionization mass spectrometry and 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https://dx.doi.org/10.22038/jmds.2015.5807 https://dx.doi.org/10.22038/jmds.2015.5807 https://dx.doi.org/10.22038/jmds.2015.5807 journal of current biomedical reports jcbior.com volume 2, number 3, 2021 eissn: 2717-1906 1 original research investigation of the effect of lactobacillus paracasei td3 on cutaneous wound healing and induction of angiogenesis on male wistar rats abolfazl dehkohneh1, ahmad jiriaie2, omid ardalani3, amin mohsenzadeh4, hoda sabati5, parvaneh jafari6,* 1department of biotechnology, faculty of advanced science and technology, tehran medical sciences, islamic azad university, tehran, iran 2microbiology department, faculty of science, islamic azad university, arak branch, arak, iran 3department of microbial biotechnology, school of biology and centre of excellence in phylogeny of living organisms, college of science, university of tehran, tehran, iran 4department of microbiology, faculty of science, ardabil branch, islamic azad university, ardabil, iran 5biotechnology and biological science research center, faculty of science, shahid chamran university of ahvaz, ahvaz, iran 6department of food safety, food security research institute, islamic azad university, arak branch, arak, iran abstract skin plays an essential role in the prevention of pathogenic microorganisms entrance. it is also considered as the first line of immune defense in our body. the therapeutic effects of probiotics on wound healing are well reported over the past decade. nevertheless, there are limited publications about the impact of probiotics on skin wound healing. here we assessed the effect of lactobacillus paracasei td3 on cutaneous wound healing in male wistar rats. during this research, thirty-six male wistar rats grouped into control positive, control negative, and trial groups, then the skins of rats were incised to make a full-thickness wound. an ointment produced from probiotic l. paracasei td3 was administrated to the trial group, and immunohistological factors of each host were evaluated and compared with control groups. the final results showed that although l. paracasei td3 could proceed the wound closure earlier than other groups and could induce angiogenesis in trial group, it could not cause any significant changes in the levels of monocytes, lymphocyte, mast cells and polymorphonuclear leukocytes in the trial group compared to control groups. this probiotic bacterium may be effective besides other probiotic bacteria. keywords: probiotics, wound healing, lactobacillus paracasei td3, skin, probiotic ointment 1. introduction skin plays an essential role in the prevention of pathogenic microorganisms’ entrance. it is also considered as the first line of immune defense in our body [1, 2]. the wound causes the skin to lose its integrity, thus activate a cascade of an event that led to wound healing. wound repair process includes three *corresponding author: parvaneh jafari, ph.d department of food safety, food security research institute, islamic azad university, arak branch, arak, iran tel/fax: +98 86 3413 2451 email: : p-jafari@iau-arak.ac.ir https://orcid.org/0000-0003-2771-9309 received: april, 25, 2021 accepted: june, 26, 2021 overlapping phases, namely inflammation, granulation, and remodeling [3, 4]. on the other hand, recently, some authors describe the four-phase concept for wound healing, including hemostasis, inflammatory, proliferation, and remodeling, respectively [5, 6]. © the author(s) 2021 https://jcbior.com/ dehkohneh et al. 2 there are several strategies to improve one or more phases to accelerate the wound healing process. since chemical agents have adverse side effects, the tendency to use a novel alternative method is increased [5, 7, 8]. an appropriate agent should accelerate the wound repair process to prevent infection as well as angiogenesis induction. more recently, some authors evaluate the effect of different strains of probiotics as a therapeutic agent. according to the world health organization (who) definition, probiotics have beneficial effects on the host when they are consumed adequately. probiotics are humanfriendly bacteria that have healthy effects on the human body, including reduction of serum cholesterol, immune-system improvement, anticancer properties, and prevention of body infection [9]. in this study, we investigated the effects of l. paracasei td3, a native iranian probiotics, on wound healing and induction of angiogenesis. moreover, we evaluated the level of production of lymphocytes, mast cells, polymorphonuclear leukocyte, and monocytes, besides examination of the epithelialization, edema, inflammation and granulation. 2. method and materials 2.1 animals and feeding thirty-six male wistar rats weighing 150-180 g and 3-4 weeks old were purchased from baqiat-allah research center. they were divided into three groups (n=12) after 10 days acclimatization, negative control (nc), positive control (pc), and trial (probiotic). the tested groups were housed under the controlled condition of temperature (22-25°c), light (12h light and 12h dark), and humidity (20%-30%). in the whole experiment period, all the rats had free access to both food and water. the rats were fed by autoclaved food, which protein (23%), salt (0.55%), and lysine (1.15%) were the most abundant ingredients on their food. after incision, they were killed at 3, 7, 14, and 21 days. our study was carried out according to the approved protocol by the institutional animal care and use committee (iacuc). 2.2 bacteria strain in this study, the lyophilized bacterium was purchased from takgen zist company (tehran, iran). to prepare the ointment, 4.8 g lyophilized bacteria were mixed with 1000 g eucerin. 2.3 anesthesia and wounding process all the rats were anesthetized with an injection of a mixture of xylazine (15mg/kg) and ketamine (20mg/kg) into the peritoneum. the dorsal area was shaved and disinfected by alcohol (70%) under anesthesia, and the skins of tested rats were incised to make a full-thickness wound approximately 1.5 cm2 by a surgical blade. after the wounding, the rats were housed in a separate sterilized cage. the wound treatments were carried out in tested groups once daily. for each wound, 1g of prepared ointment was applied to the probiotic group. for the control group, 1g of probiotic free eucerin was used. after one day the wounding; nc without any treatment, pc was treated with eucerin only and trial (probiotic) was treated with eucerin plus l. paracasei td3 strain (1012 cfu(. 2.4 ulcer size assessment assessment of the ulcer area and percentage of the ulcer healing was performed. length and width measurements of the wound (mm2) were performed in days 1, 3, 7, 14, and 21. the percentage of healing was calculated as below: percentage of healing = (ulcer area on the first day)−(ulcer area in the specific day) (ulcer area in first day) × 100 2.5 histological examination rats were sacrificed on days 1, 3, 7, 14 and 21, and tissues from the wound site was removed. the tissue samples were sectioned out and fixed overnight in 10% neutral buffered formalin, then dehydrated gradually in ethanol and embedded in paraffin. sections of 5 mm were obtained in an automated microtome. ulcerated sections were stained with hematoxylin-eosin (h&e) for histological evaluation during healing. 2.6 statistical analysis statistical analysis was performed using the anova test to determine significant differences between groups. data were analyzed using graph pad software version 6. in entire experiment the p-value less than 0.05 means significant alteration. 3. results 3.1 monocyte production monocyte production was found to decrease significantly on day 14 in the trial group compared to control negative group (p value <0.05). this change was not significant between the trial group and the dehkohneh et al. 3 control positive group. in day 3 and 21 monocyte levels in trial group was higher than both control positive and control negative group, but this change was not statistically significant. on day 7, monocyte production in the trial group was lower than two other groups, but this change was not statistically significant. data are shown in figure 1. 3.2 lymphocyte production lymphocyte production in the trial group on day 3 was higher than control negative, and on days 7, 14 and 21, this factor was lower in the trial group in comparison with the control negative group. although changes in the level of lymphocyte production were observed, these changes were not statistically significant. data are shown in figure 2. 3.3 mast cell production a gradual increase was observed in the level of mast cell production in all of the experimental groups from day 3 to day 21. the highest mast cell production belonged to control positive group on day 21. on day 7 the level of mast cell production in the trial group was higher than two control groups, but differences between groups were not statistically significant in all stages of the experiment. data are shown in figure 3. 3.4 polymorphonuclear leukocyte production a gradual decrease was observed in the level of polymorphonuclear leukocyte production in all of the experimental groups from day 3 to day 21. the lowest polymorphonuclear leukocyte production was observed in the control negative group on day 21. on day 7 the level of polymorphonuclear leukocyte production in the trial group was higher than two control groups, but differences between groups were not statistically significant in all stages of the experiment. data are shown in figure 4. 3.5 granulation a gradual decrease was observed in the level of granulation in three experimental groups from day 3 to day 21. the lowest granulation was observed in the control negative group on day 21. on day 14 the level of granulation in the trial group was higher than two control groups, but differences between groups were not statistically significant in all stages of the experiment. data are shown in figure 5. figure 1. comparison of monocyte production between three experimental groups. on day 14, the number of monocytes in control positive and the trial group was significantly lower than the control negative group. figure 2. comparison of lymphocyte production between three experimental groups. although lymphocyte production in three groups was different, these differences were not statistically significant. figure 3. comparison of mast cell production between three experimental groups. although the mast cell production increased gradually during the time in all of the experimental groups, there was no significant difference between all experimental groups. dehkohneh et al. 4 3.6 inflammation a gradual decrease was observed in the level of inflammation in three experimental groups from day 3 to day 21. the lowest inflammation was observed in the trial group on day 21. although during the experiment, the level of inflammation in the trial group was lower than the two other groups, differences between groups were not statistically significant in all stages of the experiment. data are shown in figure 6. 3.7 edema a gradual decrease was observed in the level of edema in three experimental groups from day 3 to day 21. the lowest edema was observed in the trial group on day 21. although during the experiment, the level of edema was different between three experimental groups, these differences were not statistically significant in all stages of the experiment (figure 7). 3.8 epithelialization a gradual increase was observed in the level of epithelialization in three experimental groups from day 3 to day 21. the highest epithelialization was observed in the control negative group on day 21. although, during the experiment, the level of epithelialization was different between three experimental groups, these differences were not statistically significant in all stages of the experiment (figure 8). 3.9 fibrosis a gradual increase was observed in the level of fibrosis in three experimental groups from day 3 to day 14. after that, a slight decrease was observed in all experimental groups on day 21. although, during the experiment, the level of epithelialization was different between three experimental groups, these differences were not statistically significant in all stages of the experiment. data are shown in figure 9 3.10 the progress of wound healing and angiogenesis the progress of wound healing among all experimental groups were compared. based on macroscopic closure of the wounds, the wound healing in control groups were impaired, while the wounds were fully healed in probiotics group in 21 days after incision. also, microscopic evaluation has revealed figure 4. comparison of polymorphonuclear (pmn) leukocyte production between three experimental groups. although the pmn leukocyte production decreased gradually during the time in all of the experimental groups, there was no significant difference between all experimental groups. figure 6. comparison of inflammation between three experimental groups. although the inflammation decreased gradually during the time in all of the experimental groups, there was no significant difference between all experimental groups. figure 5. comparison of granulation between three experimental groups. although the granulation decreased gradually during the time in all of the experimental groups, there was no significant difference between all experimental groups. dehkohneh et al. 5 that l. paracasei could induce angiogenesis during wound healing (supplementary figure 1, and 2). 4. discussion several approaches have been developed for wound healing acceleration [10-12]. these approaches might be involved in each of the three stages of wound healing, including inflammation, tissue formation, and tissue remodeling [13]. the therapeutic effects of probiotics on wound healing are well reported over the past decade [14-16]. nevertheless, there are limited publications about the impact of probiotics on skin wound healing. the mechanisms of action of probiotics are still unclear. moreover, it seems probiotic bacteria have more effects when they administered together. in the present study, the effect of l. paracasei td3 on skin wound healing and immunological impacts of bacterium on its host was assessed. our results showed that the administration of l. paracasei td3 would not make any statistically significant change in the wound healing process and immunological factors of its host in comparison with control negative and control positive group while it could induce angiogenesis in trial group. there are limited studies on wound healing properties of l. paracasei td3. similar to our effort, brandi et al. examined the effect of l. paracasei td3 on wound healing. their results on re-epithelialization did not show any significant change compared to control group, which was similar to our results [17]. despite our work, the effect of other members of lactobacillus on skin wounds showed an acceleration in the wound healing process. it has been reported that lactobacillus brevis, and lactobacillus plantarum might significantly accelerate wound healing and decrease inflammatory factors in rats after 21 days [18]. in another study, khodaii et al. showed that lactobacillus reuteri is capable of increasing reepithelialization in the rat model. they also reported that the probiotic group showed a lower inflammation rate compared with the control group [19]. gudadappanavar et al. also showed that lactobacillus acidophilus would accelerate wound healing by reducing epithelialization time in rat models [20]. immune regulation functions of probiotics relate to the activation of natural killer (nk) cells, dendritic cells (dc), intraepithelial γδ t lymphocytes and macrophages, which are effector cells of innate immunity important for skin [21]. there are many reports about wound healing properties of probiotics, which mainly shows they might be considered as a new therapeutic agent in wound healing treatments [17-20]. probiotics improve wound healing, while acting at the epidermis and dermis levels, where they function as signaling receptors against pathogens and activate the production of beta-defensins that increase the immunity of the skin [22]. further studies should be considered for investigation of probiotic’s safety, efficacy, and the mechanism of action in wound healing. also potential side effects include allergic reactions to inactive ingredients, bacteremia, and antibiotic resistance transfer among pathogens must be reviewed which was not studied due to time figure 7. comparison of edema between three experimental groups. although the edema decreased gradually during the time in all of the experimental groups, there was no significant difference between all experimental groups. figure 8. comparison of epithelialization between three experimental groups. although the epithelialization increased gradually during the time in all of the experimental groups, there was no significant difference between all experimental groups. dehkohneh et al. 6 constraints, materials and restrictions on access to sufficient number of rat models. todays, emerging scientific evidence has showed that probiotics may help wound healing. the present study investigated the effects of l. paracasei td3, a native iranian probiotic, on wound healing and induction of angiogenesis. despite our results suggest induction of angiogenesis by the bacterium, there is a need for further studies on the probiotic bacterium and duration of its consumption. our study about the effects of l. paracasei td3 on wound healing and immunological factors in wistar rats did not show any statistically significant result, but it might be effective on other animal models. further researches should be performed to gain a complete insight into the immunohistological and wound healing properties of l. paracasei td3. supplementary files supplementary file 1. author 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https://pubmed.ncbi.nlm.nih.gov/31915703/ https://pubmed.ncbi.nlm.nih.gov/31915703/ https://pubmed.ncbi.nlm.nih.gov/31915703/ journal of current biomedical reports jcbior.com volume 3, number 1, 2022 eissn: 2717-1906 1 original research clinical characteristics and outcomes of covid-19 patients with a history of cardiovascular disease tofigh yaghubi1, vahid shakoori2, sara nasiri2, mona keivan3, chanour tavakol4, shahin ahanjide5, ali alavi foumani1, samaneh mirzaei dahka6, mohammad sadegh esmaeili delshad1, niloofar faraji1,* 1razi clinical research development unit, razi hospital, guilan university of medical sciences, rasht, iran 2department of cardiology, guilan interventional cardiovascular research center, heshmat hospital, guilan university of medical sciences, rasht, iran 3student research committee, medical school, kermanshah university of medical science, kermanshah, iran 4school of medicine, tehran university of medical sciences, tehran, iran 5department of internal medicine, school of medicine, razi hospital, guilan university of medical sciences, rasht, iran 6student research committee, school of nursing and midwifery, guilan university of medical sciences, rasht, iran abstract new emerging severe acute respiratory syndrome coronavirus-2 (sars-cov-2) primarily affects the lungs, but the virus may cause cardiovascular disease (cvd), and a history of cvd is usually associated with comorbidities, which could increase the severity of infections. in this study, we collected demographic and clinical characteristics data from 123 patients with a history of cvd, who were confirmed to have sars-cov-2 infection by polymerase chain reaction (pcr) test in razi hospital, rasht, iran, from march 2021 to june 2021. chi-square and fisher's exact test with a significance level of p less than 0.05 was performed. all statistical analysis was performed with spss software version 26.0. among the studied patients, 99 patients were discharged and 24 of them died. 62 (50.4%) of the study population were female and 61 (49.6%) were male, and there is no significant association between gender and the outcome of patients (p = 0.159). the total mean age of patients was 68.35±12.41. statistical analysis has represented a significant relation of death outcomes in cvd patients with age 60 years and older (p = 0.001), in comparison with patients younger than 60 years. in this present study, no significant relation between underlying disease and mortality rate was reported, but in covid-19 patients with a history of cvd and age upper than 60 years, death outcome was more probable. keywords: covid-19, sars-cov-2, cardiovascular disease, clinical characteristic, underlying disease 1. introduction a mysterious outbreak of new coronavirus in 2019, december, which was named severe acute respiratory syndrome coronavirus-2 (sars-cov-2), was identified in wuhan, china [1, 2]. the most common symptom of this virus appears as mild to severe complications such as cough, fever, myalgia, *corresponding author: niloofar faraji, msc razi hospital, guilan university of medical sciences, rasht, iran tel/fax: +98 13 33550028 email: niloofarfaraji.sci@gmail.com https://orcid.org/0000-0001-5796-7157 received: october, 26, 2021 accepted: december, 25, 2021 renal failure, hypoxemia, coagulopathy, gastrointestinal, and cardiovascular complications [36]. sars-cov-2 has affected more than 238 million individuals worldwide till april 13, 2021, which was confirmed by world health organization (who). sars-cov-2 infection is triggered by the binding of the viral surface spike protein to the human © the author(s) 2022 https://jcbior.com/ yaghubi et al. 2 angiotensin-converting enzyme 2 (ace2) receptor. ace2 is highly expressed in various cell lines and organs including the intestine, lung, and heart as well, which results in some complications including hypertension, atherosclerosis, and congestive heart failure [7-9]. although much of the concentration has been on pulmonary injury, emergency clinicians need to be aware of the cardiovascular disease (cvd), which can be a noteworthy contributor to the mortality of sars-cov-2 infection [10-12]. covid-19 patients with a history of cvd (coronary heart disease, hypertension) presented more severe clinical outcomes and higher mortalities [12]. several studies have confirmed raises in cardiac enzymes and alterations in electrocardiogram (ecg) and echocardiography suggestive of acute myocardial complications in covid-19 patients [13]. furthermore, a cross-sectional study has suggested lower vascular function weeks after sars-cov-2 infection in young adults [14]. in this present study, we investigate the association of demographic data, clinical characteristics, and underlying disease with mortality in covid-19 patients with a history of cvd. 2. materials and methods a total number of 123 patients with a history of cvd were selected through the census method, who was admitted to razi hospital, rasht, iran, from march 2021 to june 2021. confirmation of sarscov-2 infection was done via polymerase chain reaction (pcr) on the nasopharyngeal and oropharyngeal swap sample, by roche rna extraction kit and pishtaz master-mix. exclude criteria were patients with negative pcr test results for sars-cov2 and inadequate medical records. this survey was in agreement with the declaration of helsinki, and the ethics committee of the guilan university of medical sciences has approved the study design [ir.gums.rec.1399.022]. written informed consent was waived by the local ethics committee due to the use of medical recodes of patients only. gender, age, history of smoking and opium, clinical symptoms included: fever, cough, myalgia, respiratory distress, impaired consciousness, hyposmia/anosmia, ageusia, anorexia, intubation needed, o2 saturation, computed tomography (ct) scan result, length of stay (los), history of underlying disease including cancer, asthma, diabetes, blood pressure disorder, chronic liver disease (cld), chronic blood disease, autoimmune disease, chronic kidney disease (ckd), chronic pulmonary disease (cpd), chronic neuropathy, and the outcome of patients were recorded as the variables for our study. the shapirowilk test was used to test the normality of data distribution. the categorical variables were presented as count and percentage. chi-square with a significance level of p less than 0.05 was performed. all statistical analysis was performed with spss software version 26.0. 3. result the statistical analysis of demographic and clinical characteristics of a total of 123 patients with a history of cvd that was confirmed as covid-19 patients, has been shown in (tables 1 and 2). 99 patients were discharged and 24 of them died. 62 (50.4%) of the study population were female and 61 (49.6%) were male, and there is no significant association between gender and the outcome of patients (p = 0.159). the total mean age of patients was 68.35±12.41. statistical analysis has represented a significant relation of death outcomes in cvd patients with age 60 and older (p = 0.001), in comparison with patients younger than 60 years. approximately, none of the patients with death outcome outcomes were under 60 years. also, between the history of smoking and using opium with death outcome, no association was reported (p = 0.381 and p= 0.959), respectively. the most common reported symptom was fever, cough, myalgia, respiratory distress, decreased o2 saturation, and ct scan positive result (p = 0.756, p = 0.133, p = 0.211, p = 0.309, p = 0.087, p = 0.054), respectively. also, some other symptoms such as hyposmia/anosmia, ageusia, and intubation needed, which were less reported in these patients, represented no significant association between the presence of these symptoms and death outcome, (p = 0.509, p = 0.605, p = 0.605, p = 857), respectively. while impaired consciousness and anorexia were rarely reported in patients, statistical analysis revealed remarkable relation between these signs and death outcomes in cvd patients (p <0.001 and p = 0.004). the most-reported underlying disease was diabetes (47.15%), and blood pressure disorder (47.96%). the statistical analysis represented no significant association between any reported underlying diseases and death outcomes, including cancer, asthma, yaghubi et al. 3 table 1. demographic and clinical characteristics data of covid-19 patients with a history of cvd demographic discharge no. (%) death no. (%) p value gender female 53 (53.5) 9 (37.5) 0.159 male 46 (46.5) 15 (62.5) age <60 32 (32.2) 0 0.001 >60 67 (67.7) 24 (100) smoking history yes 4 (4) 2 (8.3) 0.381 no 95 (96) 22 (91.7) opium yes 12 (13.1) 3 (12.5) 0.959 no 87 (87.9) 21 (87.5) clinical symptoms fever yes 53 (53.5) 12 (50% 0.756 no 46 (46.5) 12 (50) cough yes 62 (62.6) 11 (45.8) 0.133 no 37 (37.4) 13 (54.2) myalgia yes 47 (47.5) 8 (33.3) 0.211 no 52 (52.5) 16 (66.7) respiratory distress yes 59 (59.6) 17 (70.8) 0.309 no 40 (40.4) 7 (29.2) impaired consciousness yes 7 (7.1) 8 (33.3) 0.000 no 92 (92.9) 16 (66.7) hyposmia/anosmia yes 8 (8.1) 1 (4.2) 0.509 no 91 (91.9) 23 (95.8) ageusia yes 7 (7.1) 1 (4.2) 0.605 no 92 (92.9) 23 (95.8) anorexia yes 0 2 (8.3) 0.004 no 100 (100) 22 (91.7) intubation yes 5 (5.1) 1 (4.2) 0.857 no 94 (94.9) 23 (95.8) o2 saturation <93% 24 (24.2) 10 (41.7) 0.087 >93% 75 (75.8) 14 (58.3) ct scan result positive 70 (70.7) 12 (50) 0.054 negative 29 (29.3) 12 (50) los <5 day 46 (46.5) 13 (54.2) 0.219 5-10 day 38 (38.4) 5 (20.8) >10 day 15 (15.2) 6 (25) yaghubi et al. 4 table 2. comorbid underlying disease in covid-19 with a history of cvd underlying disease demographic discharge n (%) death n (%) p value cancer yes 4 (3.3) 0 0.317 no 95 (96) 24 (100) asthma yes 11 (11.1) 3 (12.5) 0.848 no 88 (88.9) 21 (87.5) diabetes yes 49 (49.5) 9 (37.5) 0.291 no 50 (50.5) 15 (62.5) blood pressure disorder yes 49 (49.5) 10 (41.7) 0.491 no 50 (50.5) 14 (58.3) chronic liver disease yes 1 (1) 1 (4.2) 0.273 no 98 (99) 23 (95.8) chronic blood disorder yes 4 (4) 1 (4.2) 0.978 no 95 (96) 23 (95.8) autoimmune disorder yes 1 (1) 0 0.621 no 98 (99) 24 (100) chronic kidney disease yes 9 (9.1) 3 (12.5) 0.614 no 90 (90.9) 21 (87.5) chronic pulmonary disease yes 2 (2) 1 (4.2) 0.541 no 97 (98) 23 (95.8) chronic neuropathy yes 2 (2) 2 (8.3) 0.118 no 97 (98) 22 (991.7) yaghubi et al. 5 diabetes, blood pressure disorder, cld, cpd, autoimmune disease, ckd, cpd, and chronic neuropathy (p = 0.317, p = 0.848, p = 0.291, p = 0.491, p = 0.273, p = 0.978, p = 0.621, p = 0.614, p = 0.541, p = 0.118), respectively. although the outcome of patients, either discharged or dead, showed no association with los (p = 0.219), among patients with higher los for more than 10 days, the mortality rate was increased. 4. discussion sars-cov-2 is a critical life-threatening issue, attracts worldwide attention, and causes lots of health and social expenses [3]. due to the susceptibility of cvd patients to severe conditions of sars-cov-2 infection, specific attention should be given to cardiovascular protection during treatment for covid-19 [15]. according to a meta-analysis study on hospitalized patients with covid-19, hypertension, cvd, diabetes, ckd, and chronic obstructive pulmonary diseases (copd) were the most prevalent underlying diseases, while cvd had the highest prevalence among diseases that put patients at higher risk of sars-cov-2 threats [16]. some studies illustrated hypertension and cvd as the most common related mortality factor in patients with cvd who were infected by respiratory viruses (middle east respiratory syndrome (mers-cov) and influenza) [17-20]. it has been reported that elderly people with comorbidities of hypertension, cvd, or diabetes, are more susceptible to severe symptoms of sars-cov-2 infection [21]. consequently, patients with a history of cvd consider a high-risk group with death determination [22-25]. similar to our report, covid19 patients aged older than 60 years with underlying cvd can aggravate symptoms and result in death outcomes [26, 27]. although, in our study, there was no statistically significant relationship between the comorbid underlying disease with death outcome in covid-9 patients with cvd, all mentioned diseases, separately, were reported as a risk factor to worsen the condition and lead to death in these patients [28-32]. among demographical and clinical characteristics, male gender, elderly age, and fever are associated with a greater risk of development of acute respiratory syndrome, severe condition, and death [31, 33]. lack of access to consumption of blood pressure and heart medications data, the impact of unwanted side effects of these medications on the worsening covid-19 condition, as well as incomplete information about the treatment methods used for these patients are some of the limitations of this study. various comorbidities like cvd, cpd, hypertension, and diabetes are risk factors for poor clinical outcomes among patients infected with sarscov-2. patients with a history of cvd had a broad range of severe conditions that led to a higher risk of development of critical or fatal covid-19 disease. according to our study, older age, anorexia, and impaired consciousness are the risk factors for covid-19 patients with a history of cvd, which could result in death outcomes. authors’ contributions concept and study design: ty, aa, sm, msd; 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https://pubmed.ncbi.nlm.nih.gov/34413640/ yaghubi et al. 7 copd: a us study in the n3c data enclave. int j chron obstruct pulmon dis. 2021; 16:2323-6. 33. wu c, chen x, cai y, xia j, zhou x, xu s, et al. risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 2019 pneumonia in wuhan, china. jama intern med. 2020; 180(7):934-43. https://pubmed.ncbi.nlm.nih.gov/34413640/ https://pubmed.ncbi.nlm.nih.gov/34413640/ https://pubmed.ncbi.nlm.nih.gov/32167524/ https://pubmed.ncbi.nlm.nih.gov/32167524/ https://pubmed.ncbi.nlm.nih.gov/32167524/ https://pubmed.ncbi.nlm.nih.gov/32167524/ https://pubmed.ncbi.nlm.nih.gov/32167524/ journal of current biomedical reports jcbior.com volume 2, number 3, 2021 eissn: 2717-1906 1 brief report prevalence and antibiotic resistance of uropathogens in children with urinary tract infections referring to abuzar hospital in ahvaz sousan akrami1,2, seyede mahla mavalizade2,3, mehran varnaseri ghandali4, aram asareh zadegan dezfuli1, zahra farshadzadeh1 , arshid yousefi avarvand3,* 1department of microbiology, school of medicine, ahvaz jundishapur university of medical sciences, ahvaz, iran 2student research committee, ahvaz jundishapur university of medical sciences, ahvaz, iran 3department of laboratory sciences, school of allied medical sciences, ahvaz jundishapur university of medical sciences, ahvaz, iran 4department of infectious diseases, razi hospital, ahvaz jundishapur university of medical sciences, ahvaz, iran abstract urinary tract infection (uti) is a prevalent disease among children. this study is an attempt to find the bacterial agents of uti and antibiotic resistance in children. a descriptive cross-sectional study was carried out on 1316 clinical samples of children at abuzar hospital in ahvaz that had positive urine culture. the strains were determined through biochemical tests and differential culture media. the pattern of antibiotic resistance of the studied strains was determined by the disk diffusion method. out of the 1316 children surveyed, 821 were girls and 495 were boys. the most isolated strains from urine cultures were escherichia coli (57.52%) followed by enterococcus (12.15%). e. coli isolates demonstrated the highest resistance to ampicillin (57.06%) and amikacin was recognized as the most effective antibiotic with a sensitivity of 91.94%. e. coli was the most common causative agent of uti in children. amikacin was recognized as the appropriate choice against urinary tract pathogens. because, in different regions and over time, the frequency distribution of antibiotic resistance varies, it is recommended to carry out periodic monitoring of antibiotic resistance for infection control. keywords: urinary tract infection, disc diffusion, antibiotic resistance 1. introduction urinary tract infections (utis), a common microbial infection, occurs at most ages, especially in children and infants [1]. these infections can be in the lower and upper urinary tract or both [2]. in general, uti in females are more prevalent (3-5%) compared to males (1%); however, boys at one year of age are at higher risk of developing the infections [3, 4]. the uti is a major cause of fever and referral to the emergency wards and hospitalization, especially in infants [5]. this disease can cause serious complications such as urinary disorders, uremia, high blood pressure, and * arshid yousefi avarvand, ph.d department of medical laboratory sciences, school of para medicine, ahvaz jundishapur university of medical sciences, ahvaz, iran tel/fax: +98 61 33738317 email: arshid.yousefi5@gmail.com https://orcid.org/0000-0002-3987-9820 received: may, 04, 2021 accepted: june, 26, 2021 even death [6]. about 150 million uti occur worldwide each year. these infections are among the main reasons for admission to hospitals and also imposing medical costs in these centers [7]. the most common etiological cause of uti is escherichia coli [8], which can be due to the greater ability of this bacterium to bind to urinary tract cells, greater resistance to antibacterial properties of human serum, production of hemolysin, and increased production of capsule antigen [9]. due to the high prevalence and serious complications, diagnosis and treatment of uti in children are necessary as soon as © the author(s) 2021 https://jcbior.com/ akrami et al. 2 possible. timely treatment with appropriate antibiotics before the urine culture and antibiogram results are very important in improving the patient and preventing kidney scarring and other complications [10]. in recent years, the overuse of common antibiotics has increased antibiotic resistance among urinary tract pathogens worldwide [10]. as a result, the most significant threat to public health is the issue of antibiotic-resistant bacteria, which can cause a high percentage of hospital deaths each year [11]. in iran, the incidence of bacterial strains resistant to antibiotics in uti is increasing and this issue is worrying [12]. it has been argued that local resistance to antibiotics should be assessed every five years for policy-making for the treatment of pediatric uti [13]. this study aims to investigate the prevalence and profile of antibiotic resistance of bacterial agents isolated from children uti of children admitted to abuzar hospital in southwestern iran. 2. materials and methods this cross-sectional descriptive study was conducted from may 2020 to march 2021 on 1316 children admitted with uti to abuzar children's hospital in southwestern iran. this study only included cases who had not received antibiotic treatment before hospitalization. samples were collected in a sterile container and sent to the laboratory. the samples were cultured using a standard loop (0.01 ml) under sterile conditions on blood agar and eosin methylene blue (emb) agar (himedia, india). after 18-24 hours of incubation at 37°c, the culture media were evaluated for growth or lack of growth. cultures with candida growth were excluded. gram-negative bacteria were identified by standard biochemical tests. gram-positive microorganisms were identified with the corresponding laboratory tests: catalase, coagulase, and esculin agar (for enterococci). the pattern of antimicrobial resistance of the studied strains was determined using the disc diffusion method by mueller–hinton medium (himedia, india). according to the edge of the zone of growth inhibition encircling the discs, and based on clinical and laboratory standards institute (clsi) recommendations [14], the results were classified as sensitive (s), intermediate-resistant (i) and, resistant (r). the antibiotics amikacin (an, 30 μg), ampicillin (am, 10 μg), ciprofloxacin (cp, 5 μg), gentamycin (gm, 10 μg), nitrofurantoin (fm, 300 μg), ceftriaxone (cro,30 μg), cefepime (fep, 30 μg), erythromycin (e, 15 μg), imipenem (imp,10 μg), cotrimoxazole (sxt, 1.25/23.75 μg), penicillin (p, 10 μg), cefoxitin (fox, 30 µg), piperacillin-tazobactam (tzp, 100/10 µg), and cc (clindamycin, 2 µg) (mast group ltd., united kingdom) were evaluated. for quality control of antibiogram steps in each series of experiments with clinical isolates, the sensitivity test was performed in the same way on the standard strains of e. coli atcc 25922, pseudomonas aeruginosa atcc 27853, staphylococcus aureus atcc 25923, enterococcus faecalis atcc 29212 and the results were compared with standard values. the results are shown as descriptive statistics in terms of relative frequency. the data were analyzed using the chi-square test in spss software version 15. statistical significance was considered as a p value <0.05. figure 1. distribution of isolated organisms in children with uti akrami et al. 3 3. results out of 1316 children with positive urine culture, 821 (62.39%) were girls and 495 (37.61%) were boys. the rate of uti was higher in girls than boys and the difference was significant (p < 0.05). the age of studied cases was up to 5 years old. the lowest incidence of uti was observed in the age group under one year (n =57, 4.33%). the most prevalent strain isolated from urine cultures was e. coli (57.52%) and the lowest strain was related to citrobacter spp. (0.15%) (figure 1). e. coli isolates showed the highest resistance (57.06%) to ampicillin. amikacin was recognized as the most effective antibiotic with 91.94% sensitivity against e. coli isolates. tables 1 and 2 shows the distribution of antibiotic resistance of isolated bacteria. 4. discussion urinary tract infections are the most common nosocomial infections, followed by surgical site infections, and lower respiratory tract infections [15, 16]. table 1. distribution of antibiotic resistance among gram-negative bacteria in children with uti * antibiotics e . c o li (n = 7 5 7 ) k le b sie lla (n = 1 2 6 ) p . a e ru g in o sa (n = 6 7 ) e n te ro b a c te r (n = 2 3 ) p ro te u s (n = 1 5 ) a c in e to b a c te r (n = 4 ) c itro b a c te r (n = 2 ) fm s 559 (73.8%) 78 (61.9%) 9 (39.1%) i 1 (0.1%) 2 (1.5%) 1 (4.3%) r 22 (2.9%) 8 (6.3 %) 5 (21.7%) an s 696 (91.9%) 90 (71.4%) 59 (88%) 15 (65.2%) 2 (50 %) 15 (100 %) 2 (100 %) i 5 (0.6%) 4 (3.1 %) 2 (2.9%) r 22 (2.9%) 23 (18.2 %) 1 (1.4%) 9 (39.1%) 2 (50 %) 2 (100 %) cro s 343 (45.3%) 69 (54.7 %) 12 (52.1%) 14 (93.3 %) 2 (100 %) i 101 (13.3%) 3 (2.3 %) r 302 (39.8%) 46 (36.5%) 12 (52.1%) 1 (6.6 %) 1 (50 %) gm s 577 (76.2%) 91 (72.2 %) 56 (83.5%) 14 (60.8%) 1 (25 %) 15 (100 %) 1 (50 %) i 4 (0.5%) r 113 (14.9%) 23 (18.2 %) 5 (7.4%) 8 (34.7%) 2 (50 %) am s 72 (9.5%) 2 (8.6%) i r 432 (57%) 15 (65.2%) imp s 678 (89.5%) 106 (84.1 %) 58 (86.5%) 23 (100%) 3 (75 %) 13 (86.6 %) 2 (100 %) i r 3 (0.3%) 2 (1.5%) 2 (2.9%) 1 (25 %) cp s 17 (13.4 %) 28 (41.7%) 6 (26%) 1 (25 %) 3 (20 %) i r 2 (1.5%) 2 (2.9%) fep s 5 (7.4%) i r 1 (0.7 %) 2 (2.9%) tzp s 16 (23.8%) i r 2 (2.9%) 1 (25 %) *results estimated based on the number of tested/total isolates an; amikacin, am; ampicillin, cp; ciprofloxacin, gm; gentamycin, fm; nitrofurantoin, cro; ceftriaxone, fep; cefepime, imp; imipenem, tzp; piperacillin-tazobactam akrami et al. 4 proper treatment for this type of infection first requires careful examination of the drug resistance of the infectious agents and then drug administration. according to research, the sex ratio varies with age and during the first trimester, uti is more prevalent in boys than in girls; while, at other ages, the prevalence of the infection is higher in girls [17]. here, the girls had the highest prevalence of uti. in the present study, consistent with other research, the most isolated strain from urine cultures was e. coli (57.52%) [18-22]. however, by comparing the findings of this study with similar studies, we found differences in antibiotic resistance patterns in different regions. isolates of e. coli, proteus and, enterobacter were mostly resistant to ampicillin. coagulase-negative staphylococci (cons) isolates were mostly resistant to erythromycin. it can be concluded that sensitivity to antibiotics has decreased due to irrational administration and use of drugs, enzymatic mutations, the transmission of resistance through genetic factors, and so on. therefore, further studies in this field, decreasing the consumption of antibiotics, and promoting rational administration by physicians are essential. various microorganisms, such as e. coli, bind together to make a colonial tool that helps biofilms formation. biofilms of microorganisms resist antimicrobial therapies. in a study by valavi et al. in ahvaz, iran, e. coli was the most frequent pathogen isolated from children with uti that showed the lowest resistance to nitrofurantoin [23]. in addition, mehri et al. concluded that e. coli was the most isolated strain followed by enterococcus; and nitrofurantoin and ceftriaxone were the antibiotics effective against bacterial agents [24]. these findings table 2. distribution of antibiotic resistance among gram-positive bacteria in children with uti * antibiotics e n te ro c o c c u s (n = 1 6 0 ) c o a g u la se -n e g a tiv e sta p h y lo c o c c i (n = 1 3 3 ) c o a g u la se -p o sitiv e sta p h y lo c o c c i (n = 1 6 ) s tre p to c o c c u s (n = 1 4 ) gm s 81 (60.9%) i 2 (1.5%) r 13 (9.7%) sxt s 13 (81.2 %) 75 (56.3%) 3 (21.4 %) i r 21 (15.7%) cp s 68 (42.5 %) 7 (43.7 %) 93 (69.9%) i 20 (12.5 %) 2 (12.5 %) 2 (1.5%) r 72 (45 %) 6 (37.5 %) 30 (22.5%) fox s 1 (6.2 %) 3 (2.2%) i r 2 (1.5%) e s 3 (18.7 %) 1 (0.7%) i r 5 (31.2 %) 30 (22.5%) 2 (14.2 %) cc s 4 (25 %) 2 (14.2 %) i r 4 (25 %) 1 (7.1 %) p s 1 (6.2 %) 1 (7.1 %) i r 1 (7.1 %) *results estimated based on the number of tested/total isolates sxt; cotrimoxazole, cp; ciprofloxacin, gm; gentamycin, e; erythromycin, p; penicillin, fox; cefoxitin, cc; clindamycin akrami et al. 5 are consistent with our work. barzan et al. in tehran reported that proteus isolates, like the isolates of the our study, showed full susceptibility to gentamicin [25]. because, in different regions and over time, the frequency distribution of antibiotic resistance varies, it is recommended to carry out periodic monitoring of antibiotic resistance to control infection. this study is the only study on the antibiotic profile of the causes of utis in children in ahvaz over the past 10 years. examination of uropathogenic agents in each region helps physicians to update their knowledge about the causative agents of uti tract infections, their susceptibility to antibiotics, and selecting an appropriate experimental treatment. in summary, e. coli was the most common causative agent of uti in children. amikacin was recognized as the appropriate choice against urinary tract pathogens. because, in different regions and over time, the frequency distribution of antibiotic resistance varies, it is recommended to carry out periodic monitoring of antibiotic resistance for infection control. also, a widespread screening program for uti in children should be introduced in order to determine the exact prevalence 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sensitivity in acute childhood diarrhoea. int j med res health sci. 2017; 6(8):65-8. 25. barzan m, hoseyni-doust r, ghalavand z. investigation of frequency and antimicrobial pattern of gram-negative bacteria isolated from urine specimens of children with urinary tract infection in tehran, iran. iran j med microbiol. 2016; 9(4):99-104. https://pubmed.ncbi.nlm.nih.gov/33093962/ https://pubmed.ncbi.nlm.nih.gov/33093962/ https://pubmed.ncbi.nlm.nih.gov/31340182/ https://pubmed.ncbi.nlm.nih.gov/31340182/ https://pubmed.ncbi.nlm.nih.gov/31340182/ https://pubmed.ncbi.nlm.nih.gov/31271618/ https://pubmed.ncbi.nlm.nih.gov/31271618/ https://pubmed.ncbi.nlm.nih.gov/31271618/ https://pubmed.ncbi.nlm.nih.gov/31271618/ http://journal.rums.ac.ir/article-1-4049-en.html http://journal.rums.ac.ir/article-1-4049-en.html http://journal.rums.ac.ir/article-1-4049-en.html http://journal.rums.ac.ir/article-1-4049-en.html http://journal.rums.ac.ir/article-1-4049-en.html 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https://www.ijmrhs.com/abstract/urinary-tract-infection-and-its-antibiotic-sensitivity-in-acute-childhood-diarrhoea-12841.html http://ijmm.ir/article-1-394-en.html http://ijmm.ir/article-1-394-en.html http://ijmm.ir/article-1-394-en.html http://ijmm.ir/article-1-394-en.html journal of current biomedical reports jcbior.com volume 1, number 1, 2020 1 original research molecular characterization of panton-valentine leukocidin positive staphylococcus aureus isolates obtained from clinical samples in isfahan, iran amirmorteza ebrahimzadeh namvar1, meisam ruzbahani2, seyed asghar havaei2,* 1 department of microbiology, school of medicine, babol university of medical sciences, babol, iran 2 department of microbiology, school of medicine, isfahan university of medical sciences, isfahan, iran abstract staphylococcus aureus is one of the main significant human pathogens which can produce various toxins such as panton-valentine leukocidin (pvl) which is known as a prominent toxin associated with s. aureus infections. pvl-positive strains can cause a wide variety of skin, soft tissue, necrotizing pneumonia, fasciitis and life-threatening infections. therefore, the aim of this study was evaluating the molecular characteristics of pvl-positive strains such as the presence of meca, sccmec types, agr types and exfoliative toxin genes. in this study, a total of 152 s. aureus strains were collected from clinical samples of patients who referred to isfahan’s alzahra hospital (iran). the isolates were confirmed phenotypically by conventional methods and then pvl-positive isolates were identified by pcr molecular test. thereafter, antibiotic resistance pattern, agr groups (i, ii, iii, and iv), exfoliative toxins (eta and etb), meca gene and sccmec various types were carried out. totally, 52 (34.2%) of strains were positive for pvl. six pvl-positive strains harbored meca gene, one strain had sccmec i, and 5 strains sccmec type iv. the highest ratio of agr groups belonged to group (i) and the (eta) gene was also detected in 18 isolates. the pvl-positive s. aureus strains can cause more serious infections, so identification of the genetic characteristics and antibiotic resistance monitoring of these strains is necessary. keywords: staphylococcus aureus, pvl, antibiotic resistance, virulence genes 1. introduction staphylococcus aureus is one of the most important nosocomial pathogens which can cause a wide range of various clinical infections. virulence factors and toxins produced by this bacterium are responsible for several infections in which, pantone valentine leukocidin (pvl) and exfoliative toxins are the most relevant. * corresponding author: dr. seyed asghar havaei, ph.d department of microbiology, school of medicine, isfahan university of medical sciences, isfahan, iran tel/fax: +98 313 7922478 email: havaei@med.mui.ac.ir https://orcid.org/0000-0002-3353-336x received: may, 28, 2020 accepted: june, 9, 2020 the pvl was first isolated from furuncles in 1936 [1]. this toxin is composed of two subunits, s and f which have synergistic activity by luks-pv and lukf-pv genes [2, 3]. injection of purified pvl, induces the liberating of histamine from basophilic granulocytes, chemotactic https://jcbior.com/ https://orcid.org/0000-0002-3353-336x ebrahimzadeh namvar et al. 2 factors (leukotriene b4 & il-8), oxygen metabolites of human neutrophilic granulocytes and enzymes [β-glucuronidase and lysozyme) [4]. in a mouse infection model, secreted pvl promotes tissue invasion, inflammatory response and necrotizing pneumonia, via mechanisms, including up-regulation of protein a and other adhesions [5]. the accessory gene regulatory system (agr) is responsible for regulating the growth, colonization, the expression of exoenzymes, toxins, surface proteins and other virulence factors [6, 7]. this system also regulates the metabolic pathways of organisms as a contributing growth factor [8]. although most human clinical s. aureus isolates are agr positive, while several reports indicate that the agr-defective mutants have been isolated from infected patients [8]. in the last two decades, the emergence of antibiotic-resistant strains, especially beta-lactam resistant has increased the clinical significance of s. aureus. the first case of methicillin-resistant s. aureus was described in the uk [9]. currently, the prevalence of methicillin-resistant staphylococcus aureus (mrsa) in some regions is about 72 up to 90% [10]. the meca gene, which is responsible for resistance to methicillin is carrying by a mobile genetic element, staphylococcal cassette chromosome mec, (sccmec) [9]. cassette chromosome recombinase (ccr) is another important part of sccmec, which is involved in the insertion and exertion of sccmec elements [11]. until now, by combining different classes of mec and ccr, at least 11 various types of sccmec elements have been identified [12]. type i, ii and iii are mainly associated with hospital-acquired mrsa (ha-mrsa), and types iv and v with community-acquired mrsa (ca-mrsa) [13]. recently, pvl-positive s. aureus strains (pvl-sa) have attracted much attention, because of the critical role of pvl in clinical infections and emerging antibiotic-resistant strains. therefore, the aim of this study was evaluating the molecular characteristics of pvl-positive strains such as the presence of meca, sccmec types, agr types and exfoliative toxin genes. 2. materials and method 2.1. isolates collection and identification a total of non-duplicate 152 s. aureus strains were collected from different specimens such as abscess (28), wound (66), respiratory tract infection (17), osteomyelitis (4), urine (16), a catheter (7) and bacteremia (14) from teaching hospitals of isfahan, iran. isolates were confirmed by phenotypic tests (gram staining, colony morphology on blood agar, mannitol salt agar, catalase, coagulase and dnase) and finally were stored at -20 ◦c in brain heart infusion broth (bhi) containing (12-15%) glycerol. 2.2. antibiotic resistance pattern antimicrobial susceptibility testing was performed using gentamicin, tetracycline, ciprofloxacin, cefoxitin, rifampin, trimethoprim/sulfamethoxazole, ampicillin and clindamycin disks (himedia) according to clsi standard guidelines [14]. s. aureus atcc 25923 and atcc 33591 were used as negative and positive controls. 2.3. dna extraction the dna was extracted using a genomic dna purification kit (fermentas, k0512) according to the manufacturer protocol recommendation. 2.4. pcr molecular test the pcr reactions were performed with specific primers that are shown in table 1.the program of amplification were as follow: initial denaturation at 95 °c for 3 minutes, followed by 35 cycles of denaturation at 95 °c for 45 seconds, primer annealing based on references for 30-45 seconds, extension at 72 °c for 45 seconds, and a final extension at 72 °c for 7 minutes. the homology sequence of agr and exfoliative products were sequenced and confirmed with the blast tools. 3. results various specimens were collected during the sampling period of time. the highest number of strains was belonged to wound 66 (43.4%) and abscess 28 (18.4%) samples. antimicrobial resistance patterns were determined by disk diffusion method. the most isolates were resistant to tetracycline (56.2%), clindamycin (35.6%) and ciprofloxacin (35.6%) whereas, there was a low resistance to gentamicin (table 2). by using pcr method, 52 isolates (34.2%) harbored pvl gene. all pvl-sa were investigated for the presence of meca, sccmec types, exfoliative toxin and accessory regulatory genes. among the pvl-sa, 18 (34.6%) strains harbored eta, 5 (9.6%) etb, and 2 (3.8%) strains carried both eta and etb genes. ebrahimzadeh namvar et al. 3 table1. the specific primers for amplification of pvl, eta, etb, agr, meca and sccmec type table 2. antibiotic resistance patterns of s. aureus isolates on the other hand, the agr i, ii, iii, iv were recognized in 39 (75%), 3 (5.7%), 7 (13.5%) and 3 (5.8%) isolates respectively. however, 46 (88.5%) strains were mssa and 6 (11.5%) strains were mrsa. finally, sccmec types i and iv, were determined in one and five. 4. discussion s. aureus is colonized in up to 25 percent of healthy people and even more common among those with skin, eye, throat and nasal mucosal surfaces. s. aureus is capable to produce various toxins in which, pvl is the most important one. the pvl-sa are more pathogenic in comparison with other strains and also can cause skin and soft tissue infections, but in some cases may lead to invasive infections including necrotizing hemorrhagic pneumonia [19]. due to the importance of these strains, evaluation of antibiotic resistance and genetic characteristics of pvlpositive strains could play an important role in determining the health policy, hence, rapid detection and monitoring of pvlsa is necessary for appropriate treatments and can be helpful in epidemiologic studies. the prevalence of pvl-strains in iran was reported 18% in 2013, although the pvl-positive strains were distinguished among blood and urine specimens other samples were negative [20]. however, in 2014, shrestha et al. reported that pvl was produced by 35.6% of s. aureus strains and was detected from bacteremia, surgical site, respiratory and urinary tract infection [2]. in the present study, 34.2% of the isolates were pvl-positive and the mentioned gene was isolated from respiratory tract infections, wound infections, abscess and osteomyelitis specimens. in addition, none of the bloodstream and urine samples were harbored the pvl gene. the relation between pvl-sa and skin, soft tissue infections and necrotizing pneumonia has been demonstrated in several studies [2, 3, 5]. bocchini et al. (2017) reported that there was a correlation between pvl toxin production and the intensity of osteomyelitis [21]. target primer sequence (5’-3’) annealing temperature reference pvl f atcattaggtaaaatgtctgcacatgatcca 51 [15] r gcatcaastgtattggatagccaaaagc eta f gcaggtgttgatttagcatt 52 [16] r agatgtccctatttttgctg etb f acaagcaaaagaatacagcg 53 r gtttttggctgcttctcttg agr pan f atgcacatggtgcacatgc 55 [17] i r gtcacaagtactataagctgcgat ii r tattactaattgaaaagtgccatagc iii r gtaatgtaatagcttgtataataatacccag iv r cgataatgccgtaatacccg meca f actgctatccaccctcaaac 55 [18] r ctggtgaagttgtaatctgg sccmec β f attgccttgataatagccytct 52 [17] α3 r taaaggcatcaatgcacaaacact ccrc f cgtctattacaagatgttaaggataat ccrc r cctttatagactggattattcaaaatat 1272 f gccactcataacatatggaa 1272 r catccgagtgaaacccaaa 5rmeca f tataccaaacccgacaactac 5r431 r cggctacagtgataacatcc antibiotics no. (%) of resistant strains total no. =52 gentamycin 2 (3.8) ciprofloxacin 5 (9.6) ampicillin 3 (5.75) clindamycin 6 (11.5) tetracycline 15 (28.8) co-trimoxazole 4 (7.6) rifampicin 4 (7.6) ebrahimzadeh namvar et al. 4 in our study, two pvl-positive strains were isolated from osteomyelitis. the osteomyelitis which is caused by pvl-sa is more complicated and similar to deep venous thrombosis or developed chronic osteomyelitis [21]. on the other hand, the pvl gene-positive mssa contains a larger number of virulence factor genes in comparison to mrsa, although they are susceptible to more antimicrobial agents [22]. numerous conducted studies have been demonstrated that the prevalence of pvl-positive strains among mssa is more than mrsa strains [23, 24]. for instance, in two separate studies by afroz et al. (2008) and vorobieva et al. (2008), all pvlpositive strains were methicillin-susceptible [23, 24]. the prevalence of pvl-positive strains, among mrsa strains in the uk and greece, have been reported 1.6% and 45%, respectively. it seems mssa strains are more involved in the spread of pvl genes among s. aureus strains. karimi et al (2017) in iran, verified that camrsa harbored sccmec iv-v and pvl gene is widely associated with the presence of sccmec iv-v [25]. moreover, a relation among camrsa, sccmec iv and pvl have been proved in study conducted by shukla et al. (2004) [26]. generally, from 40 to 90% of pvl-sa harbor sccmec iv while, only 5% of pvl-positive strains have sccmec i & iii [9]. in our study 5 (9.6%) of mrsa-pvl positive strains carried sccmec iv and only 1 (1.9%) had sccmec i. according to other virulence factors, jarraud et al. (2000) investigated that each of the agr systems is responsible for regulating certain virulence genes and noted that tsst and exfoliative producing strains mainly contain both agr group iii and iv [27]. several studies on pvlsa have shown that these strains do not belong to a specific agr group [28, 29]. in our study, 75% of pvl-sa had agr group i, while amongst mrsa strains, three strains had agr group iii and other agr groups (i and iv). agr group iii in some studies is regarded as the main one in mrsa-pvl positive strains [30]. it should be noted that agr group i was prevalent in most isolates, agr group ii was dominant in nasal swabs (30%), agr group iii was common in respiratory tract infections (26%), and agr group iv was higher in cutaneous isolates (12.1%) [31] various reported on exfoliative toxinproducing strains have suggested that the etb gene rate is lower than eta [32]. in a study performed by koosha et al (2014) in tehran, 197 isolates of s. aureus was investigated. the prevalence of eta and etb genes was reported (94.4%) and (7.6%), respectively [33]. though in our study, the prevalence of eta gene was (34.6%), whereas etb was only (9.6%). in conclusion, our findings indicate the high prevalence of pvl-sa in clinical samples. close surveillance of these strains is essential to monitoring their spread and antimicrobial resistance profiles. several studies show the increasing importance of pvl-sa in nosocomial infections. therefore, determining the frequency of virulence factors and antibiotic resistance profile can be useful in selecting the right therapeutic strategy. the indiscriminate usage of antibiotics, high ability to exchange genetic elements and various types of resistance plasmids can multiply the importance of these pathogens. our results emphasized that the susceptibility or resistance of s. aureus to antibiotics commonly used in various geographical regions is different. according to the increased resistance of s. aureus to antibiotics, 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exfoliative toxins among strains of staphylococcus aureus isolated from blood and nasal specimens. j clin microbiol. 2003; 41(4):1434-9. 33. koosha rz, fooladi aa, hosseini hm, aghdam em. prevalence of exfoliative toxin a and b genes in staphylococcus aureus isolated from clinical specimens. j infect public health. 2014; 7(3):177-85. https://pubmed.ncbi.nlm.nih.gov/17916415/ https://pubmed.ncbi.nlm.nih.gov/17916415/ https://pubmed.ncbi.nlm.nih.gov/17916415/ https://pubmed.ncbi.nlm.nih.gov/17916415/ https://pubmed.ncbi.nlm.nih.gov/17916415/ https://pubmed.ncbi.nlm.nih.gov/15986234/ https://pubmed.ncbi.nlm.nih.gov/15986234/ https://pubmed.ncbi.nlm.nih.gov/15986234/ https://pubmed.ncbi.nlm.nih.gov/15986234/ https://pubmed.ncbi.nlm.nih.gov/15986234/ https://pubmed.ncbi.nlm.nih.gov/23289889/ https://pubmed.ncbi.nlm.nih.gov/23289889/ https://pubmed.ncbi.nlm.nih.gov/23289889/ https://pubmed.ncbi.nlm.nih.gov/23289889/ https://pubmed.ncbi.nlm.nih.gov/23289889/ https://doi.org/10.1309/lmgb9gb82wkdanwf https://doi.org/10.1309/lmgb9gb82wkdanwf https://doi.org/10.1309/lmgb9gb82wkdanwf https://doi.org/10.1309/lmgb9gb82wkdanwf https://www.ncbi.nlm.nih.gov/pmc/articles/pmc153929/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc153929/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc153929/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc153929/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc153929/ https://pubmed.ncbi.nlm.nih.gov/24637043/ https://pubmed.ncbi.nlm.nih.gov/24637043/ https://pubmed.ncbi.nlm.nih.gov/24637043/ https://pubmed.ncbi.nlm.nih.gov/24637043/ journal of current biomedical reports jcbior.com volume 1, number 1, 2020 1 original research molecular investigation of extended-spectrum β-lactamases (esbls) genes in the salmonella isolates obtained from children with acute diarrhea mohsen mohammadi1, mohammad hossein tayefeh-arbab2, zohre baseri3, mojtaba taghizadeh armaki4,5, mohsen karami4,5, saman alhooei6,7, abazar pournajaf 2,4,* 1 non-communicable pediatric diseases research center, health research institute, babol university of medical sciences, babol, iran 2 department of microbiology, faculty of medicine, babol university of medical sciences, babol, iran 3 department of pathology and laboratory medicine, shariati hospital, tehran university of medical sciences, tehran, iran 4 infectious diseases and tropical medicine research center, babol university of medical sciences, babol, iran 5 department of mycology and parasitology, faculty of medicine, babol university of medical sciences, babol, iran 6 department of internal medicine, faculty of medicine, babol university of medical sciences, babol, iran 7 clinical research development unite of rouhani hospital, babol university of medical sciences, babol, iran abstract salmonellosis is an important public health concern among children in worldwide. extended-spectrum β-lactams (esbls) cause resistance to clinically important beta-lactams which are generally used to treat invasive salmonella infections. therefore, the aim of this study was to investigate the presence of shv, tem and ctx-m genes in different strains of salmonella isolated from children with acute diarrhea and to determine their resistance profile. in this cross-sectional study, 300 fecal samples were collected from children referred to the amirkola children's hospital, babol, iran. antibiotic susceptibility testing was done according to the clsi guideline. esbls-producing strains were identified using double disk synergy test method on the mueller-hinton agar plates. multiplex-pcr was performed using oligonucleotide specific primers to detect of shv, tem and ctx-m genes. in total, 7% (n; 21/300) salmonella were isolated, which 61.9%, 28.6% and 9.5% were salmonella typhimurium, salmonella enteritidis and salmonella typhi, respectively. the prevalence of the esbl-producing isolates were 52.4%. m-pcr results showed that 42.8%, 38.1% and 14.3% of isolates were carried ctxm, tem and shv genes, respectively. also, 18.2% of isolates harbored ctx-m, and tem genes, simultaneously. the high rate of esbls-producing salmonella strains in the pediatric patients is an alarm. it is also recommended that alternative drugs be used with less resistance, which requires further investigation. keywords: beta-lactamase, salmonella, diarrhea, pediatric, pcr * corresponding author: dr. abazar pournajaf, ph.d department of microbiology, faculty of medicine, babol university of medical sciences, babol, iran tel/fax: +98 11 32190181 email: abazar_pournajaf@yahoo.com http://orcid.org/0000-0002-6753-5953 received: july, 05, 2020 accepted: august, 01, 2020 https://jcbior.com/ http://orcid.org/0000-0002-6753-5953 mohammadi et al. 2 1. introduction salmonella, a member of the enterobacteriaceae family, are gram-negative bacilli and spore-free that most normally transmitted from animals to humans [1]. these intestinal organisms, which are often considered potential pathogens for humans, have more than 2,500 different serovars that are divided into three distinct species including, salmonella typhi, salmonella choleraesuis, and salmonella enterica [2]. salmonella entrica sub-species entrica serovar enteritidis (briefly s. enteritidis) is the most important cause of non-typhoidal salmonellosis. this disease is one of the most important infectious diseases between humans and animals, which is mostly related to the consumption of meat, poultry, eggs and milk [3]. so this bacterium is a food-borne pathogen. salmonellosis can develop into one of the forms of typhoid fever, septicemia, and gastrointestinal infections (gastroenteritis). gastroenteritis is the most common form of salmonellosis, often associated with symptoms such as fever, abdominal cramps, and diarrhea. the most common serotypes involved in this form is s. typhimurium and s. enteritis [4]. the course of the disease lasts 4 to 7 days and many people recover without the need for antibiotics [5]. today, one of the most important antimicrobials used in treatment of salmonellosis is β-lactam agents. unfortunately, for some reason, the addition of antibiotics to the diet of livestock, improper and arbitrary use of antibiotics, and lack of careful surveillance of drug administration have led to antibiotic-resistant strains [6]. the main problem in treating infections caused by these organisms is the emergence of multidrug resistance (mdr) strains, which often lead to prolonged hospitalization, increase mortality and mobility rates and increase the cost of treatment [7]. β-lactamases are a class of enzymes that hydrolysis the amide bond in the β-lactam ring of penicillins and cephalosporins. a common classification system is ambler system which categorizes these enzyme into 4 dissimilar groups (a, b, c, d), based on the structure. ambler type a (broadspectrum beta-lactamases (esbl), c (cephalosporinase), and d (oxacillinase) βlactamases are categorized as a serine β-lactamases because they have ser amino acid at the enzyme's active site [8]. ambler type b enzyme is classified as a metalloenzyme since the type need a divalent cations, usually zinc in active site to function. the spread of esbls is a global public health concern. esbls can develop resistance to antimicrobials such as 3th and 4th generation cephalosporins and monobactams [9]. the most common type of esbls in clinical samples is shv, whose coding gene is located on a transferable plasmid and is therefore easily distributed among bacterial strains [10]. shv β-lactamases are inhibited by clavulanic acid but not by edta. the tem-1 was the first broad-spectrum β-lactamase, found not only in enterobacteriaceae but also in pseudomonas aeruginosa [11]. among esbls, the ctx-m group has been described globally. the ctx-m gene is located on a transferable plasmid and is effective on the cefotaxime (a 3th-generation cephalosporin). these enzymes have the ability to hydrolyze cephalosporins and are inhibited by clavulanic acid, sulbactam, and tazobactam. ctx-m types are widely spread around the world. although salmonella serotypes are a significant etiologic agents of diarrhea in various parts of the world, very few reports are obtainable about the incidence of esbls among them [12]. so, the aim of the current study is to detect esbl-encoded genes (tem, shv and ctx-m) in the salmonella isolated from children with acute diarrhea and determination of the resistance pattern of these strains. 2. materials and methods 2.1. bacterial isolation in this cross-sectional study, which was performed over a period of 9 months (from the february to october 2019), in total 300 stool samples were obtained from the children's suspected to salmonellosis at the amirkola children's hospital, babol, iran. in laboratory, each sample was enriched using culturing on the selenite-f broth (mercury, germany). after incubation on the 37˚c for 24h, samples were cultured on the eosin methylene blue (emb) agar and xylose lysine deoxycholate (xld) agar (merck, germany) plates. lactose negative colonies were identified by api 20e system (biomerieux, france). all salmonella spp. isolates were serotyped with salmonella specific o and h antisera using the slide agglutination test. 2.2. antimicrobial susceptibility test in according to the standard institute of laboratory and clinical standards (clsi), antimicrobial susceptibility test was determined using agar disk diffusion method on the mueller-hinton agar (mha) (merck co, germany) for ceftazidime (30 https://www.sciencedirect.com/topics/medicine-and-dentistry/beta-lactamase https://www.sciencedirect.com/topics/medicine-and-dentistry/beta-lactamase https://www.sciencedirect.com/topics/medicine-and-dentistry/serine https://www.sciencedirect.com/topics/medicine-and-dentistry/enzyme-active-site https://www.sciencedirect.com/topics/medicine-and-dentistry/enzyme-active-site https://www.sciencedirect.com/topics/medicine-and-dentistry/divalent-cation mohammadi et al. 3 µg), aztreonam (30 µg), imipenem (10 µg), cefotaxime (30 µg), ofloxacin (5 µg), amikacin (30 µg) and tetracycline (30 µg) (himedia, india) [13]. 2.3. double disk synergy test (ddst) ddst was used for esbl-producing isolates, cefotaxime 30 µg and ceftazidime 30 µg with and without clavulanic acid 10 µg (mast, uk). the 0.5 mcfarland standard turbidity of isolates were spread on the muller hinton agar (merck, germany). ddst was done by comparing the inhibition zone of disks containing cefotaxime and ceftazidime with and without clavulanic acid. when zones were distended < 5 mm around the disk comprising clavulanic acid the strain were considered as esbl positive. klebsiella pneumoniae atcc 7006039 was used as a positive control [14]. 2.4. multiplex pcr cellular dna was extracted by the boiling way formerly defined previously [15]. the m-pcr was done using specific primers to identify shv, tem and ctx-m genes (table 1). the pcr reaction was performed at a volume of 25 μl. each pcr reaction includes, 1.5 µl of template dna, 12.5 µl of cinnagen pcr master mix, 1.0 µl of each primer, and 9 µl of ddh2o. the m-pcr reaction was performed for 33 cycles in an eppendorf mastercycle gradient thermocycler (eppendorf, germany) with the fallowing program, initial denaturation at 94°c for 6 min, denaturation at 95°c for 45 s, annealing at 57°c for 60 s, extension at 72°c for 60 s and a final extension at 72°c for 7 min. m-pcr amplicons were electrophoresed in a 1% agarose/0.5 × tbe (45 mmtris-borate, 1 mm-edta) gel stained with 0.1 μl/ml gel red™ (biotium, usa), then photographed under an uv trans-illuminator. the 100-bp dna ladder (cinnagen, iran) was used as a molecular size marker. 3. results the mean age of the patients was 13.6±1.2 years (range from 4 to 14 years), which 45.3% (n; 163) were female and 45.6% (n; 137) male. in total, 7% (n; 21/300) salmonella species were collected, which 61.9% (n; 13/21), 28.6% (n; 6/21) and 9.5% (n; 2/21) were s. typhimurium, s. enteritidis and s. typhi, respectively. antibiotic susceptibility test showed that all isolates were susceptible to imipenem (table 2). moreover, 42.8% (n; 9/21) of isolates were resistant to at least 3 different antibiotic classes and therefore considered mdr. most mdr strains belonged to s. typhimurium serotype. the results of ddst showed that 52.4% (n; 11) of isolates had esbl-positive phenotype. the results of m-pcr showed that 42.8% (n; 9), 38.1% (n; 8) and 14.3% (n; 3) of isolates were carried ctx-m, tem and shv genes, respectively. also, 18.2% (n; 2) of isolates carried genes ctx-m, and tem simultaneously. t a b le 1 . o li g o n u c le o ti d e p ri m e r se q u e n c e s u se d i n t h is s tu d y t a r g e t g e n e p r im e r s e q u e n c e s (5 ʹ→ 3 ʹ) a m p li c o n s iz e ( b p ) r e fe r e n c e s b la s h v f o rw a rd -a t g c g t t a t a t t c g c c t g t g r e v e rs e -t g c t t t g t t a t t c g g g c c a a 7 4 7 [1 6 ] b la t e m f -t c g c c g c a t a c a c t a t t c t c a g a a t g a r -a c g c t c a c c g g c t c c a g a t t t a t 4 4 5 [1 7 ] b la c t x -m f -a t g t g c a g y a c c a g t a a r g t k a t g g c r -t g g g t r a a r t a r g t s a c c a g a a y c a g g g 5 9 3 [1 7 ] mohammadi et al. 4 4. discussion diarrheal diseases caused by salmonella are a worldwide health problem. most people improve without specific treatment. antibiotics are usually used only to treat cases suffering from severe infection. patients should drink extra liquids as long as diarrhea lasts. in some cases, diarrhea may be so severe that the person needs to be hospitalized. occasionally, infection may spread from the intestines to the bloodstream, and then to other parts of the body. in these cases, salmonella can cause death unless the person is treated promptly with antibiotics. βlactamases are extensively used in the treatment of salmonellosis [1]. in the present study, out of 300 stool samples, 7% salmonella spp. were obtained, which is consistent with the study directed by nodeh farahani et al. [18]. this contradiction can be related to the number of samples and the year of study. in agreement with soltan dallal et al. [19], slide agglutination test results showed that 61.9%, 28.6% and 9.5% of isolates were belonged to the s. typhimurium, s. enteritidis and s. typhi serotypes, respectively. this variation may be resulted from distribution of different serotypes in various geographical areas. antimicrobial susceptibility test showed that all strains were susceptible to the imipenem, which was fully consistent with the study of eshraghi et al. [20]. also, 80.9% of isolates were resistant to tetracycline which was consistent with findings of siourimè et al. [21]. in our study, with the exception of only one strain (s. enteritidis), all of them (20 isolates; 95.2%) were sensitive to ciprofloxacin, which is almost consistent with the results of eshraghi et al. [20]. the researchers showed that the favorable effect of quinolones in combination with cephalosporins. spiliopoulou et al. showed that all isolates were sensitive to ceftriaxone and ciprofloxacin, which is consistent with the present study [22]. this agreement could be due to the lack of need for antibiotics in the treatment of salmonellosis and its self-limitation. in the ddst test, 52.4% of isolates were esbls producing phenotype. also, the prevalence of ctx-m, tem and shv genes were 42.8%, 38.1%, and 14.3%, respectively. these data are inconsistent with the study of tajbakhsh et al., since that all 174 salmonella species were negative for tem, ctx-m and shv genes [23]. these results could be due to the geographical distance and the presence of other resistance mechanisms such as, efflux pumps, chromosomal beta-lactamase and porin loss. of 138 s. enterica, 29.6% (n; 40) esbl-producing isolates were collected by ranjbar et al. [24]. the frequency of ctxm, tem and shv genes was 12.3% (n; 17), 29.9% (n; 40) and 2.9% (n; 4), respectively, which was different with the present study. these results are important as an alarming in the development of resistance genes to the other susceptible strains and special attention to these strains. all 92 salmonella spp. collected by boisramé-gastrin et al. were positive for tem gene. so, 22.8% (n; 21/92) and 5.4% (n; 21/92) of isolates were harbored shv and ctx-m genes, respectively [25]. the present study provided information on the frequency and antimicrobial susceptibility profile of salmonella isolates in iran. the high rates of esbls positive-salmonella strains collected from pediatric table 2. antibiotic susceptibility profile antimicrobial agents s. typhimurium (n=13) s. enteritidis (n=6) s. typhi (n=2) s i r s i r s i r ceftazidime 3 (23.1) 1 (7.7) 9 (69.2) 1 (16.6) 0 5 (83.3) 0 0 2 (100) aztreonam 4 (30.7) 1 (7.7) 8 (61.5) 2 (33.3) 0 4 (66.6) 0 0 2 (100) imipenem 13 (100) 0 0 6 (100) 0 0 2 (100) 0 0 cefotaxime 4 (30.7) 0 9 (69.2) 2 (33.3) 0 4 (66.6) 0 0 2 (100) ofloxacin 8 (61.5) 1 (7.7) 4 (30.7) 4 (66.6) 1 (16.6) 1 (16.6) 1 (50) 1 (50) 0 amikacin 2 (15.4) 2 (15.4) 9 (69.2) 5 (83.3) 0 1 (16.6) 0 0 2 (100) tetracycline 3 (23.1) 0 10 (76.9) 1 (16.6) 0 5 (83.3) 0 0 2 (100) ceftriaxone 12 (92.3) 0 1 (7.7) 5 (83.3) 0 1 (16.6) 2 (100) 0 0 ciprofloxacin 12 (92.3) 0 1 (7.7) 6 (100) 0 0 2 (100) 0 0 results presented as no. (%) abbreviations: s; susceptible; i: intermediate-resistant; r: resistant mohammadi et al. 5 cases is alarming and indicates a necessity to substitute the cephalosporins with an appropriate alternative. current limitations include the limited number of serotypes identified and resistance genes. it is also suggested that other resistance genes and other types of beta-lactamases should be identified. accurate diagnosis and rapid identification of esbls-producing salmonella strains from pediatric patients is very important. therefore, continuous monitoring of these resistances, especially at the endemic region, is an obvious thing that can prevent the spread of resistance to other strains and reduce the cost of treatment. acknowledgments a special note of thank goes to amirkola children's hospital for providing patients specimen, as well as epidemiological and demographic data. author contributions all authors contributed similarly to this work, and confirm the final version of manuscripts. conflict of interests authors declare there is no conflict of interest. ethical declarations all samples were obtained from patients as parts of routine sampling during their hospitalization period, so the regional ethical committee waived the need for informed consent. financial support self-funding. references 1. gordon ma, feasey na, graham sm. 45 nontyphoid salmonella disease. in: magill aj, hill dr, solomon t, ryan et, editors. hunter's tropical medicine and emerging infectious disease (ninth edition). london: w.b. saunders; 2013. p. 462-7. 2. gast, rk and porter, re., jr. salmonella infections. in diseases of poultry. 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https://pubmed.ncbi.nlm.nih.gov/21803770/ https://pubmed.ncbi.nlm.nih.gov/21803770/ journal of current biomedical reports jcbior.com volume 3, number 3, 2022 eissn: 2717-1906 1 review machine learning, infection, microbial toxins profile and health monitoring pre/post general surgeries during covid-19 pandemic mohammad javad mohammadi1, kiana aslanimehr2, arash barghi3, alisam rezaee4, mahdis mirkazemi5, sakineh mazloom6, erfan ghanbarzadeh7, amir rigi8, aylar moqaddasi1,*, sahar heidary9 1medical school, islamic azad university, sari branch, sari, iran 2qazvin university of medical sciences, qazvin, iran 3kurdistan university of medical sciences, sanandaj, iran 4science and research branch, islamic azad university, tehran, iran 5tehran university of medical sciences, tehran, iran 6department of nursing, health clinical sciences research center, zahedan branch, islamic azad university, zahedan, iran 7universal scientific education and research network (usern), guilan, iran 8bachelor student of nursing and young researchers and elite club, zahedan branch, islamic azad university, zahedan, iran 9urmia medical science university, urmia, iran abstract although almost 2 years have passed since the beginning of the coronavirus disease 2019 (covid-19) pandemic in the world, there is still a threat to the health of people at risk and patients. specialists in various sciences conduct various research in order to eliminate or reduce the problems caused by this disease. surgery is one of the sciences that plays a critical role in this regard. both physicians and patients should pay attention to the potent steps of different infections’ key-points during pre/postgeneral surgeries in the case of preventing or accelerating the healing process of nosocomial acquired covid-19. the relationship between covid-19 and general surgical events is one of the factors that could directly or indirectly play a key role in the body's resilience to covid-19. in this article, we introduce a link between pre/post-general surgery steps, human microbial toxin profiles, and the incidence of acquired covid-19 in patients. in linking the components of this network, artificial intelligence (ai), machine learning (ml) and data mining (dm) can be important strategies to assist health providers in choosing the best decision based on a patient’s history. keywords: covid-19, general surgeries, microbial toxins, machine learning 1. introduction in the two years since the coronavirus disease 2019 (covid-19) pandemic, elective and emergency surgeries have been influenced by various health protocols [1]. there is frequent asking question that says “is it safe to have surgery after covid-19 infection?” and studies suggest that elective surgeries *aylar moqaddasi, md medical school, islamic azad university, sari branch, sari, iran tel/fax: +98 936 773 1444 email: alr.mqdsi@gmail.com http://orcid.org/0000-0001-7089-719x received: october, 18, 2021 accepted: january, 12, 2021 should be delayed, when possible. it is now obvious that the lasting effects of covid-19 can affect human health in different ways via different routes and including body maintenance and reactions to surgery. in this case, the changes could be significant. studies have shown a substantial increment in the risk of postoperative death and pulmonary complications © the author(s) 2022 https://jcbior.com/ https://jcbior.com/ 2 (six weeks after symptomatic and asymptomatic covid-19 infection). in fact, if we look more closely at the issue of infection in surgeries, we will see that this is not a new issue at all. in fact, even before the onset of the covid-19 pandemic, surgical wound infection was an important issue for medical systems. the infectious agents can infect the patient before, after or during surgery. in hospitalized patients, wound infection is the second most prevalent cause of nosocomial infections [2, 3]. the risk of these infections ranges from 2 to 20%, depending on the facility, the patient, the condition, and the kind of operation [2]. surgical wound infection can increase the time of patients hospitalization. approximatly, 6080% of infections are related to the wound site. infections are often diagnosed using one of the following criteria: 1) wound discharge that is purulent, 2) the presence of microorganisms from the wound discharge and 3) removal of purulent discharge from the wound site [4]. according to a global standard, wounds are classified into four groups, depending on the extent of the infection at the time of surgery: 1) a clean wound: non-infectious and non-inflammatory, 2) clean but infected wound, 3) infected wound: which is fresh wounds, is open or due to accident trauma and 4) dirty infectious wounds: old wounds caused by trauma with dead tissue or perforated viscera [5]. wound classification can also be a predictor of wound infection and is therefore very important [6]. past studies have shown that in 30% of surgeries, glove rupture occurs, which in turn leads to wound infection originating from the operating room [6]. age, surgery in more than one anatomical location, increased duration of surgery, longer surgical wound length, surgery due to malignancy or rupture of viscera, history of hypertension, history of diabetes mellitus, using narcotic substances (drug), consumption of alcohol, recent steroid use, recent history of chemotherapy, low serum hemoglobin and albumin are among the predisposing factors for wound infection in patients have been mentioned [7]. in this study, we look at several microbial agents and their byproducts that might cause infection before, after, or during surgery. we believe that machine learning will be a beneficial methodology for categorizing, diagnosing, or forecasting surgical wound infection as a novel strategy. 2. surgery site infection, microorganisms and their secondary metabolites various microorganisms are known to cause surgical wound infections. the most prevalent isolated organisms are a group of bacteria like staphylococcus aureus, coagulase-negative staphylococci, enterococcus spp., escherichia coli, acinetobacter spp., klebsiella pneumoniae, proteus spp., morganella spp., citrobacter spp., some anaerobic isolates and pseudomonas aeruginosa (in the case of burned wound infection) [2, 8]. many of these bacteria are resistant to antibiotics, which is an important problem in treating wound infections [9]. many of these bacteria complicate the healing process by forming protective structures such as biofilms [10]. on the other hand, the production of bacterial secondary metabolites such as toxins, along with antibiotic resistance, is another fatal blow that bacteria inflict on humans during wound infection [11]. another very important point to consider in the management of wound infection is bacterial toxins. unfortunately, not many studies have been done in this regard, and the mechanism of toxicity of the bacteria that cause wound infection has many unknowns; this is a very important issue. it is critical to note that killing the bacteria does not necessarily mean removing its toxin from the body. for example many gram-negative bacteria have toxins such as endotoxin (lipopolysaccharide = lps) that even if killed with the correct sensitive selected antibiotic, their toxin is still released and will have systemic effects on patients [12]. many common woundinfecting bacteria's toxin, such as s. aureus, is also a super-antigen and may cause deadly shock effects in a short period of time and at extremely low concentrations. this means that even the slightest level of wound infection should not be taken lightly [13]. some bacteria, such as p. aeruginosa and e. coli, also have great diversity in some secondary and toxic metabolites, such as pigments and cytotoxic toxins. some of these toxins are even resistant to the heat that normally kills bacteria [14]. therefore, it is recommended to pay attention to the symptoms of microbial toxicity in the patient along with the symptoms of general bacterial infection in order to achieve effective treatment in surgical wound infection. for better clarification, we are briefly reviewing the pathogenesis steps of bacteria: 1) binding of 3 bacteria to cellular receptors (commonly referred to as colonization), 2) proliferation (at this stage, bacteria have different defense and invasive strategies such as biofilm production or secrete toxins), 3) dissemination (proliferated bacteria or produced microbial toxins will distribute systemically in the body) and 4) transmission (at this stage the bacterium is able to transfer to a new host) [15]. at different stages of wound infection, bacteria and their toxins can cause different clinical effects in patients [16].the type of clinical effects observed in patients can depend on several factors and depend on patient or pre/post operation different risk factors. some of the most important items in this case include: 1) the type of bacteria and toxin produced, 2) the active (vegetative) or inactive form of the bacterium (spore) that was initially colonized in the wound, 3) the mechanism of action of the toxin released from bacteria at the site of infection (neurotoxic, cytotoxic, enterotoxic, etc.), 4) the strength and readiness of the patient's immune system, 5) careful selection of effective and sensitive antibiotics before and after surgery, 6) regular and accurate monitoring of the patient after surgery, 7) poor nutritional state and habits of the patient's daily life like being current smoker or level of personal hygiene by the patient , 8) extremes of age and occupation of the patient, 8) presence of underlying diseases or use of immunosuppressive drugs, 9) proper nursing care of the wound, 10) observance of aseptic conditions before, during or after surgery or at the time of nursing care of the wound after surgery or while surgery draining [17]. depending on each of the above, different clinical symptoms can be seen in patients. clinical signs of a surgical site infection usually present 5-7 days after surgery, but they might appear up to 3 weeks later (especially in the case of the prosthesis insertion). the most prevalent are: spreading erythema, localized discomfort, pus or discharge from the incision. wound dehiscence and persistent pyrexia, as previously reported [18]. the majority of surgery site infections (ssis) are superficial, however some are deeper and can result in significant wound collapse. fortunately, in clinical practice, the requirement for debridement and open wound treatment is uncommon [19]. aseptic wound swabs should be collected for culture at the wound site for any ssi infection, especially if a purulent discharge is evident. it is critical to prevent wound edge culturing wherever feasible in order to reduce skin flora contamination. it is also critical to do blood tests for infection indicators (fbc, crp) [20]. it is advised to be used if there is any sign of systemic involvement or sepsis. inflammatory cytokines (il-6, il-1, tnf-a) and platelet indicators such as platelet count, mean platelet volume (mpv), and mean distribution weight (mdw) might help predict the likelihood of endotoxemia and septic shock [21]. 3. applied machine learning (ml) choosing the best diagnostic method is always one of the major concerns of medical systems [22]. a good way to predict the severity or occurrence of an infection is to categorize clinical and laboratory data and information using computer knowledge. artificial intelligence (ai), in this situation, machine learning (ml) and data mining (dm) are three multipotentially relevant methodologies. these sciences can be summarized as follows: ai as a subset of ml, is an area of computer science that employs automated computing processes, reasoning, and inferencee by computers. main application of ml is to converts data into information and makes logical decisions through this conversion. the most important algorithms in these conversions are as follows: classification, clustering, feature selection and prediction. on the other hand, dm is a way to extract information from a big data. it is not a technical discipline but uses different algorithms related to natural language processing (nlp), ml and ai. dm have the ability to search and compare different data of obtained from different programs, texts bodies, summaries and create a unique question-answer systems for categorizing and classifying data to make reasonable conclusion [23]. we can utilized these computer sciences to write specific algorithms and help computers to behave intelligently to perform various tasks. as mentioned previously, ml has the potential to generalize data from a lot of information and can utilize calculations to recognize connections between data. dm, ai, and ml are three sciences that, can be of great help for physicians in the manner of diagnosis and prediction process if they are given the right data and asked the right request based on a logical algorithm [24]. by the use of state of the art lab technologies, modern medicine produces mass data obtained from 4 infection cases. ml and dm has the ability to analyze raw/multidimensional data of wound abnormalities, microbial toxicity, tissues charges and patient lab information that is available at databases of hospitals or clinical labs. after targeted classification, these information and data could present regular patterns involved in ssi development. yielded computing data is useful to introduce and determine correlations between different characteristics such as patients' personal data, disease symptoms or even disease predictions. one of the most important aspects of ml in the case of data analysis is helping physicians to diagnose infection symptoms more accurately and choose the appropriate treatment for patients with significant changes in their surgery site morphology and histology (figure 1) [21, 25]. today, the use of technology and science of ml in the prevention, early diagnosis and treatment of many infectious disease like sepsis and many neurological diseases such as alzheimer's, dementia or parkinson's is underway [26]. for example sepsis, consider as the most important disease at the first 28 days of life and is one of the main causes of neonatal death in the intensive care units [18]. this neonatal infection is known as a nosocomial infection. because most of these infections are resistant to antibiotics, they can be a major cause of clinical non-response to treatment and the rapid spread of sepsis and septic shock, multiple failures, and increased mortality in hospitals. acinetobacter is an important opportunistic bacterium that is widely distributed in hospitals and is a major cause of nosocomial infections. because it quickly becomes resistant to major groups of antibiotics, such as penicillin, it is difficult to treat. given the importance of rapid diagnosis of neonatal sepsis, the researchers conducted a study of sepsis with an infectious agent that examined acinetobacter in neonates admitted to the intensive care unit using ml modeling. to conduct this study, they first searched for databases, clinical findings, laboratory findings, arterial blood gas, vital signs, comorbidities, and sepsis invasive procedures. after extracting this data, the list containing 75 features of more than 4,000 infants was extracted and analyzed using machine learning [27]. figure 1. application of ml in covid-19 ssi prediction. 1) ml models will develop for analyzing classified and categorized different libraries of patients disease information include: 2) covid-19 symptoms, sepsis and bacterial toxemia markers, hospitalacquired infections criteria, 3, 4) ssi and other postoperative infections indexes, microbiological test results, molecular biologic test results, blood and other clinical samples biochemistry results, types of antimicrobial prescription and microbial resistance, monitoring and tracking of site-specific infections, 5) finding explanations based on analysis and going through predictions based on analysis results and finally, 6) held best decision. 5 in other studies, other factors such as gestational age and vital signs, positive blood culture, lactate, systolic blood pressure, gestational age, birth weight, mechanical ventilation, complete feeding and blood transfusion, lethargy and malnutrition and finally sex of the baby applied to predict sepsis in infants [27]. ml is also used in ssi evaluation and prediction. for example a simple algorithm for monitoring women after cesarean section has been written. machine learning uses the score obtained from each of the questions asked of the patient and can be very helpful in predicting infection at the surgical site. such an application and achievement of machine learning in rural areas, which are far from many medical centers, and the world today, which is involved with the covid-19 pandemic, is significant and very important [6, 18]. in another study, ml was used to assess the severity of infection progression in superficial ssi, organ/space ssi, and total ssi. in this investigation, the following factors were significant for assessing the identification of superficial, organ, and total ssi: ssi-related imaging, ssi-related treatment, ssi-related international classification of diseases (icd), corynebacterium, enterococcus, staphylococcus, streptococcus, superficial ssi antibiotic, organ ssi antibiotic, fluid, abscess, wound minimal partial thromboplastin time, systolic blood pressure, and temperature [21]. text mining (tm) and ml were employed to predict ssi in another study published by silva et al [23]. in their study, descriptive datasets included: the number of patients, the mean (and standard deviation) of patients' ages, the average number of surgeries per patient, female patients, the mean (and standard deviation) of female patients' ages, male patients, the mean (and standard deviation) of male patients' ages, and the average number of surgeries per patient. the number of surgical procedures, the number of elective procedures, the number of urgent procedures, the number of emergency procedures, and the average (and standard deviation) of the surgical team. in this investigation, the following algorithms performed well in predicting ssi: logistic regression (lr), multinomial naive bayes (mnb), nearest centroid (nc), random forest (rf), stochastic gradient descent (sgd), support vector classification (svc), and linear svc are all examples of support vector classification algorithms [23]. ml even have used for screening covid-19 as a quick and efficient tool based on subjects symptoms and exploration of covid-19 mortality risks [21]. scientist believe that early mortality prediction using noninvasive ml models are a promising revolutionary method. on the other hand, the application of ml in connection with the diagnosis of ssi is developing every day and these methods could be very useful in telemedicine systems during covid-19 pandemic. 4. conclusion if the infection at the surgical site is associated with a covid-19 infection, the treatment becomes more complicated. a successful covid-19-ssi ml project requires the collaboration of physicians, nurses, laboratory personnel, microbiologists, microbial toxicologists, computer engineers, and programmers. another important factor in the proper implementation of a ml project related to ssi is the existence of complete information, high sample size and the existence of powerful computer servers for data analysis. these are the things that will increase the accuracy of the results obtained. in the sample studies cited in the text, a number of bacterial factors were examined to assess and predict ssi deterioration. it is suggested that future researchers also use factors related to the clinical effects of toxins produced by bacteria to increase the accuracy of their prediction results. researcher could consider different variables during intraoperative, post-operative or pre-operative phases as critical stages of infectious agent or bacterial toxins entrance to the surgery sites that lead to better variables selection. ultimately, the progression of telemedicine by developing ml for medical care presents potential, and having a positive impact on ssi care and surgical quality improvement especially in rural area during covid-19 pandemic. authors' contributions mj: collecting and summarizing machine learning articles. ka, sh: collecting and summarizing infection articles. ab: collecting and summarizing microbial toxins profile articles. ar: collecting and summarizing health monitoring articles. mm: drafting, endnote library preparation and referencing manuscript body. sm: collecting and summarizing both microbial toxins and health monitoring articles. egh, ar, am: edit files based on reviewers comments, suggesting the idea of article, writing first 6 draft of article, scientific editing and native language editing. conflict of interests the authors reported no potential conflict of interest. ethical declarations this review article has no ethical declarations. financial support financial support of this review article has based on personal foundation. references 1. liu z, zhang y, wang x, zhang d, diao d, chandramohan k, et al. recommendations for surgery during the novel coronavirus (covid-19) epidemic. indian j surg. 2020;82(2):124–8. 2. cooper ra. surgical site infections: epidemiology and microbiological aspects in trauma and orthopaedic surgery. int wound j. 2013;10(s1):3–8. 3. thompson rl, cabezudo i, wenzel rp. epidemiology of nosocomial infections caused by methicillin-resistant staphylococcus aureus. ann intern med. 1982;97(3):309–17. 4. nagachinta t, stephens m, reitz b, polk bf. risk factors for surgical-wound infection following cardiac surgery. j infect dis. 1987;156(6):967–73. 5. ambrose np. use of pre-operative procalcitonin and c-reactive protein measurements as biomarker of post-operative infective complications of pancreaticoduodenectomy. christian medical college, vellore; 2020. 6. zhu y, simon gj, wick ec, abe-jones y, najafi n, sheka a, et al. applying machine learning across sites: external validation of a surgical site infection detection algorithm. j am coll surg. 2021;232(6):963–71. 7. haridas m, malangoni ma. predictive factors for surgical site infection in general surgery. surgery. 2008;144(4):496–503. 8. lee hg, jang j, choi je, chung dc, han jw, woo h, et al. blood stream infections in patients in the burn intensive care unit. infect chemother. 2013;45(2):194–201. 9. estahbanati hk, kashani pp, ghanaatpisheh f. frequency of pseudomonas aeruginosa serotypes in burn wound infections and their resistance to antibiotics. burns. 2002;28(4):340–8. 10. hedayati ch m, abedinzade m, khanaki k, khakpour tleghani b, golshekan m, mohammadi e. comparative protective effects of viola spathulata, urtica dioica, and lamium album on endoplasmic reticulum (er) stress in rat stroke model. casp j neurol sci. 2021;7(3):172–9. 11. panayidou s, ioannidou e, apidianakis y. human pathogenic bacteria, fungi, and viruses in drosophila: disease modeling, lessons, and shortcomings. virulence. 2014;5(2):253–69. 12. bone rc. gram-negative sepsis: a dilemma of modern medicine. clin microbiol rev. 1993;6(1):57–68. 13. elek sd, conen pe. the virulence of staphylococcus pyogenes for man. a study of the problems of wound infection. br j exp pathol. 1957;38(6):573. 14. setlow p. spore resistance properties. microbiol spectr. 2014;2(5):2–5. 15. arciola cr, campoccia d, montanaro l. implant infections: adhesion, biofilm formation and immune evasion. nat rev microbiol. 2018;16(7):397–409. 16. atiyeh bs, costagliola m, hayek sn, dibo sa. effect of silver on burn wound infection control and healing: review of the literature. burns. 2007;33(2):139–48. 17. owens cd, stoessel k. surgical site infections: epidemiology, microbiology and prevention. j hosp infect. 2008;70:3–10. 18. fletcher rr, olubeko o, sonthalia h, kateera f, nkurunziza t, ashby jl, et al. application of machine learning to prediction of surgical site infection. annu int conf ieee eng med biol soc. 2019 jul;2019:2234-2237. 19. stevens dl, bisno al, chambers hf, everett ed, dellinger p, goldstein ejc, et al. practice guidelines for the diagnosis and management of skin and soft-tissue infections. clin infect dis. 2005;41(10):1373–406. 20. gingold mj, bystrzycki ab, cicuttini fm. textbook of adult emergency medicine. 21. mahdavi m, choubdar h, zabeh e, rieder m, safavi-naeini s, jobbagy z, et al. a machine learning based exploration of covid19 mortality risk. plos one. 2021;16(7):e0252384. 22. maglogiannis i, kontogianni g, papadodima o, karanikas h, billiris a, chatziioannou a. an integrated platform for skin cancer heterogenous and multilayered data management. j med syst. 2021;45(1):1–13. 23. da silva da, ten caten cs, 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https://pubmed.ncbi.nlm.nih.gov/33404959/ https://pubmed.ncbi.nlm.nih.gov/31834905/ https://pubmed.ncbi.nlm.nih.gov/31834905/ https://pubmed.ncbi.nlm.nih.gov/31834905/ https://pubmed.ncbi.nlm.nih.gov/10225344/ https://pubmed.ncbi.nlm.nih.gov/10225344/ https://pubmed.ncbi.nlm.nih.gov/33198233/ https://pubmed.ncbi.nlm.nih.gov/33198233/ https://pubmed.ncbi.nlm.nih.gov/34356267/ https://pubmed.ncbi.nlm.nih.gov/34356267/ https://pubmed.ncbi.nlm.nih.gov/34356267/ https://pubmed.ncbi.nlm.nih.gov/34356267/ https://pubmed.ncbi.nlm.nih.gov/17208641/ https://pubmed.ncbi.nlm.nih.gov/17208641/ https://pubmed.ncbi.nlm.nih.gov/17208641/ https://pubmed.ncbi.nlm.nih.gov/17208641/ https://pubmed.ncbi.nlm.nih.gov/17208641/ journal of current biomedical reports jcbior.com volume 3, number 1, 2022 eissn: 2717-1906 1 review brain neural network, development, microbiome, microbial toxins and covid-19 ali samrezaee1, maryam doustmehraban2, naimeh ghadimi3, sedigheh bahador4, arash barghi5, erfan ghanbarzadeh6, amir rigi7, mojtaba hedayati ch8,*, atiyeh kimiaeifar9 1school of basic medical sciences, academy of medical science, zhengzhou university, zhengzhou, china 2student research committee, school of medicine, qazvin university of medical sciences, qazvin, iran 3student research committee, school of medicine, ardabil university of medical sciences, ardabil, iran 4student research committee, school of medicine, esfahan university of medical sciences, esfahan, iran 5student research committee, school of medicine, kurdistan university of medical sciences, sanandaj, iran 6student research committee, school of medicine, guilan university of medical science, rasht, guilan, iran 7microbial toxins physiology group, universal scientific education and research network, rasht, iran 8department of microbiology, virology and microbial toxins, school of medicine, guilan university of medical sciences, rasht, , iran 9iran university of medical sciences, tehran, iran abstract although almost 2 years have passed since the beginning of the coronavirus disease 2019 (covid-19) pandemic in the world, there is still a threat to the health of people at risk and patients. specialists in various sciences conduct various researches in order to eliminate or reduce the problems caused by this disease. neural network science plays a vital role in this regard. it is important to note the key points of neuro-microbial involvement in the diagnosis and management of covid-19 therapy by physicians and patients whose nervous systems are challenged. the relationship between covid-19, microbiome and the profile of microbial toxins in the body is one of the factors that can directly or indirectly play a key role in the body's resistance to covid-19 and changes in the neural network of the brain. in this article, we introduce the relationship and behavioral and mood problems that can result from neuronal changes. in linking the components of this network, artificial intelligence (ai), machine learning (ml) and data mining (dm) can be important strategies to assist health providers to choose best decision based on patient’s history. keywords: covid-19, brain, neural network, microbial toxins, machine learning 1. introduction coronavirus disease 2019 (covid-19), known as significant public health challenge worldwide and is the causative agent of severe acute coronavirus respiratory syndrome. since december 2019, the covid-19 outbreak has become a significant universal pandemic threat for almost all people and specially patients with specific disabilities *corresponding author: mojtaba hedayati ch, ph.d department of microbiology, school of medicine, guilan university of medical sciences, rasht, iran tel/fax: +98 13 33326061/+98 13 33326061 email: mhedayatich@gums.ac.ir https://orcid.org/0000-0002-8277-9841 received: october, 10, 2021 accepted: january, 12, 2021 [1, 2]. in pandemic situation, we are in constant struggle to stop virus infection or attenuate severity of disease every day [3]. scientists in different fields tend to do their best, monitoring and managing disease. because of complicated nature of neural network abnormalities and coinfections, it seems that there is a need for multidisciplinary research and performance based on complex systems biology of human neural © the author(s) 2022 https://jcbior.com/ samrezaee et al. 2 network-microbiome interaction in patient suffered from covid-19 [4]. we are focouseing on human neural network, microbial toxins profile, and computer science-based research as a comprehensive monitoring strategy package against covid-19. for this purpose, it is necessary to explain a series of concepts to understand the network relationship between these sciences, which we will describe as follows. 2. brain neural network development, microbiome, microbial toxins and covid-19 since the clinical symptoms of covid-19 focus more on respiratory symptoms, its consequences on the central nervous system (cns), brain neural network development (bnn) have received less attention. however, in some cases, respiratory failure may occur following infection of the brainstem and affecting the respiratory centers [5]. the exact mechanism and relationship of neurological consequences in patients with coronavirus is not known. some receptors for the virus, such as angiotensin-converting enzyme 2 (ace2), appear to be less abundant in the brain, causing peripheral nerves to become infected more rapidly than cns [4]. neural pathways and neural networks are gateways for viruses to enter cns. viruses can internalize to cell via antrograde or retrograde neurotransmitters movement by infecting the base of sensory or motor nerve terminals [5].the olfactory nerve terminals which are coming from the frontal sinus are considered high-risk routes virus gate to cns [6]. this route is converted to a conduit between the nasal epithelium and the cns due to the unique structure of the olfactory nerves and olfactory bulb in the nasal cavity and frontal lobe of the brain. coronaviruses can thus reach the brain through the nose and the olfactory cavity. within 7 days, viruses that infect nasal cells can reach the brain and cerebrospinal fluid (csf) via the olfactory nerve and produce demyelination owing to inflammation and response [7]. the detection of high levels of the virus in the brainstem after the onset of coronavirus also indicates the transmission of the virus to the cns through the respiratory tract, which is associated with neurodegenerative diseases. involvement of this area of the brain indicates the role of respiratory and cardiac control centers in covid19-induced acute respiratory syndrome [8]. the growth and development of the neural network (nn) is one of the most known important requirements for human physiological stability [9]. this evolution could depend on different variables like genetic, microbiome, total microbial toxins profile, bodies regulatory systems and so on. maturation of human nn might affected during microbiome dysbiosis that itself, caused mood changes and sickness behaviours [9, 10]. even it shows that difference in gut-brain axis between individuals and its microbiota composition diversity could lead to changes in human personality traits. the composition and diversity variations in the gut microbiota are linked to personality differences, including sociability and neurological disorders [11, 12]. it has been shown that social interactions can increase the diversity of the gut microbiome and the nn, and people with higher stress or anxiety have lower microbiome diversity. this discussion is very important in relation to some neurological diseases such as autism and can be examined in relation to other neurological diseases as well [11]. nutrition can also play an important role in regulating nn growth by affecting the microbiome. for example, studies have shown that adults who were not breastfed as children had fewer microbiomes as adults [13]. studies showed that, proper formation and ratio of the microbial population in life is indispensable for approporiate nn development [14]. in addition to the microbiome, microbial secondary metabolites, such as microbial toxins, can affect the physiology and behavior of the neural network [15]. occupational exposure, daily routine, food consumption cultures and environmental factors in many places of the world are effective in the formation of different human microbiota and total microbial toxins volume in different forms. it is very important to monitor the relationship between nn development, microbiome and their toxins (such as lipopolysaccharide; lps) especially in covid-19 patients. because direct or indirect effects of microbiota and microbial toxins volume on bnn development is unavoidable [16-18]. previous studies have shown that lps (as a systemic inflammatory toxin), could lead to synaptic loss and cognitive decline in both animal models and human [5]. part of this is due to the effect of lps on stimulating the secretion of inflammatory cytokines, and part is related to the activation of microglia and the population of blood samrezaee et al. 3 cells such as platelets [5, 19]. nowadays in some studies, developing brain is considering as an innate immunity key regulator and central player in braindevelopmental origin immune cells interaction of the body [20]. therefore, it makes sense to consider acute, chronic, and even fetal stimuli by immune stimulants (such as microbial toxins) as an interfering factor in this sensitive pathway. based on “leaky gut-brain hypothesis”, many microbial toxins will affect the growth and development of the neural network and the brain through this mechanism [21, 22]. many studies have been done so far that show that in various neurological diseases, this microbial-microbial toxin association can be used for therapeutic purposes in many diseases. a therapeutic applications of some bacterial protein like: clostridium botulinum botulinum neurotoxin (bont), bacillus anthracis lethal toxin (lf), bordetella pertussis pertussis toxin (ptx), escherichia coli cytotoxic nectorizing factor1 (cnf1) and chlorotoxins have previusely presented as an immunotoxins or other form to attenuate a series of disease [12, 23]. another members of bacterial toxins like neurotoxins are multipotential toxins which could consider as interacting agents with nn and neuronal cells. they can target different nn include: motoneurons, inhibitory interneurons, hippocampal neurons, enterocyte neurons, enterochromaffin cells and vagal nerve, enterochromaffin cells and enteric neurons [24, 25]. during infection with covid-19, the microbiome, microbial populations, and the volume and type of microbial toxins in the body can change. these changes or accidental coinfection of individuals can have different effects on the growth and function of the brain's neural network [26]. these effects are very diverse and can range from stimulation of the vomiting center in the brain to stimulation of glutamate (glu)-acetyl cholin (ach)-gama amino butyric acid (gaba)-glycin (gly) release and apoptosis of brain neurons. a series of neurotransmitters and neuropeptides realease or blocking also could affect during exposure with some bacterial neurotoxins. the most important neurotransmitters in this case include: ach, glu, aspartate (asp), gaba, gly, dopamine (dopn), adrenalin (adrln), noradrenalin (nadrln), seretonin (or 5-ht), adenosine triphosphate (atp) corelease with ach and nicotinamide adenine dinucleotide (nad), calcitonin gene-related peptide (cgrp), vaso intestinal peptide (vip), neuropeptide y, sp and nitric oxide (no) [24]. one of the problems in studying the biology of systems related to microbiome-microbial toxins and their effects on neural networks is the existence of many variables. if there is a way to classify or categorize these variables and find their relationships, the possibility of predicting, preventing, and treating management is not unexpected. 3. machine learning as a promising strategy different methods have been used till now to increase the effectiveness of covid-19 prophylaxis or treatment include personal hygiene prevention, nutrition, lifestyle changes and vaccinations [27]. however, no interdisciplinary viewpoint has been established to assess the acquired data. we suggest artificial intelligence (ai), machine learning (ml), and data mining (dm) as multipotentially relevant ways for integrating the components of the microbiome, microbial toxins, nn, and covid-19 network. in brief, ai utilized automated ways of computing, reasoning and inference by computers. ml is a subset of ai. it converts data into information and makes decisions based on this conversion. most indispensable algorithms in this case are: classification, clustering, feature selection and prediction. on the other hand, dm do extracting information from a massive data. dm is not a technical discipline but uses different algorithms related to natural language processing (nlp), ml and ai. it is capable to search within different programs, manuscript abstracts and create question-answer systems to classify data and make conclusion [28]. these computer sciences help computers to behave intelligently and perform various tasks. ml has the capacity to generalize data from expansive sums of information and can utilize calculations to recognize connections and d esigns between comes about of dm, get valuable comes about, and take modern activities [29]. dm, ai, and ml are three sciences that, in expansion to their contrasts, are moreover straightforwardly related to each other [30]. modern medicine produces a lot of data obtained from covid-19 cases. ml and dm has the ability to analyze raw/multidimensional data of brain abnormalities, microbial toxicity, microbiome chages samrezaee et al. 4 and covid-19 patient lab information that is available at medical or clinical databases or hospitals. after targeted classification, these information and data could present regular patterns involved in brain disease development. yielded computing data is useful ti introduce and determine correlations between different characteristics such as patients' personal data, disease symptoms or even diseae predictions. one of the most crucial aspects of ml in the case of data analysis is helping physicians in more accurate diagnosis of diseases and choosing the appropriate treatment for patients with significant changes in their microbiome golden ratio or microbial toxins profiles that can lead to neuronal dysfunctions[31]. today, the use of technology and science of machine learning in the prevention, early diagnosis and treatment of many alzheimer's, dementia, and parkinson's disease are all on the rise [32]. better early prognosis improves the qualification of preventing or theurapetic decisions for physicians, minimizing different medical errors, and could decline the cost and time of diagnosis, or indeed rescues and fullfill patients [33]. natural language, network diffusion algorithm, image processing and volumetric information, time-series data, graph data, support vector machine, and general data and multimodal data are some of the different types of ml data categories and methos that are now used to select the best ml model for a given problem based on different neurodegenerative diseases [32]. scientists use 2d, 3d, graph convolutional neural network models, specialized natural language processing tools (bert, spacy, corenlp, syntaxnet, etc.), hierarchical basin models (svms, for example), random forests, and variations-as many ml models use an autoencoder to diagnose alzheimer's disease (ad), parkinson's disease (pd), or to assess dementia categorization. we also used a cutting-edge machine learning (ml) model to predict the development of molecular fingerprints in amyotrophic lateral sclerosis (als) and the side effects of various medicines [32, 34]. different neurodegenerative disease-related data types (neuroimaging, cognitive performance tests, transcriptomic data, biomarker data, motor performance test and metabolomics data) now conventionally used by machine learning algorithms for disease progression evaluation [32, 35]. ml has also been found to be beneficial in identifying microbial and microbial toxin-related diseases, including as septicemia, nosocomial infections, surgical site infections (ssis), and other postoperative infections, as well as evaluating and forecasting microbial test findings. increase sitespecific infections such as general infections, musculoskeletal infections, antibiotic prescriptions, deep fungal infections, and urinary tract infections (uti) are monitored and tracked [27]. machine learning can be used in many areas because of the infectious nature of covid-19 and the effect that microbial profiles and microbial toxins can have on a patient's brain-neural network. medical imaging, drug discovery, vaccine development, molecular biology and neuro-infection control are all commonalities between neuropsychiatric and infectious diseases that can be practiced under the supervision of machine learning monitoring [36]. 4. comprehensive conclusion with the emergence of the covid19 pandemic, human populations today are experiencing unprecedented health protocols. some of these precautions have become commonplace. like the constant use of hand sanitizers and new eating habits. these procedures can affect the amount and type of microbiota and microbial noxins volume in the body. for example, it is expected that as a result of overusing of hand sanitizers and antibiotics, the golden ratio of the human microbiome changes, so does the total available lps of the body. the presence of such microbial toxins, which are themselves the cause of neural degeneration and one of the most prevalence natural leaders of neurodegenerative diseases in the body, is a very important caution alarm. because lps is not the only microbial toxin that is expected to increase in the body during the covid-19 pandemic. undoubtedly, such changes are being experienced chronically every day in human populations, and their effects on the body's defense systems and the growth of brain nn will be seen in humans. our hypothesis is that the use of various information related to neuropsychiatric problems in neurodegenerative diseases and its association with microbial profiles and microbial toxins in covid-19 patients will be a new promising and highly advanced approach in the diagnosis and treatment of the disease in the future. by monitoring and analyzing microbial toxin and considering neurological disease data via ml models, a new approach may be taken to reduce the potential samrezaee et al. 5 of neurodegeneration in human body during the covid-19 pandemic. authors’ contributions as: collecting and summarizing neurologyrelated articles. md: collecting and summarizing neurology-related articles. mgh: collecting and summarizing both neurology and machine learningrelated articles. sb: collecting and summarizing both neurology and microbial toxins-related articles. egh: endnote library preparation and referencing manuscript body. ab: collecting and summarizing microbiome-related articles. ak: collecting and summarizing microbiome-related articles. ar: journal finding and edit files based on reviewers comments. mh: suggesting the idea of article, writing first draft of article, scientific editing. all authors read and approved the final version of paper. conflict of interests none to declare. ethical declarations this review article has no ethical declarations. financial support financial support of this review article has based on personal foundation. references 1. stawicki sp, jeanmonod r, miller ac, paladino l, gaieski df, yaffee aq, et al. the 2019-2020 novel coronavirus (severe acute respiratory syndrome coronavirus 2) pandemic: a joint american college of academic international 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detection of extended-spectrum beta-lactamase genes among escherichia coli isolates from urinary tract infection in mashhad maryam hafiz1, gholamreza hashemi tabar1,*, mehrnaz rad1 1department of pathobiology, school of veterinary medicine, ferdowsi university of mashhad, mashhad, iran abstract urinary tract infections (utis) are known as one of the most important infections around the world, and escherichia coli is the most important cause of uti. also, the empiric treatment and misusing of antimicrobial agents has led to increasing multi-drug resistance around the world which is a worldwide concern. extended-spectrum beta-lactamase (esbls) is an enzyme group that is produced by the enterobacteriaceae family. the three main esbls enzyme are as follow: blactx-m, blatem, and blashv, additionally, there are several types of each of them by the same mechanism. this study was conducted to evaluate the prevalence of esbl genes among e. coli isolated from uti patients. a total of 105 isolates were collected from uti patients at two hospitals in mashhad from 2017 to 2019. bacterial identification was performed by standard microbiologic methods. the assessment of antimicrobial susceptibility was accomplished by the disk diffusion method. the presence of esbl genes was investigated by multiplex-pcr. the prevalence of uti, among females, was identified more than males. furthermore, the blatem and blactx-m genes were detected in all isolates, but only six isolates (5.7%) were harboring blashv. the considerable role of e. coli in uti infection, as well as the presence of esbl genes in e. coli strains, emphasize the need for surveillance of antimicrobial therapy to prevent the extension of resistance among clinical strains. keywords: escherichia coli, esbl, urinary tract infection, multiplex-pcr 1. introduction urinary tract infections (utis) happen through community-acquired or nosocomial-acquired, which are one of the most prevalent types of human infection [1, 2]. escherichia coli is known as the most important pathogen in utis, among both outpatient and inpatient [3, 4]. on the other hand, the empiric of treatment and misusing of antimicrobial agents such as, beta-lactams, beta-lactamase inhibitor, fluoroquinolones, and carbapenems, has led to increasing multi-drug resistance around the world which is a worldwide concern [5, 6]. extendedspectrum beta-lactamase (esbls) is an enzyme group *corresponding author: gholamreza hashemi tabar, ph.d azadi square, mashhad, razavi khorasan province, iran, p.o. box: 9177948974 tel/fax: +98 51 38836056 email: hashemit@um.ac.ir https://orcid.org/0000-0001-9760-1096 received: june, 14, 2021 accepted: july, 12, 2021 that is produced by the enterobacteriaceae family, which are mostly isolated from inpatient cases [7]. the esbl positive bacteria are able to inactivate several antimicrobial agents including an oxyimino-group such as cephalosporins (e.g. ceftazidime, and ceftriaxone), monobactam (e.g. aztreonam), but not able to inactivate cephamycin, and carbapenems [7, 8]. the three main esbl enzymes are blactx-m, blatem, and blashv [9, 10]. these enzymes are encoded generally by genes located on plasmids. actually, the mutation in these genes (particularly blatem and blashv) led to the change in the form of the enzyme near its active site to increment the affinity and © the author(s) 2021 https://jcbior.com/ hafiz et al. 2 hydrolytic capability of beta-lactamase for oxyimino compounds [11]. also, some of them are encoding on transposons which could transmit between organisms [8]. the growing up of esbl producing strains brings up some problems including the transmission of resistance to other organisms, refusing treatment, and increase in mortality rate. so, more surveillance is needed to identify the rate of the problem and help to select appropriate antimicrobial agents to treat the infection. this study was conducted to evaluate the prevalence of blatem, blactx-m, and blashv genes by multiplex polymerase chain reaction (m-pcr) among e. coli isolated from uti patients. 2. materials and methods 2.1 sample’s collection a total of 105 e. coli isolates were collected from uti patients at ghaem and emamreza hospitals in mashhad from 2017 to 2019. also, the basic information of patients was gathered, including age and gender, uti history, hospital visit reports, and reports of taken antimicrobial agents from patients. isolates cultured on selective-differential media, macconkey agar (mac), and eosin methylene blue agar (emb), were incubated overnight at 37°c. then, microscopically examination was done by gram staining from lactose-positive colonies. likewise, biochemical profiling, such as oxidase/catalase activity, motility, indole, urease, triple sugar iron agar (tsi), and methyl red-voges proskauer test (mr-vp) has been studied. 2.2 antimicrobial susceptibility test the assessment of antimicrobial susceptibility was accomplished by the disk diffusion method recommended by the clinical and laboratory standards institute (clsi, 2020) (13)[12], against 15 antibiotic disks (padtanteb, iran) as follow: ceftazidime (30 μg), imipenem (10 μg), amikacin (30 μg), nalidixic acid (30 μg), ampicillin (10 μg), ciprofloxacin (5 μg), cefixime (5 μg), cefazoline (30 μg), ceftriaxone (30 μg), meropenem (10 μg), gentamicin (10 μg), nitrofurantoin (300 μg), cefepime (30 μg), cefotaxime (30 μg) and co-trimoxazole (1.25/23.75 μg). e. coli atcc 25922 was used as a positive control for antimicrobial susceptibility tests. 2.3 pcr amplification dna extraction was done by the boiling method as described earlier by nazari et al. [13]. the prepared dna samples were assessed for prevalence of blatem, blashv, and blactx-m genes by m-pcr by using the mj mini thermal cycler (bio-rad, hercules, ca, usa) [14]. the pcr reaction was conducted in 25 volumes including 12.5 μl of pcr 2x mastermix (parstous company, iran) containing taq dna polymerase, reaction buffer, dntps mixture, a protein stabilizer, and the convenience for use was optimized by adding table 1. the information of oligonucleotides used as primer in multiplex-pcr primer name primer sequence (5’-3’) amplicon size (bp) reference blatem164.se tcg ccg cat aca cta ttc tca gaa tga 445 14 blatem164.as acg ctc acc ggc tcc aga ttt at blashv.se atg cgt tat att cgc ctg tg 724 blashv.as tgc ttt gtt att cgg gcc aa blactx-m. u1 atg tgc agc acc agt aaa gtg atg gc 593 blactx-m. u2 tgg gta aag taa gtg acc aga atc agc gg table 2. the frequency of uti in different age groups age groups <10 11-20 21-30 31-40 41-50 51-60 >60 female 8 3 9 5 5 10 16 male 5 0 2 3 6 5 28 total a 13 3 11 8 11 15 44 a results presented as number of cases hafiz et al. 3 sediment for electrophoresis and 2x solution of loading dye, 1 μl of each primer (10 pm), 2 μl of dna template (100 ng/reaction) and 4.5 μl of nuclease-free water. pcr conditions were as follow: initial denaturation at 95°c for 15 seconds; followed by 35 cycles of denaturation at 94°c for 30 seconds, annealing at 61°c for 40 seconds, and extension at 72°c for 2 minutes with a final extension at 72°c for 10 minutes [15]. electrophoresis of amplicons was performed using 1.5% agarose gels. the primers' information is listed in table 1. also, e. coli atcc 35218 and distilled water were used as the positive and negative control, respectively. 3. results 3.1 prevalence of uti in different ages and genders based on the results, it was determined that the prevalence of uti among females with 56 (53.3%) cases was more than males with 49 (46.7%) cases. most isolates were recovered from patients with >60 ages, and the lowest rate was observed in 11-20 age groups. the information of patients’ age groups was listed in table 2. 3.2 antimicrobial susceptibility test according to the results, amikacin (98%), meropenem (93%), nitrofurantoin (89%), and imipenem (88%) revealed the highest activity against isolates, respectively. all results are listed in table 3. 3.3 pcr results the analysis of pcr products on gel electrophoresis revealed that the most prevalent esbls among studied isolates were blatem, and blactxm genes (100%). while only six (5.71%) isolates were carried the blashv gene. 4. discussion uti is known as one of the most highlighted healthcare infections. our finding notes that women have a higher rate of uti than men. this fact confirms earlier findings of the stress uti in females [16-19]. also, the most prevalent was observed in the >60 age group, which was similar to previous surveys [20, 21]. however, this result was in contrast with other observations, which reported the highest prevalence in 2-10 and 20-29 age groups, respectively [17, 22]. our results revealed the highest resistance rate for t a b le 3 . t h e r e su lt s o f a n ti m ic ro b ia l su sc e p ti b il it y t e st ( t o ta l n = 1 0 5 ) p h e n o ty p e c a z im p a m k n a l a m p c ip c f m m e m c f z c r o g e n n it f e p c t x s x t s u sc e p ti b le 5 4 (5 1 .4 ) 9 3 (8 8 .5 ) 1 0 3 (9 8 .1 ) 6 7 (6 3 .8 ) 1 8 (1 7 .1 ) 5 0 (4 7 .6 ) 5 5 (5 2 .4 ) 9 8 (9 3 .3 ) 4 4 (4 1 .9 ) 4 5 (4 2 .8 ) 8 7 (8 2 .8 ) 9 4 (8 9 .5 ) 5 8 (5 5 .2 ) 4 1 (3 9 .1 ) 3 1 (2 9 .5 ) in te r m e d ia te r e si st a n t 0 0 0 4 (3 .8 ) 0 0 0 0 0 0 0 0 5 (4 .7 ) 0 0 r e si st a n t 5 1 (4 8 .6 ) 1 2 (1 1 .5 ) 2 (1 .9 ) 3 4 (3 2 .4 ) 8 7 (8 2 .9 ) 5 5 (5 2 .4 ) 5 0 (4 7 .6 ) 7 (6 .7 ) 6 1 (5 8 .1 ) 6 0 (5 7 .2 ) 1 8 (1 7 .2 ) 1 1 (1 0 .5 ) 4 7 (4 4 .1 ) 6 4 (6 0 .9 ) 7 4 (7 0 .5 ) a b b re v ia ti o n : c a z , c e ft a z id im e ; im p , im ip e n e m ; a m k , a m ik a c in ; c ip , c ip ro fl o x a c in ; c f m , c e fi x im e ; m e m , m e ro p e n e m ; c f z , c e fa z o li n e ; c r o , c e ft ri a x o n e ; g e n , g e n ta m y c in ; n it , n it ro fu ra n to in ; f e p , c e fe p im e ; c t x , c e fo ta x im e ; s x t , tr im e th o p ri m /s u lf a m e th o x a z o le https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=3&cad=rja&uact=8&ved=0ahukewjjhcq7-orwahulasakhubdbpaqfggymai&url=https%3a%2f%2fwww.researchgate.net%2fpost%2fnuclease_free_water_vs_ultra_pure_water_dnase_rnase_free&usg=afqjcne5yjnndf3z5jkvfq_d39lqgecoua https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=3&cad=rja&uact=8&ved=0ahukewjjhcq7-orwahulasakhubdbpaqfggymai&url=https%3a%2f%2fwww.researchgate.net%2fpost%2fnuclease_free_water_vs_ultra_pure_water_dnase_rnase_free&usg=afqjcne5yjnndf3z5jkvfq_d39lqgecoua hafiz et al. 4 ampicillin (82.8%) and cotrimoxazole (70.4%) which was consistent whit previous surveys [17, 23-25]. however, another study was recorded a lower resistance rate for ampicillin and cotrimoxazole, 67% and 45%, respectively [26]. on the other hand, the highest susceptibility was observed against amikacin, meropenem, nitrofurantoin, and imipenem. while the lower susceptibility for amikacin (61%) and nitrofurantoin (72%) has been reported in an earlier survey [27]. however, our finding for nitrofurantoin susceptibility was close to other studies [17, 28]. also, the susceptibility rate of isolates to imipenem and amikacin in the current study was in line with previous surveys [17, 23, 29]. according to earlier reports, the high rate of esble. coli was noted from uti among both inpatient and outpatient, whereas it was more significant among inpatient [24, 30]. generally, the highest presence of esbls was recorded for blatem and blactx-m, which was in line with earlier results [9, 31-34]. although, some studies reported a lower rate of blactx-m, 28%, and 32%, respectively [35, 36]. this discrepancy can be explained by different geography and the increase of genes transferring during the time. on the other hand, blashv was detected in only six isolates. our experiment about the prevalence of the blashv gene was close to previous survey [33]. according to these results, the blashv gene is not predominant as the two other genes. however, new studies revealed a high frequency of blashv that might increase in the future [11, 37]. certainly, further tests such as confirmatory esbl test and statistical analysis are required to determine the relationship between these genes and resistant phenotype. the considerable role of e. coli in uti infection, as well as the presence of esbl genes in e. coli strains, emphasize the need for surveillance of antimicrobial therapy to prevent the extension of resistance among clinical strains. acknowledgments the authors would like to thank all ghaem and imam reza hospitals staff, which collaborating in collecting isolates. author contributions all authors contributed equally to each part of the manuscript and approved the final version. conflict of interests the authors claim that there is no conflict of interest. ethical declarations this study was in accordance with the declaration of helsinki and ethical permission was sought from the institutional ethics committee of ferdowsi university of mashhad, mashhad, iran. however, because we only used leftovers from clinical specimens, the local ethics committee waived the need for informed consent. financial support this study was funded by ferdowsi university of mashhad, mashhad, iran (grant number 109). references 1. sedighimehr n, dehghan manshadi f. electrical stimulation for lower urinary tract dysfunction in people with multiple sclerosis: a systematic review. j clin physiother res. 2018; 3(2):48-53. 2. haghighatpanah m, mojtahedi a. characterization of antibiotic resistance and virulence factors of escherichia coli strains isolated from iranian inpatients with urinary tract infections. infect drug resist. 2019; 12:2747-54. 3. larramendy s, gaultier a, fournier jp, 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jcbior.com volume 3, number 3, 2022 eissn: 2717-1906 1 review imaging reality: a review of covid-19 pandemic mohammad mahdi hanaei1, seyed mehdi mousavi2, nazanin ebrahimi ghasem abadi3, chanour tavakol4, seyedeh maral mousavi5,*, behnaz ganbar-moghaddam5,* 1department of internal medicine, school of medicine, shahid beheshti university of medical sciences, tehran, iran 2department of cardiology, cardiology research center, heshmat hospital, guilan university of medical sciences, rasht, iran 3department of anesthesiology, school of medicine, tehran university of medical sciences, tehran, iran 4student research committee, school of medicine, tehran university of medical sciences, tehran, iran 5razi clinical research development unit, razi hospital, guilan university of medical science, rasht, iran abstract recently, coronavirus disease 2019 (covid-19) has been the main universal health concern and the most important challenge to the world. this disease affects all age groups, genders and, races. due to lack of definitive medication detection of the disease in an early stage and prevention of its transmission plays an important role in its control. transcription polymerase chain reaction (rt-pcr) and chest computed tomography (ct) are the most common diagnostic methods for covid-19. besides a review on the general founding of the covid-19 pandemic, we tried to collect a hand on report focusing on radiological knowledge and its applications, limitations, and instructions. keywords: covid-19, radiology, pandemic, computed tomography 1. introduction recently, coronavirus disease 2019 (covid-19) has been the main universal health concern and the most important challenge to the world. it reached several countries in about 30 days and became known as one of the most dangerous pandemics. after reaching 200 countries, it became a global threat which led to the death of many individuals in the entire world [1-4]. this enveloped single-stranded rna virus is distinctly present in both animals and people and can be easily transmitted from human to human, the way this condition spreads from person to person has made it a public threat. the severe acute respiratory syndrome coronavirus 2 (sars‑cov‑2) is almost 50% genetically identical to the middle east respiratory syndrome coronavirus (mers-cov) and about 79% *behnaz ganbar-moghaddam, ph.d razi hospital, guilan university of medical science, rasht, iran tel/fax: +98 13 33542460 email: behnaz.moghaddam@gmail.com https://orcid.org/0000-0002-8382-2661 *seyedeh maral mousavi, md mousavi.maral1993@gmail.com https://orcid.org/0000-0001-5252-4592 received: april, 01, 2022 accepted: september, 05, 2022 identical to sars-cov. initial studies revealed bats were the key reservoirs of coronavirus in china [5]. unfortunately, this virus has the potential of significant genetic mutations that is unknown to the innate immune system of the human body and therefore could have an extensive impact on human health [1, 3, 6]. lack of information and also accurate treatments resulted in the death of many people around the world [1, 6]. 2. epidemiology as of early october 2020, 66 countries recorded less than one death per 100,000 population (including 21 mostly small countries without any deaths), while 17 countries exceeded 50 deaths per 100,000 [7]. based on the world health organization (who) report until november 10th, 2020, there have been © the author(s) 2022 https://jcbior.com/ hanaei et al. 2 50,676,072 confirmed cases of covid-19, including 1,261,075 deaths. some piece of evidence showed death rates were higher in men in comparison with women [8]. climatic-environmental and non-environmental conditions such as family, social structure, body immunity, rainfall, temperature and humidity, air pollution, cigarette smoking, or drinking alcohol are factors that would affect the morbidity and mortality of covid-19 [9]. this respiratory virus is highly active between 5 to 20 °c that indicates the temperature is an effective factor in the virus transmission. a higher prevalence of coronavirus was observed in north america and southern europe relative to southeast asia and the western pacific. the temperature is considered one of the effective factors in this issue [3, 9]. there are contradictory pieces of evidence about the role of humidity. according to the who, humidity between 40 and 80% is most likely to increase virus transmission. increasing the size of droplets in higher humidity will increase the probability of their deposition. therefore, it is expected that higher humidity decreases the transmission of the virus. this concept is true in some parts of iran, spain, russia, and turkey, where high humidity has reduced virus transmission, but in parts of the united states and northern europe it has been so different, there may be a link between the prevalence and mortality of coronavirus with smoking and air pollution [9]. tourism and social scientific, religious or political ceremonies may also contribute to the global spread of the virus [10]. 3. morbidity and transmission this infection can be highly contagious and be mainly transmitted through inhalation of contaminated droplets, close contact, contaminated surfaces, and hands in contact with the eyes, and mouth [1, 11]. in newborns, it can be transmitted vertically [12]. gastrointestinal transmission is still under investigation, although this virus has been found in the feces and urine of infected individuals [1, 12]. it can be transmitted to an average of 2-3 people from asymptomatic or symptomatic patients and even from individuals in their incubation period, and people close to these patients, such as healthcare staff and their family members, are at risk of catching the disease [6]. national limitations can play an important role in breaking the covid-19 transmission chain [13]. covid-19 can cause mild to severe clinical symptoms such as pneumonia, respiratory failure, and death [5, 6]. the disease affects all age groups and genders, but deaths are more common in people between the ages of 50 and 80 [3]. patients with older ages and underlying diseases are at higher risk of severe to fatal disease. in children under the age of 19, this rate was very low at about one percent [14]. a meta-analysis study showed that the most prevalent comorbidities associated with covid-19 were hypertension, diabetes, cardiovascular disease, liver disease, lung disease, malignancy, cerebrovascular disease, copd and asthma. of which, hypertension 46% (37%-55%) and diabetes 21% (16%-27%) were the most prevalent, respectively [15]. the clinical presentations of this disease vary; it may initially be similar to the flu and pneumonia and present with symptoms such as fever, shortness of breath, myalgia, fatigue, headache, sore throat, dry cough loss of smell and taste. large-vessel stroke was reported among hospitalized covid-19 patients younger than 50 years old [15, 16]. the digestive system may be a potential route for covid-19 infection. based on zhou et al. results, 26% of covid-19 patients experienced gastrointestinal (gi) symptoms. the frequency of gi symptoms in female patients was significantly higher than in male patients. higher clinical manifestations such as sore throat, dizziness, and fatigue have been reported in patients suffering gi symptoms. hemoglobin level in the gi symptom group was also significantly lower than in the non–gi symptom group [17]. stool samples with positive covid-19 nucleic acid were reported among patients which suggests the possibility of transmission through feces [18]. sleep dysfunction is frequent and neurological manifestations (infrequent) of covid-19 in patients and even health care workers who are directly involved in patient care [19, 20]. the most common complication of covid-19 was pneumonia which may develop into severe respiratory conditions, arrhythmia, and shock. this complication can be seen in more severe covid-19 cases to a great extent, acute respiratory distress syndrome (ards) has been found in covid-19 patients undergoing autopsy, this condition can cause hanaei et al. 3 organ failure and may be life-threatening due to its association with low blood oxygenation levels [4]. 4. diagnosis the synthesis of a double-stranded dna molecule from an rna mold via transcription polymerase chain reaction (rt-pcr) is the standard laboratory method for detecting covid-19 coronavirus. this technique uses swabs taken from the nasopharynx and/or oropharynx. for patients with pneumonia, lower respiratory tract secretions may also be tested. the sputum specimens are more reliable and have a lower false-negative rate for covid-19 detection than throat swabs [21, 22]. the sensitivity varies depending on the timing of specimen collection during illness [2325]. the sensitivity of throat samples is reported as 60% at the initial phase of illness. higher sensitivity has been observed in patients with mild (82%) to severe (89%) conditions [25]. currently, radiographic imaging techniques are extensively used in covid-19 diagnosis. chest x-ray is used as a basic tool for covid-19 diagnosis. although some signs of illness might fade by the overlying tissue, it is still valuable especially in following illness in severe cases [26]. the false-negative rate is high using chest radiographs, especially in early and mild cases [27]. chest computed tomography (ct) is generally feasible, reliable, and rapid. it has high sensitivity with a very low rate of missed diagnosis. [28-31]. ai et al. studied 1014 patients' data; taking rt-pcr results as a reference, chest ct predicted 97% of covid-19 suspicious cases correctly [32]. fang et al. based on 51 patients’ data evaluated the relative sensitivity of ct and rt-pcr as 98% and 71% respectively. generally, rt-pcr and chest ct are comparable in covid-19 diagnosis and no significant difference has been found between them [27]. however, ct findings are nonspecific and overlap with other viral infections. a study of 62 healthcare centers in 34 countries showed that chest ct is used often (76%) for covid-19 assessment rather than initial diagnosis (22%) [33]. most organizations do not recommend diagnostic imaging when rt-pcr and immunoassays are available and patients have mild disease. but in the absence of these methods, chest radiographs or ct are so valuable in patients with moderate to severe clinical features of covid-19 infection [34]. kim et. al. studied the diagnostic value of chest ct and rt-pcr through meta-analysis. based on their finding the pooled sensitivity was 94% for chest ct and 89% for rt-pcr. they evaluated the pooled specificity of chest ct as 37%. the positive predictive value (ppv) for chest ct ranged from 1.5% to 30.7% and the negative predictive value (npv) ranged from 95.4% to 99.8%. for rt-pcr, the ppv ranged from 47.3% to 96.4%, while the npv ranged from 96.8% to 99.9%. due to the low ppv of ct, the application of this screening method may cause unnecessary public exposure. the sensitivity of the chest ct depends on disease severity, comorbidities, and the proportion of asymptomatic patients. the rt-pcr requires swab samples taken from the nasopharyngeal, throat, or sputum. it is difficult and prone to error in elderly patients. therefore the sensitivity of rt-pcr is lower when the proportion of elderly people is higher [35]. 5. radiographic features the most common ct findings reported in covid-19 pneumonia are bilateral, multi-lobar, subpleural areas of ground glass opacity (ggo) with or without consolidations affecting the lower lobes [23, 29, 36, 37]. ggo has also been frequently reported to have round morphology [36]. in the intermediate phase of infection (4–14 days from symptom onset), a so-called “crazy-paving pattern” is seen. peak radiologic abnormalities occur at around day 10, followed by gradual regression starting two weeks after symptom onset [23]. bronchovascular thickening in lesions is common. there are no particular differences in ct features between adults and pediatrics. generally, covid-19 signs in pediatric cts are milder except for bronchial wall thickening, which was more common in pediatric patients [38]. evidence that shows a single fraction of low dose whole lung irradiation (0.3 – 1.5 gy) delivered at the middle of mediastinum via two parallel opposed anterior-posterior external radiation fields) would improve respiratory symptoms of covid-19 in patients with moderate pulmonary involvement [39-41]. 6. ct protocol a low-dose, single-phase (inspiratory breath-hold if possible), fast, non-contrast ct of the entire chest (apex to the base) is sufficient for the evaluation of most patients with covid-19 infection [33, 34]. contrast may disturb the ggo interpretation. it should be considered that generally, patients are hanaei et al. 4 suffering from shortness of breath or cough. therefore, fast scanning protocols will decrease the probability of repetitive scanning. although highresolution computed tomography (hrct) is used widely for covid-19 diagnosis [42-44] no specific benefit or need for it has been reported [34]. generally, the radiographic examination should prescribe in pregnant patients with specific cautions. firstly, the patient should be informed about possible risks and benefits. the ct scan request, patient file, and clothing must be marked in red as a label for highrisk patients. during covid-19 pandemic specific disinfection strategies should be considered. disinfection protocols are more important in case of the pregnant patients. personal protection equipment for the corona virus-like masks, gloves, gowns, goggles, and disinfectants must be available for the patients. preventing exposure to other patients reduces the risk of covid-19 transmission [45]. strict infection control must consider by the radiology department to ensure the prevention of transition. depending on the degree of exposure, personnel should use protective equipment properly. types of equipment include a surgical cap, goggles, a surgical or n95 face mask, a filtering facepiece respirator (n95), gloves, a fluid-impermeable gown, coveralls, and shoe covers. hand hygiene is necessary before and after each examination [46]. 7. conclusion as a global health issue, covid-19 has attracted lots of attention all around the world. despite plenty of unknowns, our knowledge about the virus is increasing day by day. in this review, we tried to collect a brief and hand on report of founding this pandemic. authors' contributions study design and supervision: mehdim, maralm, bg. data collection and interpretation: mh, ne, ct. drafting: mh, ne, ct, maralm, bg. critical revisions; mehdi m, bg. all authors read and approved the final version of manuscript. conflict of interests none. ethical declarations not applicable. financial support self-funded. references 1. fadaka ao, sibuyi nrs, adewale ob, bakare oo, akanbi mo, klein a, et al. understanding the epidemiology, pathophysiology, diagnosis and management of sars-cov-2. j int med res. 2020; 48(8):0300060520949077. 2. fan j, liu x, pan w, douglas mw, bao s. epidemiology of coronavirus disease in gansu province, china, 2020. emerg infect dis. 2020; 26(6):1257-65. 3. bhattacharya s, basu p, poddar s. changing epidemiology of sars-cov in the context of covid-19 pandemic. j prev med hyg. 2020; 61(2):e130-e6. 4. geier mr, geier da. respiratory conditions in coronavirus disease 2019 (covid-19): important considerations regarding novel treatment 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https://pubmed.ncbi.nlm.nih.gov/32652455/ https://pubmed.ncbi.nlm.nih.gov/32652455/ https://pubmed.ncbi.nlm.nih.gov/32652455/ journal of current biomedical reports jcbior.com volume 3, number 3, 2022 eissn: 2717-1906 1 © the author(s) 2022 original research health protocol compliance in the prevention of covid-19: comparison of healthcare workers and ordinary people groups shahla ashraf 1, nazanin noori roodsari2, tahereh zeinali3, niloofar faraji3, seyyed mahdi zia ziabari1,2,* 1clinical research development unit of poursina hospital, guilan university of medical sciences, rasht, iran 2department of emergency medicine, school of medicine, guilan university of medical sciences, rasht, iran 3gastrointestinal and liver disease research center, guilan university of medical sciences, rasht, iran abstract coronavirus disease 2019 (covid-19) is a type of acute respiratory syndrome that due to its high prevalence, mortality, and the lack of appropriate treatment; health protocol compliance has the most important role in controlling this disease. therefore, this study aimed to compare the frequency of health protocol compliance in the prevention of covid-19 in two groups of healthcare workers and ordinary people. in this cross-sectional study, individuals were randomly selected from two groups of healthcare workers and ordinary people in the community. information was completed in the form of an online questionnaire and in-person interview. a total number of 246 ordinary people and 216 healthcare workers were studied. the mean age of ordinary people was 35.53±10.16, and the mean age of healthcare workers was 34.16±8.74 years. the effect of age, gender, education level, occupation, contact with a suspected individual in the group of ordinary people, severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection among family members and relatives in the group of healthcare workers, and travel history in both groups on the score of health protocol compliance was significant. the effect of habitat, underlying disease, medication history, history of sars-cov-2, family member infection, or death, was not significant. healthcare workers were more knowledgeable, had more positive attitudes, and their higher sense of total well-being was seen to be more critical to enhancing compliance. keywords: covid-19, sars-cov-2, prevention, health protocol compliance, healthcare workers 1. introduction coronaviruses are a great family of viruses with four types (α-coronavirus, β-coronavirus, δcoronavirus, γ-coronavirus), and various sub types and species [1]. human coronaviruses were first identified in the mid of 1960s. among 40 different species of the coronavirus family, seven of them have been identified that can transmit to human cells and cause diseases such as colds. sometimes some coronaviruses attack the respiratory system and sometimes they show their symptoms in the intestines and stomachs of people. symptoms of coronavirus in *corresponding author: seyyed mahdi zia ziabari, md department of emergency medicine, school of medicine, guilan university of medical science, rasht, iran tel/fax: +98 13 33333448 email: mahdi.zia.ziabari@gmail.com https://orcid.org/0000-0002-3708-7723 received: february, 16, 2022 accepted: april, 30, 2022 the lungs usually appear in common colds and a secretory infection called pneumonia, which is usually associated with a mild cold in most individuals. four coronaviruses have been detected in this category, which cause infections of the respiratory system in children and adults [1-3]. although, other types of the virus are associated with more severe symptoms; severe acute respiratory syndrome coronavirus (sars-cov), which consider transmitted by bats in china, caused a global severe acute respiratory syndrome (sars) epidemic in 2002 that led to 800 known death. sars, scientifically known as sarshttps://jcbior.com/ https://jcbior.com/ ashraf et al. 2 cov, caused acute and severe respiratory syndrome in patients [4]. another one is middle east respiratory syndrome coronavirus (mers-cov); a newer strain of the virus was discovered in september 2012 in a 60year-old man in saudi arabia resulting in patient death. the man had traveled to dubai a few days earlier and the second case was in a 49-year-old man in qatar who also died. the virus was first discovered in the health protection agency laboratory in london. this coronavirus is known as the mers-cov and leads to 858 death in middle east [5]. the last but not least, severe acute respiratory syndrome coronavirus 2 (sars-cov-2); is a coronavirus that causes the coronavirus disease 2019 (covid-19) pandemic in 2020 till now [6]. symptoms can range from the common cold to fever, cough, fatigue, myalgia, headaches, loss of taste or smell, sore throat, and acute respiratory problems. the patient may also have a cough for several days, apparently for no apparent reason. acute gastrointestinal problems such as diarrhea, acute respiratory failure, coagulation disorders, and renal failure have also been reported, which may require hemodialysis [7, 8]. characteristics of this disease such as incubation period, asymptomatic few days, rapid transmission, and lack of response to treatment in some patients have forced health systems to face complex challenges [9]. covid-19 symptoms usually begin a few days after infection. but in some patients, the symptoms may appear a little later. according to several studies, symptoms include fever, dry cough, dyspnea, fatigue, myalgia, and diarrhea. the incubation period is 2-14 days and with an average of 4 days. ground-glass opacity is seen in 56.4% of patients’ chest computed tomography (ct) scans. 17.9% of patients with non-severe symptoms and 8.9% of patients with severe symptoms showed any problems in their radiology or ct scan result. lymphocytopenia or a decrease in the number of circulating lymphocytes was observed in 83.2% of patients at the time of admission [10, 11]. covid-19 symptoms are various from none to severe, which result in serious problems such as pneumonia, lack of oxygen, and even death. these symptoms are more common in people with underlying diseases [12]. the common way of coronavirus transmission is by respiratory drop. aerosol transmission can occur under certain conditions, especially in closed, crowded, and inadequately ventilated areas. the virus can be spread through the mouth or nose of an infected person in small liquid particles when coughing, sneezing, talking, or breathing heavily [13, 14]. simultaneously with the official announcement of the spread of sars-cov-2 in iran, a large number of people paid attention to the observance of hygienic principles and regular handwashing with soap and water and other disinfectants [15]. after china and several other asian countries, iran was one of the first countries to be exposed to this deadly virus. according to official iranian news, the first case of covid-19 was reported in qom on february 20, 2020, then in guilan and several other cities. guilan province was one of the first provinces to be affected by this virus in the country [16]. due to the lack of appropriate treatment for sars-cov-2 infection, this study was designed to compare the prevalence of compliance to the health protocol in the prevention of covid-19 among two groups of healthcare workers and ordinary people. 2. methods 2.1 participants in this cross-sectional study, 462 individuals were selected randomly among the ordinary people group and healthcare workers in healthcare centers in guilan, iran, july-december 2020. to determine the sample size, the formula is proportional to the difference between the mean of the two communities and the health protocol variable. considering the statistical power of 80%, the error level of 0.05 and the standard deviation were 2.71 (healthcare workers) and 2.60 (ordinary people group). the questionnaire was designed; the validity was confirmed based on the lavashe table, and the reliability was checked by cronbach's alpha. the content validity ratio (cvr) and content validity index (cvi) for all questions were 0.84 and 1.00, respectively. the cronbach’s alpha for all questions was 0.71. the information according to the questionnaire was collected through online and inperson methods. all the participants gave their informed consent to participate in the study. the study design was approved by the ethical committee of guilan university of medical sciences [ir.gums.rec.1399.486]. 2.2 statistical method statistical methods including, mean and standard deviation, frequency and percentage have been used to describe the obtained data in this study. also, to ashraf et al. 3 investigate the normality of the studied data, the shapiro–wilk test, and kurtosis and skewness indices were used. then, the homogeneity of variance of the studied groups was evaluated by levon's test. to analyze the research data, independent t test, anova, pearson correlation, mann whitney test, kruskal wallis, mann-whitney test with bonferroni correction, spearman correlation test, and linear regression were performed through spss.24. the significance level in all tests is considered 0.05. 3. results in this investigation, 246 ordinary people group and 216 healthcare workers were studied. the mean age of the ordinary people group was 35.53 ±10.16 and in healthcare workers was 34.16 ± 8.74 years. in the ordinary people group and healthcare workers 149 (69%), 177 (72%) were female; and 196 (79.7%), 194(89.8%) were urban, respectively. the frequency of education levels and occupations in the two groups is illustrated in table 1. in the ordinary people group, a significant relationship was observed between age (p <0.001), gender (p = 0.019), level of education (p <0.001), occupation (p <0.001) with health protocol compliance. in neither group, there was no significant relationship between habitat and health protocol compliance (p >0.05), table 2. correspondingly, the frequency of clinical characteristics (underlying disease, medication history, travel history, history of sars-cov-2 infection, sars-cov-2 infection among family members and relatives, death outcome of sars-cov-2 infection, contact with suspected individuals) illustrated in ordinary people group and healthcare workers in table 1. based on the results in ordinary people group and healthcare workers no significant relationship was observed between the underlying diseases, history of medication, history of sars-cov-2 infection, and death outcome of sarscov-2 infection with the score of health protocol compliance (p >0.05) table 2. in both groups, a significant relationship was observed between travel history and health protocol compliance score (p <0.019 and p <0.022). so that the average score of health protocol compliance was lower in people with a history of travel to suspicious areas. the score of health protocol compliance was lower in healthcare workers than among family members and relatives who had sars-cov-2 infection (p = 0.012). the score of health protocol compliance was lower in the ordinary people group who had contact with suspected individuals (p = 0.005). the comparison of other variables in the two groups is demonstrated in table 2. 3.1 determining the synchronized effect of variables on the score of health protocol compliance as with table 3, the relationship between age (p = 0.001), gender (p = 0.040), education (p = 0.007), and group (p <0.001) was significant with the score of health protocol compliance. based on demographic variables, between the two groups, the ordinary people group had a higher score in health protocol compliance (p <0.001) and was most associated with age (p = 0.001). there was the highest correlation between age and health protocol compliance score (p = 0.001), so increasing age, increases the score of health protocol compliance. likewise, the effect of travel history (p = 0.003), contact with suspected individuals (p = 0.005), and group (p = 0.004) on the score of health protocol was significant. people with a history of travel and contacting suspected individuals had a lower score of health protocol compliance. also based on clinical variables healthcare workers had a higher score than the ordinary people group (p = 0.003), table 3. 4. discussion due to our study, healthcare workers exhibited a significantly higher score of healthcare protocol compliance compared to the ordinary people group (p = 0.014), this is also in line with previous studies [17, 18]. this indicates that healthcare workers pay more attention to healthcare protocol compliance than ordinary people group. also, our findings indicated that upper age was associated with healthcare protocol compliance (r=0.244, p <0.001), which is similar to previous studies [19, 20], while, this association was not significant among healthcare workers (p = 0.104), this can be justified by the constant and close contact of healthcare workers with patients. the female gender represented a higher score for health protocol compliance, compared to the male gender in the ordinary people group (p <0.001, table 2). this could be due to behavioral differences between females and males. females are more likely to stay home than males, leaving homeless, regular hand washing, and surface cleaning, while, this relationship was not significant in healthcare workers (p = 0.307). padidar et al. reported that females are more cooperating with ashraf et al. 4 public health protocol than males (p<0.001), particularly with protecting themselves from covid19 [21]. table 1. characteristics of two studied groups variables healthcare workers (216) n (%) ordinary people (246) n (%) gender male 67 (31) 69 (28) female 146 (69) 177 (72) age mean ± sd 34.16±8.74 35.53±10.16 habitat urban 194 (89.9) 196 (79.7) rural 22 (10.2) 50 (20.3) education elementary 0 (0) 10 (4.1) m school 0 (0) 6 (2.4) diploma 10 (4.6) 34 (13.8) upper diploma 18 (8.3) 15 (6.1) bachelor 79 (36.6) 101 (41.1) master 36 (16.7) 52 (21.1) phd 73 (33.8) 28 (11.4) occupation self-employee 54 (22) clerk 72 (29.3) housewife 67 (27.2) unemployed 10 (4.1) student 43 (17.5) travel history no 136 (63) 100 (40.7) yes 80 (37) 146 (59.3) underlying disease no 184 (85.2) 218 (88.6) yes 32 (14.8) 28 (11.4) medication history no 140 (64.8) 163 (66.3) yes 76 (35.2) 83 (33.7) history of sars-cov-2 infection no 137 (63.4) 204 (82.9) yes 79 (36.6) 42 (17.1) sars-cov-2 infection among family members and relatives no 84 (38.9) 117 (47.6) yes 132 (61.1) 129 (52.4) death outcome of sars-cov-2 infection no 197 (91.2) 232 (94.3) yes 19 (8.8) 14 (5.7) contact with suspected individuals no 69 (31.9) 164 (66.7) yes 147 (68.1) 82 (33.3) ashraf et al. 5 table 2. comparison of the demographic and clinical variables in healthcare workers and ordinary people groups study groups variable p value relationship between gender and health protocol compliance score male female ordinary people 21.57±4.06 22.83±2.52 0.019 healthcare workers 22.85±2.89 23.26±2.44 0.307 relationship between habitat and health protocol compliance score urban rural ordinary people 22.29±3.20 23.24±2.42 0.052 healthcare workers 23.19±2.46 22.63±3.57 0.919 relationship between education and health protocol compliance score elementary middle school diploma upper diploma bachelor master phd ordinary people 23.60±2.27 22.66±5.24 23.11±2.3 22.00±2.32 22.98±2.87 22.34±3.24 20.00±3.22 <0.001 healthcare workers 23.80±2.44 24.11±1.45 23.20±2.46 22.88±2.61 22.86±2.92 0.408 relationship between occupation and health protocol compliance score self-employee n=54 clerk n=72 housewife n=67 unemployed n=10 student n=43 ordinary people 22.35±3.47 22.73±2.94 23.47±1.97 22.40±2.41 20.69±3.63 <0.001 relationship between underlying disease and health protocol compliance score no yes ordinary people 22.53±2.86 22.10±4.45 0.826 healthcare workers 23.29±2.46 22.21±3.12 0.071 relationship between medication history and health protocol compliance score no yes ordinary people 22.74±2.67 21.97±3.72 0.098 healthcare workers 23.03±2.70 23.32±2.39 0.429 relationship between travel history and health protocol compliance score no yes ordinary people 23.01±2.49 22.12±3.38 0.019 healthcare workers 23.44±2.28 22.61±2.99 0.022 relationship between sars-cov-2 infection and health protocol compliance score no yes ordinary people 22.69 ± 2.72 21.47 ± 4.34 0.087 healthcare workers 23.15 ± 2.53 23.11 ± 2.72 0.915 ashraf et al. 6 the ordinary people group with a phd degree showed the highest scores of disobediences among the other levels of educational groups (p<0.001). this relationship probably depends on the type of different occupations according to the level of education in ordinary people group. that is, due to the increase in the level of education, the type of occupation was such that they were less exposed to the sars-cov-2 virus, and as a result, they followed the healthcare protocol less. therefore, due to the low-risk and high-risk occupation environments in relation to covid-19, compliance health protocol has been lower in people with higher education. data from an ongoing cohort study in zurich, switzerland on 737 individuals, has indicated that compliance with covid-19 public health protocol was lower among males, those with a higher education level [22]. contradictory to this, other studies showed that people with high education showed the highest scores of adherence to quarantine restrictions [17, 23]. among the ordinary people group, students showed a significantly lower scale of health protocol compliance compared to employees (p<0.004) and housewives (p=0.004). lack of awareness denied the importance of health protocols, and more desire to return to normal life, are some of the reasons that can decrease the tendency to follow health protocols, which is consistent with the findings of a previous study [17, 24] . in general, there was no significant relationship between the two groups for health protocol compliance scores in terms of habitat, since almost everyone's access to global media and news related to health protocols in the prevention of covid-19. yue et al. have indicated that urban and rural residents had a positive attitude toward covid19. they found that all the respondents had recognized the seriousness of the disease and were concerned about the deterioration of the epidemic. the great majority of the residents believed that preventive study groups variable p value relationship between an infected family member and health protocol compliance score no yes ordinary people 22.82±2.86 22.17±3.24 0.094 healthcare workers 23.65±1.98 22.81±2.87 0.012 relationship between the death of a family member and health protocol compliance score no yes ordinary people 22.54 ± 3.07 21.50 ± 3.15 0.219 healthcare workers 23.14 ± 2.59 23.05 ± 2.67 0.903 relationship between contacting suspected individuals and health protocol compliance score no yes ordinary people 22.91±2.64 21.62±3.67 0.005 healthcare workers 23.56±2.09 22.93±2.78 0.068 table 3. determining the synchronized effect of variables on the score of health protocol compliance variables p value b sd beta d e m o g ra p h ic age 0.001 0.04 0.01 0.14 gender 0.040 0.59 0.29 0.09 habitat 0.893 0.05 0.40 0.007 education 0.007 -0.30 0.11 -0.14 group <0.001 0.97 0.27 0.16 c li n ic a l travel history 0.003 -0.81 0.27 -0.14 underlying disease 0.143 -0.59 0.40 -0.07 medication history 0.831 0.06 0.29 -0.01 sars-cov-2 infection 0.898 -0.04 0.32 -0.006 family member infected 0.096 -0.47 0.28 -0.08 death of a family member 0.970 0.02 0.52 0.002 contacting suspected individuals 0.005 -0.82 0.29 -0.14 group 0.004 0.82 0.28 0.14 ashraf et al. 7 measures could prevent the infection of covid-19 and thought that people should immediately report or cut off contact with people around them returning from wuhan or its surrounding areas [25]. on the contrary, callaghan et al. have studied rural and urban different scores in covid-19 prevention behaviors. they found no statistically significant differences based on rural status for staying 6 feet apart, washing hands, and canceling social events [26]. neither in the healthcare workers group, nor ordinary people group, there was no significant relationship between underlying disease and medication history in terms of health protocol compliance. as one study concluded that people with mental health problems have lower adherence to precautionary measures against covid19 [27]. the score of health protocol compliance was lower in people with a history of travel to suspicious areas. some reasons such as, not observing a safe distance, not finding a suitable place to wash hands during the trip, not having enough disinfectants, masks and, gloves, using public vehicles, etc., can reduce health protocol compliance, which has been observed in both health workers and ordinary people group. the health protocol compliance score was not related to a person's history of coronavirus infection, but was lower in healthcare workers whose families and acquaintances were infected with the coronavirus (p=0.012). infection of family members may be related to a group of healthcare workers who were less likely to follow a health protocol at home or thought they would be permanently protected against future coronavirus infections. the attitude toward the history of sars-cov-2 infection in friends or family members and compliance with covid-19 preventive measures was similar amongst healthcare professionals and non-healthcare professionals, likely due to the fear of infection [18]. in the group of ordinary people, the health protocol compliance score was lower in the case of contact with suspected individuals (p= 0.005). this can be justified in the group of ordinary people compared to healthcare workers due to less exposure and lack of knowledge about how the virus is transmitted from person to person. many studies and surveys are being carried out by countries to understand people’s attitudes and perceptions of covid-19 and their association with knowledge, protective behaviors and practices [18, 28]. however, very few studies and surveys have been conducted at a global level to understand the factors related to compliance towards various public health measures and differences in perceptions and practices between those that work in health services compared to other sectors. because of the compliance of health protocol in the ordinary people group, including students, males lower than females, it is possible to reduce the severity of this pandemic to some extent by providing more education and emphasizing compliance with health protocol. the control of responsible agencies in the continuation of travel restrictions and unnecessary traffic, due to the reduction of health protocol during the travel, can to some extent interrupt the chain transmission of the sars-cov-2. 5. conclusion healthcare workers were more knowledgeable, and had more positive attitudes, their higher sense of total well-being was seen to be more critical in enhancing compliance. therefore, focusing on the well-being of the general population would help to enhance their compliance with the preventive measures for covid-19. besides, given the charged partisan discourse surrounding covid-19, having conservative voices encourage humans about the importance of health protocols and other preventive behaviors could help change behavior and slow the covid-19 pandemic. acknowledgments we would like to thank all hospital staff and the specialist for assistance with conforming and recording cases. authors’ contributions concept and study design: nn, sz. methods, data collection and experimental work: sa, nn, sz. results analysis and conclusions: sa, tz, nf. manuscript preparation and editing: tz, nf. all authors read and approved the final version of manuscript. conflict of interests the authors declare no competing interests. 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file:///c:/users/elecom93/appdata/roaming/microsoft/word/mehr.tums.ac.ir/main%20pdf file:///c:/users/elecom93/appdata/roaming/microsoft/word/mehr.tums.ac.ir/main%20pdf file:///c:/users/elecom93/appdata/roaming/microsoft/word/mehr.tums.ac.ir/main%20pdf file:///c:/users/elecom93/appdata/roaming/microsoft/word/mehr.tums.ac.ir/main%20pdf https://pubmed.ncbi.nlm.nih.gov/32112714/ https://pubmed.ncbi.nlm.nih.gov/32112714/ https://pubmed.ncbi.nlm.nih.gov/32112714/ https://pubmed.ncbi.nlm.nih.gov/32112714/ https://pubmed.ncbi.nlm.nih.gov/18387216/ https://pubmed.ncbi.nlm.nih.gov/18387216/ https://pubmed.ncbi.nlm.nih.gov/18387216/ https://pubmed.ncbi.nlm.nih.gov/18387216/ https://pubmed.ncbi.nlm.nih.gov/32757087/ https://pubmed.ncbi.nlm.nih.gov/32757087/ https://pubmed.ncbi.nlm.nih.gov/32757087/ https://pubmed.ncbi.nlm.nih.gov/32757087/ https://pubmed.ncbi.nlm.nih.gov/33619836/ https://pubmed.ncbi.nlm.nih.gov/33619836/ https://pubmed.ncbi.nlm.nih.gov/33619836/ https://pubmed.ncbi.nlm.nih.gov/34385242/ https://pubmed.ncbi.nlm.nih.gov/34385242/ https://pubmed.ncbi.nlm.nih.gov/34385242/ https://pubmed.ncbi.nlm.nih.gov/34385242/ https://pubmed.ncbi.nlm.nih.gov/34385242/ https://pubmed.ncbi.nlm.nih.gov/32576418/ https://pubmed.ncbi.nlm.nih.gov/32576418/ https://pubmed.ncbi.nlm.nih.gov/32576418/ journal of current biomedical reports jcbior.com volume 2, number 4, 2021 eissn: 2717-1906 1 original research evaluation of sequential organ failure assessment (sofa) score efficiency in predicting the mortality of intensive care unit admitted covid-19 patients mohammad haghighi1, hossein khoshrang1, siamak rimaz1, tofigh yaghubi kalurazi2, zahra atrkar roushan3, samaneh ghazanfar tehran1,*, paniz rezaei1 , niloofar faraji4 1anesthesiology research center, department of anesthesiology, alzahra hospital, guilan university of medical sciences, rasht, iran 2department of health, nutrition and infectious disease, school of medicine, guilan university of medical sciences, rasht, iran 3department of biostatistics, faculty of medicine, guilan university of medical sciences, rasht, iran 4razi clinical research development unit, razi hospital, guilan university of medical sciences, rasht, iran abstract assessing the severity of the disease at the time of hospitalization can reduce the mortality of patients with coronavirus disease 2019 (covid-19). for this stance, various scoring systems have been described to predict mortality rates. sequential organ failure assessment (sofa) is one of the scoring systems which have been used in this study. in order to calculate the sofa score, demographic and characteristics information, clinical status, and laboratory findings were recorded from 154 patients with covid-19, who have been admitted to the intensive care unit (icu) for a period of 6 months. sofa score was calculated in three time periods at the time of hospitalization, 72 hours after hospitalization, and the last day of hospitalization. based on the outcome of the disease (death or recovery), patients were divided into two groups, and the results were analyzed in both groups. statistical analysis has represented that the sofa score was significantly higher in patients who died to compare with recovered ones in all time periods. our findings suggest that sofa scoring system can be used to predict mortality rate in icu admitted covid-19 patients. keywords: covid-19, sofa score, icu, mortality 1. introduction in december 2019, several cases of pneumonia with unknown etiology were reported in wuhan city, hubei province, china. in the early stages of pneumonia, symptoms of acute respiratory infection occurred, and some patients progressed rapidly to acute respiratory failure and other serious complications [1]. on february 11, 2020, the world health organization (who) officially named the new *corresponding author: samaneh ghazanfar tehran, md anesthesiology research center, department of anesthesiology, alzahra hospital, guilan university of medical sciences, rasht, iran tel/fax: +98 911 2318819 email: tehranisamaneh88rasht@gmail.com http://orcid.org/0000-0002-9910-2394 received: august, 21, 2021 accepted: september, 21, 2021 coronavirus infection coronavirus disease 2019 (covid-19), and the international committee of the red cross study group named it as severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [2]. four months since the advent of covid-19 in china, the disease has spread rapidly to other parts of the world and has become an international threat [3]. the mortality rate in patients with covid-19 with severe conditions has been reported to be fluctuated © the author(s) 2021 https://jcbior.com/ haghighi et al. 2 between 11% and 61% [1, 4-6]. various studies have shown that covid-19 affects different organs and its complications include pneumonia, liver and kidney disorders, heart damage, immunodeficiency, and coagulation disorders, which are in the result of cell and organotropism [7, 8]. sepsis is a life-threatening complication of infection and occurs in the form of physiological, pathological, and biochemical disorders [9, 10]. one of the key strategies to improve the treatment of sepsis is to prioritize patients in terms of the risk of adverse outcomes. this can help physicians to prioritize initial treatments such as antibiotics and fluid therapy [11]. in critically ill patients, early medical interventions to reduce mortality depend on effective early assessment methods. a scoring system could be helpful to determine the prognosis and accelerate practical evaluation by physicians to identify these patients and could be useful in providing information to relatives of patients, treatment decisions, and guidance in allocating resources [12]. however, there is currently no specific scoring system for evaluating patients with covid-19. the organ failure assessment score (sofa) system can be used to determine prognosis and predict mortality. sofa was first described in 1996, and it was used as a tool to evaluate organ failure in sepsis and also to evaluate the effects of treatments such as mechanical ventilation and vasopressor on organ dysfunction [13]. sofa describes dysfunction of various organs through several parameters, including oxygenation index (ratio of arterial oxygen pressure to the percentage of inhaled oxygen), mean arterial pressure, glasgow coma scale, creatinine or urine volume, bilirubin, and platelets, respectively for the respiratory system, circulation, neurology, renal, hepatic and coagulation. each of these parameters is assigned 0 to 4 points, and the sum of these points is 0 to 24, which determines the overall sofa score. the higher score indicates the worsening of the organ disorder. numerous studies have shown that sofa can predict morbidity and mortality and has been used for evaluation in critically ill patients and in the intensive care unit (icu) [12, 14-16]. this study has been conducted since there is no special scoring system in patients with covid-19, and on the other hand, there is no performed research on sofa score in patients with covid-19, who have been admitted to the icu in iran. this present study to investigate the clinical characteristics, medical records and laboratory results of icu admitted covid-19 patients to further evaluation for the role of sofa score in predicting the complexity of the disease in three different time periods and help to identify the related factors. 2. materials and methods 2.1 study design this present retrospective analytical crosssectional study was performed on patients with covid-19, who have been admitted to the icu of razi hospital in rasht, iran, after receiving the approval of the vice-chancellor for research and the code of ethics number ir.gums.rec.1399.509. inclusion criteria included all patients with a definitive diagnosis of covid-19 based on a positive nasopharyngeal pcr test, hospitalization in the icu of razi hospital in rasht, iran, from april 1 to september 22, 2020. exclusion criteria included patients with insufficient information about their condition, and also patients who had been admitted to the icu before april 1, 2020 or were still admitted to the icu after september 22, 2020. in this regard, the checklist has been used with the following items: age, sex, underlying disease history, clinical status, and laboratory results, which have been documented in the medical records of icu, admitted covid-19 patients, to calculate the sofa score. the sofa score of patients in three time periods was calculated at the time of admission to the icu, 72 hours after admission, and the last day of hospitalization in the icu. based on the outcome of the disease (death or discharge), patients were divided into two groups. 2.2 data analysis data analysis was done through spss software version 22 (spss inc., usa). for variables with normal distribution, descriptive analysis was performed using mean and standard deviation. number and percentage were used to report qualitative variables. chi-square test, independent ttest, logistic regression, and mann whitney test were used to evaluate the study's objectives. 3. results demographic information, including age, sex, and comorbidities in the two groups of discharged and deceased patients, is given in table 1. in general, the mean age of the patients was 60.07±15.73 years, with haghighi et al. 3 53.2% of males. the chi-square test has shown no statistically significant relationship between gender and deceased or recovered patients with covid-19 disease (p = 0.54). however, there was a remarkable relationship between the two groups in terms of age, so that the mean age in the deceased patients was higher than the other group (p= 0.002). also, there was a noteworthy relationship between the presence of underlying disease in patients admitted to icu and the outcome of treatment, but there was no difference in the distribution of underlying disease in hospitalized patients (p> 0.05). sofa score analysis with mann whitney u test has represented a significant difference between sofa score values in the three time periods in the two groups (table 1). logistic regression on sofa score at the time of admission in the icu as an intervening variable in predicting patient mortality has illustrated that increasing each sofa score at the time of hospitalization can increase the probability of mortality by 1.27 times (p<0.001). also, logistic regression on sofa score at 72 hours after admission in the icu as an intervening variable in predicting patient mortality has illustrated that increasing each sofa score at 72 hours after admission can increase the probability of mortality by 1.47 times (p<0.001). logistic regression on sofa score on the last day of hospitalization in the icu as an intervening variable in predicting patient mortality has illustrated that increasing each sofa score on the previous day of hospitalization could increase the probability of mortality by 1.9 times (p<0.001), table 2. mann whitney u test has illustrated that there was a significant difference between the values of sofa score at the time of admission, 72 hours after admission, and the last day of admission according to the presence of underlying diseases in the studied patients (p = 0.02, p =0.007, p=0.01). among the underlying diseases, there was a significant difference between the values of sofa scores in the studied periods only in heart, kidney, and liver diseases. sofa score in heart disease at 72 hours postoperatively, in kidney disease in all stages, and in liver disease in the first and last day of hospitalization was significantly higher than the other group (table 3). 4. discussion the covid-19 disease can easily lead to acute respiratory distress, multiorgan dysfunction, acute heart damage, acute kidney damage, and even death in the case of severity [17]. during the corona pandemic, the increase in the number of critically ill patients and the limited medical resources leads to global concern worldwide [18, 19]. therefore, the initial evaluation of patients with severe covid-19 disease is crucial for monitoring and early medical interventions. consequently, the important duty of physicians is to screen patients with a high mortality rate among very critically ill patients [20]. a scoring system can help physicians to be quick and accurate diagnosis to make treatment decisions. however, a simple and effective way to assess the severity and outcome of patients with covid-19 is challenging for physicians [17]. the sofa score is one of the scoring systems that is used to assess organ failure and can predict the severity and outcome of the disease. for this purpose, the sofa scale is used in the health care system, which has high accuracy, but on the other hand, it’s complicated and time-consuming because it requires the evaluation of several parameters [17, 21]. a study has reported that modified sequential organ failure assessment (msofa) score also can be useful as well as the sofa and it’s easier to implement in resource-constrained settings [22]. organ dysfunction is associated with a high mortality rate and a large percentage of the budget in the icu. the sofa score can be used to assess organ dysfunction or failure and morbidity. however, this system was first developed to describe organ function rather than predict patient outcomes. studies have shown a remarkable association between organ function discrepancy and mortality [23]. since there is no special scoring system for risk assessment in patients with covid-19, we conducted this study to evaluate the effectiveness of the sofa score in critically ill patients to identify an effective risk scoring system in covid-19 patients. this present study has represented that the sofa score was higher in patients who died to compare with recovered ones in all time periods. also, based on logistic regression, the sofa score was identified as a predictor of mortality in critically ill patients in the icu. haghighi et al. 4 a study by liu et al. [24] has evaluated the efficacy of sofa compared to quick sofa (qsofa) score, which identifies patients outside the icu with suspected infection that are at a high risk for inhospital mortality and is composed of 3 variables (respiratory rate, systolic blood pressure, and altered mental status), in predicting the outcome of patients with covid-19, sofa≥3, and qsofa≥1 are related to mortality in severe covid-19 disease. in addition, sofa is a very sensitive marker in diagnosing inhospital mortality and is preferred to qsofa for predicting outcomes [21]. another study by raith et al., which has been conducted to evaluate the power of sofa, qsofa, and systemic inflammatory response syndrome (sirs) criteria, which assessment includes 4 clinical variables (temperature, heart rate, respiratory rate, and white blood cell count), in predicting the outcome of patients suspected of infection admitted to the icu, has reported that increasing sofa score is more than twice as strong predictor for mortality in comparison to qsofa and sirs criteria [10]. yang et al. by considering 60-day mortality, have reported the patients with a higher sofa score (5≤) had a higher risk of mortality than patients with a lower sofa score (5≥), which is similar to our study [17]. also, in the study of chen et al., sofa score, along with old age and high underlying disease, were identified as two independent risk factors for death in patients with covid-19 [12]. however, a study by raschke et al., which examined the accuracy of the sofa score to determine the possible mortality of patients with covid-19 pneumonia requiring mechanical ventilation, showed that the power of sofa to predict mortality in patients with covid-19 pneumonia was weak and significantly less valuable than the age factor [25]; they justified the results by referring to the previous studies, which stated sofa score in 3 subgroups (pulmonary, hepatic and renal disease) and the relation with mortality in covid-19 patients [25, 26]. our study showed that there was a statistically significant relationship between sofa score and underlying disease at baseline, 72 hours after hospitalization, and the last day of hospitalization. table 1. demographic, clinical findings and sofa values of hospitalized patients by treatment outcome variables death recovery p value gender male 45 (54.9) 37 (45.1) 0.54 female 43 (59.7) 29 (40.3) age (mean ± sd) 63.45 ± 14.78 55.57 ± 15.94 0.002 underlying disease yes 73 (62.4) 44 (37.6) 0.019 no 15 (40.5) 22 (59.5) underlying disease diabetes 24 (47.1) 27 (52.9) 0.49 high blood pressure 22 (34.4) 42 (65.6) 0.07 heart disease 16 (36.4) 28 (63.6) 0.30 respiratory disease 8 (36.4) 14 (63.6) 0.50 kidney disease 5 (33.3) 10 (67.7) 0.43 liver disease 3 (100) 0.26 neurological disease 4 (36.4) 7 (63.6) 0.65 malignancy 6 (42.9) 8 (57.1) 1.00 rheumatic disease 3 (60) 2 (40) 0.65 sofa score at the time of admission 8.76 ± 3.69 6.12 ± 2.87 0.0001 72 hours after admission 9.92 ± 3.9 5.6 ± 2.76 0.0001 last day of admission 11.07 ± 3.92 4.65 ± 2.09 0.0001 table 2. comparison of sofa score predictive power by measurement times variables time of admission incidence rate (or) confidence interval (ci) p value sofa score at the time of admission 1.27 1.13-1.42 <0.001 72 hours after admission 1.47 1.27-1.70 <0.001 last day of admission 1.90 1.55-2.34 <0.001 haghighi et al. 5 sofa levels in patients with underlying disease were higher in all stages than in patients without underlying disease. studies have shown that patients with a history of previous underlying disease are at table 3. comparison of sofa score scores in the studied time periods according to the presence or absence of underlying disease in hospitalized patients variables sofa score underlying disease mean ± sd p value underlying disease at the time of admission yes 7.95 ± 3.58 0.02 no 6.59 ± 3.53 72 hours after admission yes 8.33 ± 4.05 0.007 no 6.5 ± 3.67 last day of admission yes 8.74 ± 4.45 0.01 no 7.0 ± 4.7 high blood pressure at the time of admission yes 8.04 ± 3.77 0.26 no 7.33 ± 3.47 72 hours after admission yes 8.73 ± 3.81 0.10 no 7.48 ± 4.14 last day of admission yes 8.9 ± 4.27 0.09 no 7.91 ± 4.73 diabetes at the time of admission yes 7.8 ± 3.42 0.54 no 7.54 ± 3.7 72 hours after admission yes 7.89 ± 3.63 0.52 no 7.8 ± 4.2 last day of admission yes 8.29 ± 3.8 0.49 no 8.33 ± 4.91 heart disease at the time of admission yes 8.02 ± 3.48 0.27 no 7.47 ± 3.65 72 hours after admission yes 9.12± 4.12 0.01 no 7.36 ± 3.9 last day of admission yes 9.45 ± 4.81 0.05 no 7.87 ± 4.39 respiratory at the time of admission yes 7.36 ± 4.06 0.49 no 7.67 ± 3.53 72 hours after admission yes 8.05 ± 5.18 0.83 no 7.79 ± 3.83 last day of admission yes 8.0 ± 4.54 0.68 no 8.37 ± 4.57 kidney disease at the time of admission yes 11.0 ± 3.96 0.001 no 7.26 ± 3.38 72 hours after admission yes 10.84 ± 4.01 0.005 no 7.47 ± 3.89 last day of admission yes 3.4± 13.11 0.01 no 8.02 ± 4.49 liver disease at the time of admission yes 14.00 ± 1.73 0.01 no 7.50 ± 3.52 72 hours after admission yes 14.00 ± 7.07 0.10 no 7.72 ± 3.92 last day of admission yes 17.66 ± 1.15 0.004 no 8.13 ± 4.4 neurologic disease at the time of admission yes 7.81 ± 2.89 0.61 no 7.61 ± 3.66 72 hours after admission yes 6.8 ± 4.26 0.37 no 7.92 ± 4.01 last day of admission yes 7.54 ± 4.82 0.65 no 8.38 ± 4.55 malignancy at the time of admission yes 8.0 ± 3.78 0.62 no 7.59 ± 3.6 72 hours after admission yes 8.66 ± 4.79 0.45 no 7.74 ± 3.94 last day of admission yes 9.35 ± 5.25 0.37 no 8.22 ± 4.49 rheumatic disease at the time of admission yes 6.4 ± 2.5 0.55 no 7.67 ± 3.63 72 hours after admission yes 5.66 ± 1.15 0.41 no 7.88 ± 4.05 last day of admission yes 5.2 ± 3.76 0.07 no 8.42 ± 4.55 haghighi et al. 6 higher risk of death [27]. among the underlying diseases, liver disease was one of the cases that showed a significant relationship with sofa levels at the beginning of hospitalization and also on the last day of hospitalization. patients with covid-19 who had a history of liver disease had a higher sofa score than patients without liver disease at the time of admission. patients with liver disease, especially uncompensated cirrhosis due to immune dysfunction, may be more susceptible to infection with sars-cov-2 [28]. however, due to primary reports, 2-11% of covid-19 patients had a history of liver disease, but the true prevalence of covid-19 among liver disease remains unknown [28, 29]. these patients show poor prognosis when admitted to the icu, especially in the case of dysfunction or multiple organ failure [30]. among the underlying diseases, kidney disease was one of the cases that had a significant relationship with sofa levels at the beginning of hospitalization, 72 hours after hospitalization, and the last day of hospitalization in our survey. patients with covid-19 with a history of chronic kidney disease had higher sofa scores than patients without kidney disease at all stages of the measurement. having chronic underlying kidney disease is associated with a higher rate of in-hospital mortality. also, patients with dialysis-dependent renal failure have the highest risk of in-hospital death compared to patients who do not undergo dialysis [31]. a study by goswami et al. was conducted to evaluate the effectiveness of sofa and other scoring systems on the outcome of patients with chronic kidney disease admitted to the icu, which has represented that each sofa score increase mortality significantly in 30-day period. also, the sensitivity and specificity of sofa ≥10.5 in predicting 30-day mortality are 85% and 100%, respectively [32]. heart disease was another underlying disease in patients with covid-19 that had a statistically significant relationship with sofa levels in 72 hours after hospitalization and the last day of hospitalization. according to new studies, the sofa score is a strong predictor in the cardiac intensive care unit [33]. studies have shown that the clinical outcome is severe in patients with covid-19 with a history of cardiovascular disease. a history of cardiovascular disease and the presence of cardiovascular risk factors make patients with covid-19 vulnerable. in addition, covid-19 can exacerbate underlying cardiovascular disease and even predispose these patients to new cardiac complications [34, 35]. although several studies have been performed to evaluate the efficacy of the sofa scoring system in patients with covid-19 disease, few studies have systematically evaluated the accuracy of sofa in diagnosing the severity of covid-19 disease and its predictive value. also, according to our investigation, this is the first study that evaluated the sofa score in 3 time periods in critically ill patients with covid-19, which is the strength of our study. however, this study had some limitations; one of these limitations was the study's sample size. certainly, studies with a large number of critically ill patients with covid-19 can more accurately assess the efficiency of the sofa scoring system. this study was a single-center study. due to limited treatment resources and the impossibility of providing an intensive care bed for all critically ill patients, many of these patients were not admitted to the icu. extra multicenter studies should be done in longer time periods to enhance the sofa score's efficiency. also, other scoring systems are widely used in the icu. it is recommended that the effectiveness of these scoring systems in patients with covid-19 be evaluated in future studies. in summary, the sofa scoring system at the beginning of hospitalization and 72 hours after hospitalization can be used to predict mortality in critically ill patients with covid-19 who have been admitted to the icu. acknowledgments we would like to thank all individuals who volunteered to participate in this study. furthermore, thank you to razi hospital staff and specialists for assistance with conforming and recording cases. author contributions concept and study design: mh, hkh, ty. methods and experimental work: sr, zar, st. results analysis and conclusions: pr, mh, hkh, nf. manuscript preparation: mh, st, nf, zar. all authors read and approved the final version of manuscript. conflict of interests no potential conflict of interest was reported by the authors. ethical declarations haghighi et al. 7 all subjects gave their informed consent to participate in the study, and the study design was approved by the ethical committee of guilan university of medical sciences by the code of ethics number ir.gums.rec.1399.509. financial support self-funding. references 1. chen n, zhou m, dong x, qu j, gong f, han y, et al. epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study. lancet. 2020; 395(10223):507-13. 2. guo yr, cao qd, hong zs, tan yy, chen sd, jin hj, et al. the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak an 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school of nursing and midwifery, guilan university of medical sciences, rasht, iran 2social determinants of health research center (sdhrc), guilan university of medical sciences, rasht, iran 3burn and regenerative medicine research center, guilan university of medical sciences, rasht, iran 4department of medical nanotechnology, school of advanced technologies in medicine, mazandaran university of medical sciences, sari, iran 5department of medical-surgical nursing, school of nursing and midwifery, kashan university of medical sciences, kashan, iran abstract nursing students experience a number of emotional challenges during their educations, leading to high levels of stress among them. one of the strategies of stress management in nursing students is the use of mindfulness. this study aimed to assess mindfulness and related factors among iranian nursing students. in a cross-sectional study, 100 nursing students studying at nasibeh faculty of nursing and midwifery affiliated to mazandaran university of medical sciences, iran was enrolled. data were collected via census sampling from may to june 2018. data were collected using a three-part questionnaire including demographic characteristics, kentucky mindfulness skills scale, and spielberger state anxiety scale. data analysis was performed using descriptive statistics, t-test, chisquared, and anova tests. the mean score of mindfulness of nursing students was 117.35 ± 12.89. the mean scores of mindfulness in the dimensions of observation, description, act with awareness, and acceptance without judgment was 36.33 ± 6.0, 25.68 ± 4.91, 28.17 ± 4.49, and 27.17 ± 3.80, respectively. the mean score of anxiety in nursing students was 14.17 ± 3.78. there was a significant relationship between age and dimension "description" of mindfulness (r = 0.22, p = 0.029). there was a significant relationship between dimension "observation" of mindfulness and experience stressful events during the last six months (t = 2.327, df = 96, p = 0.022). therefore, due to the importance of mindfulness among nursing students, it is possible to reduce the risk factor by creating the necessary conditions such as continuing education. keywords: mindfulness, nursing students, anxiety, iran 1. introduction nursing students experience a number of emotional challenges during their educations, which lead to high levels of stress among them [1]. high levels of perceived emotional stress and anxiety cause a disorder in learning, problem-solving, and coping skills among nursing students, which ultimately *corresponding author: mohammad javad ghazanfari, msc department of medical-surgical nursing, school of nursing and midwifery, kashan university of medical sciences, kashan, iran tel/fax: +98 930 2426919 email: javad.ghazanfari12@yahoo.com http://orcid.org/0000-0003-3555-6044 received: september, 20, 2021 accepted: november, 11, 2021 hinders learning, retention, and application of knowledge in clinical and academic settings [1-3]. on the other hand, previous evidence has shown that multiple stressors such as academic education and presence in clinical settings lead to high levels of stress among nursing students [2, 4-6]. time constraints, caring for critically ill or dying patients, fear of making © the author(s) 2021 https://jcbior.com/ karkhah et al. 2 mistakes in practice, criticism and inappropriate behavior of professors, and discrimination in clinical settings are some of the other stressors for nursing students [6-9]. finally, persistent and excessive tolerance of these stressors leads to psychological problems such as stress, anxiety, and depression [3, 10]. based on a systematic review and meta-analysis, the prevalence of depression among nursing students was 34% [11]. another study in canada showed that the prevalence of mild to severe depression, anxiety, and stress among nursing students was 33%, 39%, and 38%, respectively [12]. increased anxiety and depression among nursing students can increase their risk of suicidal behaviors. hence, a study in portugal found that 5% of nursing students had suicidal behaviors [13]. a study in iran showed that 38.7% of nursing students suffer from mild to severe depression [14]. meanwhile, mindfulness-based training is an effective strategy for managing stress and anxiety among nursing students [1, 15-17]. mindfulness is defined as conscious attention to the present, a certain quality of consciousness, and attention to the moment of life. in fact, mindfulness is a clear awareness of the present reality and awareness of personal emotions [10, 18]. today, it is believed that conscious attention to the present increases a person's well-being [19]. previous evidence has shown that there is a positive relationship between conscious attention to the present and factors such as negative emotions, anger, social anxiety, self-help, positive emotions, optimism, and self-esteem [6, 20]. in addition, mindfulness can be effective in promoting emotional intelligence [21], reducing stress, anxiety, depression [22-24], reducing negative thoughts, improving sleep, increasing concentration and accuracy [2], and relaxation [25]. thus, it is clear that effective cognition and intervention in mindfulness can help reduce stress, anxiety, and depression and improve clinical and educational skills, quality of life, well-being, empathy, and control of individual emotions among nursing students [26]. however, implementing mindfulnessbased interventions requires understanding the extent and dimensions of mindfulness. therefore, due to the importance of mindfulness among nursing students and limited studies in this field, the present study aimed to assess mindfulness and related factors among iranian nursing students. 2. materials and methods 2.1 study design and sample in a cross-sectional study, 100 nursing students studying at nasibeh faculty of nursing & midwifery affiliated to mazandaran university of medical sciences, iran was enrolled. data were collected via census sampling from may to june 2018. nursing students from 2 to 6 semesters were included in the present study. students of 1 (due to lack of familiarity with the concept of mindfulness), 7 and 8 semesters (due to lack of access to them), and participants who were not satisfied were excluded from this study. 2.2 data collection data were collected using a three-part questionnaire including demographic characteristics, kentucky mindfulness skills (kims) scale, and spielberger state anxiety scale. 2.2.1 demographic characteristics demographic characteristics of nursing students such as age, sex, place of residence, marital status, semester, grade point average, experience stressful events during the last six months, smoking, perceived academic difficulty, the economic situation, satisfaction from their performance, and perceived communication problems were collected. 2.2.2 kentucky inventory of mindfulness skills this 39-item questionnaire was designed by beer et al. (2004) to assess four components of mindfulness including observing (12 items), describing (8 items), acting with awareness (10 items), and accepting without judgment (9 items). participants responded to the items in this questionnaire using a five-point likert scale from never (score of 1) to always (score of 5) [27]. this scale is scored from 39 to 195. in iran, this tool was confirmed by narimani et al. with cronbach's alpha of 0.82 [28]. 2.2.3 a short form of spielberger state anxiety scale this 6-item questionnaire assesses levels of anxiety. participants responded to the items in this questionnaire using a four-point likert scale from at all (score of 1) to many (score of 4). this scale is scored from 6 to 24. this scale assesses anxiety at three levels: 1) mild (6 to 11), 2) moderate (12 to 17), and 3) severe karkhah et al. 3 (18 to 24). in a similar study, the correlation coefficient between the full form and the shortened form of the instrument was 96% [29]. 2.4 ethical consideration this research was approved by the ethics committee of mazandaran university of medical sciences. the objectives of the present study were explained to the participants and their informed consent was obtained. questionnaires were completed by nursing students, independently. the questionnaires were anonymous to ensure the confidentiality of the information. 2.5 data analysis spss for windows, version 16.0 (spss inc., chicago, il, usa) was used for statistical analysis. continuous and categorical variables were presented using mean (standard deviation) and frequency (percentage). the shapiro-wilk test was used to assess the normality of data distribution. due to the normal distribution of data, t-test and anova were used to assess differences between the groups. also, the chisquare test was used to assess the relationship between nursing students' mindfulness and study variables. statistical significance was considered p <0.05. 3. results 3.1 participants characteristics a total of 100 nursing students were included in the present study. of the participants, 56% were female, 71% were single, 75% were indigenous, 10% were smokers, 37% were in good financial condition, 27% had experienced stressful events during the past six months, and 44% had communication problems. the mean age and grade of nursing students were 21.8 ± 2.6 and 16.42 ± 1.40, respectively. 3.2 mindfulness and related factors among nursing students the mean score of mindfulness of nursing students was 117.35 ± 12.89. the mean scores of mindfulness in the dimensions of observation, description, act with awareness, and acceptance without judgment was 36.33 ± 6.0, 25.68 ± 4.91, 28.17 ± 4.49, and 27.17 ±3.80, respectively (table 1). the mean score of anxiety in nursing students was 14.17 ± 3.78. there was a significant relationship between age and dimension "description" of mindfulness (r = 0.22, p = 0.029). the mean score of mindfulness was higher in women compared to men (p> 0.05). the mean scores of "act with awareness" and "acceptance without judgment" were higher in women compared to men (t = 0.239, df = 98, p = 0.811). the mean scores of "observation" and "description" dimensions of mindfulness were higher in men compared to women (p> 0.05). the mean scores of mindfulness in the dimensions of "observation", "act with awareness", and "acceptance without judgment" were higher in single students compared to married students (p> 0.05). however, married students in the "descriptive" dimension of mindfulness had higher scores than single students (p> 0.05). as shown in table 2, there was no significant relationship between mindfulness and experience stressful events during the last six months (p = 0.295). however, there was a significant relationship between dimension "observation" of mindfulness and t a b le 1 . m in d fu ln e ss a n d i ts d im e n si o n s v a r ia b le m in im u m m a x im u m m e a n ( ± s d ) o b se rv in g 2 6 .0 0 4 9 .0 0 3 6 .3 3 ± 6 .0 0 d e sc ri b in g 1 4 .0 0 4 0 .0 0 2 5 .6 8 ± 4 .9 1 a c t w it h a w a re n e ss 1 9 .0 0 4 2 .0 0 2 8 .1 7 ± 4 .4 9 a c c e p t w it h o u t ju d g m e n t 1 6 .0 0 3 4 .0 0 2 7 .1 7 ± 3 .8 0 t o ta l 8 8 .0 0 1 4 7 .0 0 1 1 7 .3 5 ± 1 2 .8 9 karkhah et al. 4 experience stressful events during the last six months (t = 2.327, df = 96, p = 0.022). there was no significant relationship between mindfulness and communication problems (p = 0.648). however, there was a significant relationship between the "observation" and "description" dimensions of mindfulness and communication problems (t = 2.185, df = 98, p = 0.031). there was no significant relationship between mindfulness and smoking (p = 0.212). however, the mean score of the "act with awareness" dimension of mindfulness was higher in non-smokers compared to smokers (p <0.05). also, non-smokers had less anxiety compared to smokers (p <0.05). there was no significant difference between mindfulness scores and anxiety levels (p = 0.4). however, there was a significant relationship between levels of anxiety and the "accept without judgment" dimension of mindfulness (p = 0.043). scheffe's post hoc test showed that "accept without judgment" reduces anxiety levels from mild to severe. 4. discussion the findings of the present study showed that nursing students had a high level of mindfulness. inconsistent with this finding, a study in the usa found that nursing students had a moderate to low level of mindfulness [2]. this discrepancy may be due to differences in organizational contexts and academic settings. on the other hand, the level of mindfulness of last semester students is lower compared to firstsemester students, which can be due to higher expectations, focus on goals, and advances in clinical education [30]. in this regard, first semester students have attention deficit disorder due to problems such as stress, distraction, and high memory impairment which can be one of the reasons for a low level of mindfulness in them [31]. nursing educators have an important role to play in reducing these problems for nursing students. identifying the causes of these problems in students and implementing effective interventions can reduce psychological distress such as stress, anxiety, and depression in nursing students [32]. hence, the findings of a systematic review and meta-analysis showed that mindfulness-based interventions can play an important role in reducing anxiety, depression, and distraction in nursing students [32]. based on the findings of this study, nursing students with communication problems had a low level of mindfulness. this finding was consistent with the results of studies in the usa, norway, and canada [33-35]. nursing students with communication problems have behavioral problems such as table 2. the relationship between mindfulness and demographic characteristics among nursing students (n = 100) variable mean ± sd df t-test p value sex 98 0.239 0.811 male 117.00 ± 14.39 female 117.62 ± 11.70 marital status 98 0.599 0.551 single 117.84 ± 12.99 married 116.13 ± 12.80 place of residence 98 0.814 0.418 dorm 115.32 ± 10.96 with family 117.97 ± 13.56 smoker 98 -1.257 0.212 yes 112.50 ± 12.91 no 117.88 ± 12.85 experience stressful events during the last six months 96 1.052 0.295 yes 119.70 ± 13.64 no 116.61 ± 12.69 communication problem 98 -0.457 0.648 yes 116.68 ± 12.55 no 117.87 ± 13.25 sd: standard deviation; df: degrees of freedom karkhah et al. 5 inattention to others, impulsivity, and negative personality traits, which can lead to a low level of mindfulness [35]. consistent with this finding, a study in the usa found that there was a positive association between mindfulness and the tendency to communicate [36]. as the present study showed, there was a significant relationship between age and dimension "description" of mindfulness. this finding was consistent with the results of a study in the usa [37]. however, another study in the usa showed that there is no significant relationship between age and mindfulness [38]. these differences may be due to differences in personality traits and social and educational desirability in different societies [39]. in this study, the mean score of mindfulness was higher in women compared to men, which was confirmed by a study in iran [40]. inconsistent with this finding, a study in the usa found that mindfulness was higher in men than in women [41]. these contradictions can be due to differences in individual characteristics, cultural and social conditions [42], and religious conflicts and tendencies in different societies [43]. however, these variables can affect people's mental health and ultimately affect mindfulness. therefore, it is recommended that well-designed studies in the future be conducted in this area. in the present study, half of the nursing students suffer from academic stress. academic stress can be due to the variety of curricula and different educational policies in different faculties that affect the level of mindfulness of nursing students [40]. however, identifying different educational aspects such as new educational ideas, playing a role in presenting lesson plans, and creating diversity in the classroom can potentially be effective in improving the mindfulness of nursing students [44]. nursing students face higher stress compared to students in other disciplines. hence, academic stress leads to adverse outcomes for physical and mental health, absenteeism, and poor academic performance in nursing students [45]. therefore, it is suggested that nursing educators provide coping strategies for nursing students to improve resilience and reduce perceived stress. in the present study, there was no significant difference between mindfulness scores and anxiety levels. however, there was a significant relationship between levels of anxiety and the "accept without judgment" dimension of mindfulness. also, "accept without judgment" reduces anxiety levels from mild to severe. inconsistent with this finding, a study in iran showed that there was a significant and negative relationship between mindfulness and anxiety [46]. another study in iran showed that a mindfulnessbased stress reduction program (mbsr) was effective in improving students' mindfulness, expression, and anxiety [29]. however, previous evidence in the united kingdom [47], south korea [16], usa [48], portugal, and brazil [49] have shown that mbsr is effective in reducing stress, anxiety, and depression among nursing students. obviously, mbsr can be used as a promising way to reduce stress in nursing students. this study had several limitations. the main limitation of the present study was the lack of generalizability of the findings to all nursing students due to the limited sample size. also, the self-report nature of the questionnaires may not adequately reflect mindfulness. overall, the findings of the present study showed that the mindfulness of nursing students was at a high level. therefore, due to the importance of mindfulness among nursing students and its relationship with variables such as age, marital status, the experience of stressful events, communication problems, and anxiety, it is possible to reduce the risk factor by creating the necessary conditions such as continuing education. due to the importance of mindfulness among nursing students, it is recommended that more attention be paid to evidence-based interventions in this area. also, considering a mind-based program in nursing students' curriculum as a way to improve work memory capacity and ensure success in emotion regulation is inevitable. it is recommended that future studies assess mindfulness, emotion regulation, and working memory capacity among nursing students. nursing students typically experience high levels of stress in educational and clinical settings. this stress leads them to anxiety or depression and reduced academic performance. meanwhile, nursing educators must prioritize 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https://pubmed.ncbi.nlm.nih.gov/26778081/ https://pubmed.ncbi.nlm.nih.gov/26778081/ https://pubmed.ncbi.nlm.nih.gov/26778081/ journal of current biomedical reports jcbior.com volume 3, number 2, 2022 eissn: 2717-1906 1 review exosomal micrornas released from tumor cells can play a role as reliable non-invasive biomarkers in the diagnosis and prognosis of breast cancer mahnaz kiyanjam1, ghazal soleymani1, fatemeh khaloozadeh1, fatemeh rouhollah1,* 1department of cellular and molecular, faculty of advanced science and technology, tehran medical sciences, islamic azad university, tehran, iran abstract breast cancer is one of the leading causes of death in women. breast cancer develops from the milkproducing cells of the breast tissue. exosomes from breast cancer cells have been linked to proliferation, metastasis, chemoresistance, and carcinogenesis, according to research. exosomes are membranebound extracellular vesicles that can be secreted from cancer cells and contain a variety of contents such as nucleic acid, protein, lipid, etc. micrornas, a component of exosomes, are small non-coding rnas that dysregulate in various cancers and can function as oncogenic or suppressive rna. mirnas influence cancer progression by binding to the 3' or 5' utr (untranslated region) of their target. because of their important role in cancer tumorigenesis, progression, and metastasis, some novel diagnostic and prognosis tools have been developed. in this study, we list some prominent exosomal microrna, the key function of some specific exosomal mirna, and their ability to act as a biomarker or a new therapy that they have been researching since 2020. keywords: biomarkers, breast cancer, mirna, exosomal mirna 1. introduction breast cancer (bc) is one of the most common malignant tumors [1]. although some treatments, such as surgery, radiotherapy, chemotherapy, and endocrine, can help to increase the long-term survival rate, the overall rate remains low. as a result, scientists are looking for genetic and molecular determinants, such as non-coding rnas, to aid in early diagnosis. micrornas are single-stranded rnas that are twenty-two nucleotides long and are one of the noncoding genes [2, 3]. mirnas act as transcription regulators because they can form hydrogen bonds with mrna (utr3), altering gene expression. as a result, dysregulated mirna expression can result in *corresponding author: fatemeh rouhollah, ph.d department of cellular and molecular, faculty of advanced science and technology, tehran medical sciences, islamic azad university, tehran, iran tel/fax: +98 21 22006660/+98 21 22008049 email: frouhollah@iautmu.ac.ir https://orcid.org/0000-0001-5243-129x received: september, 05, 2021 accepted: april, 17, 2022 uncontrolled cell proliferation and growth, which can lead to cancer [3], making micrornas a good target for diagnosis, prognosis, and treatment. biopsies, which are collected non-invasively from largely blood and other liquids, are needed to detect micrornas (urine, tear, etc.) [3]. exosomes are vesicles released by cells that are absorbed by a bilayer membrane made up of supermolecules containing cell-specific proteins, lipids, and nucleic acids (including mirna and lncrna) [4]. current exosomal mirnas show promise in cancer detection and treatment [5], but plasma mirna measurement cannot replace exosomal mirna measurement [6]. as a result, each © the author(s) 2022 https://jcbior.com/ https://jcbior.com/ kiyanjam et al. 2 plasma sample contains a unique stable microrna expression pattern [7]. 2. exosomal mirna exosomes can serve as a vehicle for mirna exchange between living things ("a message in a bottle") and a form of living thing communication. however, it is unknown which mirnas are modified as payloads via cancer-derived exosomes in order to advance neoplasm proliferation and metastasis. they investigated the features and useful non-uniformity of exosomes produced from cancer cells with diverse pathologic process abilities in the show contemplate. they anticipated that mirnas modified by exosomes may be important in implementing epigenetic changes in recipient cells, and they identified the crucial cancerderived exosomal mirnas. furthermore, in this manner, these forms of thoughts it was confirmed that cancer-derived exosomal mirnas in plasma might serve as a biomarker for assessing carcinoma spread. as a result, scientists believe it will assist to rationalize the processes of carcinoma metastasis interfered by exosomes, as well as provide more avenues for prospective therapeutic treatment [8]. this study, to which we also refer, looked at the huge quantity of exosomes generated by various metastatic cancer patients discovered that the invasion and migratory capabilities of exosomes is dependent on the cell from which they were produced. to investigate the role of active cancer-associated fibroblasts (cafs) in cancer progression, western blot and immunofluorescence were employed to detect active caf markers such as transforming growth factor-β (tgf-β), il-6, cxcl12, and platelet-derived growth factor (pdgf). mir-146a was discovered as the promoter of nuclear factor-to-cancer-associated fibroblasts (nf-to-caf) transition by exploring the mirnas profile. mir-146a can negatively target thioredoxin interacting protein (txnip) in order to stimulate the wnt/-catenin signaling pathway and activate wnt genes such as axin2 and dkk1 [9]. the functions of differentially expressed mirnas has been shown in table 1. to make advancement in breast cancer therapy, openness and accurate determination are critical. agent imaging methods used for breast evaluation include mammography, breast ultrasound, and breast attractive radiation introduction. furthermore, mammography has a reduced affectivity among younger women and women with large breasts. mammography approaches have been enhanced by breast ultrasound and attractive reverberation imaging. in any case, these options are not without drawbacks. as a result, various recent initiatives have been made to identify high-risk people for breast cancer. fluid biopsies have been utilized to help in the identification of breast cancer in the early stages. fluid biopsies have the advantage of being noninvasive for early cancer diagnosis. it also allows for different redundancy and simple illness tests [10]. the activation of the pi3k/akt pathway can result in tumor improvement and movement, as well as a problem with dna repair, which may also contribute to gi. later studies have shown that supportive mitogen-activated protein kinase signaling unwinds cell cycle checkpoints and allows cells to avoid prolonged g2 arrest by activating the gathering of the promitotic kinase, finally improving gi. dna damage response (ddr) assures the assistance of gi, and a recent study found that score can be a coordinate poor regulator of ddr. the remember became accomplished in multicenter cohorts, including tcga-bc, gse22220, gse73002, gse41922, and inhouse medical exosome cohorts. in the disclosure cohort, the genome instability-derived mirnas signature (migisig) became noteworthy. to validate its worth with the forecast and backbone of bc, the migisig became linked to an inner approval cohort and distinct outside validation cohorts [11, 12]. 2.1. exosomal mirnas in tnbc this study looked at exosomal mirnas in triple negative breast cancer (tnbc), and we think they employed bioinformatics to identify certain mirnas and compare their expression to healthy controls. the results revealed that some mirnas were elevated while others were downregulated, indicating that there are changes in mirna expression between healthy controls and tnbc. furthermore, they employed qrt-pcr to demonstrate the potential of certain mirna as a predictive biomarker [13]. 2.2. urine-derived exosomes as a non-invasive biomarker recent research analyzed urine-derived exosomes to identify mir424, mir660, mir423, mir125b, mir194, mir17, let7a, let7d, let7f, and let7i as a noninvasive biomarker to diagnose bc. mir-424 has been kiyanjam et al. 3 identified as a potential biomarker, with dysregulation reported in serum and plasma levels. it has a suppressive role, as does the mir424/503 complex, and its function is opposite to its increased expression. mir-424 influences cancer development and malignancy. mir-660, let7-i, let7-f, let7-d, and mir423 have been downregulated and are oncogenic regulators. the increased expression of mir-125b influences malignant development as well as the responsiveness and prognosis to bc treatment [3]. mir-222, mir-194, let7-a, let7-e, and mir-17 have all been shown to be overexpressed. they proposed that a panel of four mirna types, mir-424, mir-423, mir660, and let7-i, might be used as a highly specific combinatory biomarker tool in the discrimination of bc based on urine samples [14]. 2.3. mir-1246 and mir-155 many of the research conducted in 2020 evaluated two chosen mirnas from microarray profiling in trastuzumab resistance in her-positive early-stage and metastatic bc patients. they deliberately studied mir-1246 and mir-155. it has been observed that high expression of these mirnas is related to trastuzumab effect in metastatic patients, poor efs in early-stage patients, and poor dfs in metastatic patients. furthermore, they show that mirnas can predict poor trastuzumab response in both early-stage and metastatic patients. there was n2.4. mir-204-5po discernible relationship between mirnas and os [15]. 2.4. mir-204-5p they employed 293t cell lines to manufacture exosomes in investigations on 293t cell lines to show exosome-mediated mir-204-5p administration inhibiting tumor development in bc. instead of anticancer medications, mir-504-5p was enhanced to develop a novel therapeutic. this exosomal mirna has been found to enhance 5-fu-induced apoptosis and thereby boost 5-fu cytotoxicity [16]. 2.5. mir-7641 qrt-pcr and xenograft assays were used by several researchers to investigate exosomal mir-7641. among other mirnas, mir-7641 was found to be the most differentially expressed in the metastatic patient. through exosomes, mir-7641 can transmit its tumorpromoting capability to recipient cancer cells. patients with distant metastases had higher levels of mir-7641 than those without. furthermore, bioinformatics analysis revealed a link between this mirna and bc survival, as well as several biological processes such as apoptotic mitochondrial alterations, cytosolic trafficking, and cytokine production [17]. furthermore, they have shown that the tumorpromoting power of mir-7641 appears to be transmitted to recipient cancer cells via exosomes, and mir-7641 also increased neoplasm growth in vivo. the study found that individuals with distant metastasis of cancer had significantly higher levels of mir-7641 in their plasma than those without metastasis. furthermore, bioinformatics research has revealed table 1. the functions of differentially expressed mirnas name function reference mir-223 mir-223 is a coordinator of breast cancer progression 21 mir-1246 mir-382-3p, -598-3p, -1246, and -184 are all implicated in breast cancer development and are possible biomarkers for breast cancer diagnosis mir-206 mir-206 promotes cancer development in breast cancer by targeting the fulllength neurokinin-1 receptor mir-24 elevated mir-24-3p has a prognostic effect in breast cancer and is associated with the metastatic process mir-373 mir-373 has been linked to cancer formation mir-21 in breast cancer, circulating mir-21 has diagnostic and prognostic value mir-6875 the discovered combination of mir-1246, mir-1307-3p, mir-4634, mir-68615p, and mir-6875-5p may be useful for early diagnosis of breast cancer mir-202 mir-202 was shown to be highly increased in whole blood samples from patients with early-stage breast cancer mir-219b gga-mir-219b inhibits the proliferation, migration, and invasion of the marek's disease tumor cell msb1 kiyanjam et al. 4 that mir-7641 is related to cancer survival, and mir7641 is tightly related to a number of other essential cellular and natural forms [18]. 2.6. mir-500a-5p in our investigations, we discovered that the majority of researchers that employed next-generation sequencing to screen mirnas in caf-derived exosomes discovered that mir-500a-5p was upregulated. exosomes with overexpressed mir-500a5p were identified and delivered to bc cells. this mirna has been shown to stimulate the development and proliferation of bc cells. it was proposed that mir500a-5p be inhibited to limit bc development and metastasis [19]. 2.7. hsa-mir-4235p a research on the expression level of hsa-mir-4235p in plasma, as well as pooled ribonucleic acid sequencing in duplicate, was carried out in five groups of patients. it was discovered that has-mir-423was overexpressed in patient liquid biopsies. as a consequence, has-mir-423p was established as a bc biomarker [20]. furthermore, because hsa-mir-423-5p expression is highly associated to the disease, abnormal hsa-mir423-5p overexpression might be one of the earlywarning signals given out by breast cancer cells [21]. in silica, the hsa-mir-423-5p-regulated coding characteristics are widely distributed in tumor-related signaling pathways [22]. 2.8. mir-128-1, mir-128-2, and mir-421 overexpression of migisig increases genomic instability by activating association s part capture and enhances the occurrence of cancer cells. because multi-nuclei and micro-nuclei are indicators of genotoxicity and bodily insecurity, they found the recurrence of multi-nuclei and micro-nuclei when the three mirnas were overexpressed. they first identified the expression levels of mir-128-1, mir128-2, and mir-421 in bc cells and solid human exocrine gland animal tissue cells, 184a1 and mcf10a, and then designated the genomically unstable mda-mb-231 cell line with low-level expression of the three mirnas to perform the taking once tests. mirna mimics were transfected into mda-mb-231 cells and refined for 48 hours, during which time the expression levels of mir128-1, mir-128-2, and mir421 were significantly increased. using a high-content framework, we discovered that overexpression of mir128-1, mir-128-2, and mir-421 dramatically increased the number of multinuclei and micronuclei inside the mda-mb-231 cell line [23, 24]. 3. mirnas and the genes that they target 3.1. mir-455-5p/ cdkn1b & mir-1255a/ smad4 the tcga sequencing database was utilized by scientists to discover two exosomal mirnas, mir455-5p and mir-1255a, as therapeutic targets for breast cancer. increased levels of mir-455-5p have been demonstrated to inhibit cdkn1b. cdkn1b, also known as p27kip1, is a cyclin-dependent kinase that suppresses the cell cycle, and its downregulation in bc cells causes tumor development, mitosis, and poor prognosis. mir-1255a also has an oncogenic function in bc by downregulating smad4, altering the tgfsignaling pathway. by reducing their targets, cdkn1b and smad4, both mirnas may have an effect on nearby or distant non-malignant recipient cells. nonetheless, the connection between mir-455-5p and mir-1255a between cdkn1b and smad4 has yet to be established [25]. researchers employed bioinformatics to identify mir27a-3p, a novel implicated mirna in bc that can allow cancer cells to avoid the immune system. the affinity of mir-27a-3p was confirmed using a dualluciferase reporter assay. endoplasmic reticulum stress has been shown to boost the expression and release of this mirna. furthermore, bc cells transmitted mir-27a-3p to macrophages, causing the magi2/pten/pi3k axis to be targeted for immune evasion. magi2 is a target for mir-27a-3p, an important protein for pten that upregulates to inhibit the pi3k/akt signaling pathway. mir-27a-3p promotes pd-l1 expression in macrophages by inhibiting magi2 [26]. 3.3. mir-1910-3p and mtmr3 exosomal mir-1910-3p has been studied by scientists. by overexpressing this mirna, they were able to demonstrate proliferation and migration of bc cells. it was discovered that boosting mir-1910-3p decreases apoptosis while increasing autophagy, implying that mir-1910-3p has a role in decreasing apoptosis. this mirna's gene target is myotubularin-related protein 3 (mtmr3), which is expressed at a reduced level in bc cells. by targeting mtmr3, mir-1910-3p can kiyanjam et al. 5 activate the nf-b signaling pathway, boosting proliferation and migration, inducing autophagy, regulating the expression of wnt 2, wnt 3, wnt 5, wnt 10b, -catenin, and activating the wnt/-catenin signaling pathway. furthermore, exosomes enriched in mir-1910-3p were shown to move to the bc microenvironment and stimulate migration and proliferation. because of its specificity and sensitivity, mir-1910-3p is a promising biomarker [27]. 3.4. mir-181d-5p and hoxa5 the researchers employed chip and dual-luciferase reporter assays to investigate the interaction between mir-181d-5p and hoxa5. their findings show that mir-181d-5p is highly expressed in bc cells, but cdx2 and hoxa5 are downregulated. cdx2 can bind to the hoxa5 promoter, increasing its expression; moreover, cdx2 is a target for mir-181d-5p. overexpression of hoxa5 can raise e-cadherin and vimentin levels while decreasing n-cadherin, slug, snail1, twist1, zeb1, and zeb2 levels, indicating inhibition of cell proliferation, invasion, migration, and emt while promoting cell death in breast cancer mir-181d-5p, through downregulating cdx2, can reduce hoxa5 levels, boosting emt, proliferation, invasion, and migration while suppressing apoptosis in breast cancer cells [28]. 3.5. mir-3613-3p and socs2 scientists investigated caf exosomes containing mir3613-3p to demonstrate the role of this mirna in bc. they employed real-time pcr and microarray to discover the overexpression of mir-3613-3p, which inhibited socs2. socs2 can be activated by cytokines and regulated by mirna. socs2 has the ability to modulate several signaling pathways that contain growth hormone signaling relevant to cell growth. overall, it has been demonstrated that mir-3613-3p functions as an oncogenic mirna by downregulating socs2 expression, resulting in metastasis and cell proliferation. it has been proposed that mir3613-3p can be employed as a nonspecific biomarker for bc and as a possible biomarker for prognosis prediction [29]. 3.6. mir-4443 and timp2 exosomal mir-4443, which enhances tumor metastasis in bc by down-regulating timp2, was examined by scientists using tagging and transfected exosomes. timp2 (a member of the timp family) can limit metastasis by blocking mmp (zinc-dependent endopeptidases), which breakdown extracellular matrix, allowing invasion and migration to occur. this mirna is produced by bc cells in the microenvironment and promotes liver metastasis [30]. 4. bc and immunocytes researchers employed mirna microarrays to investigate the relationship between bc and immunocytes. the il-6 high breast cancer exosomederived mir-9 and mir-181a regulate socs3 and pias3 post-transcriptionally, respectively, activating the jak/stat signaling pathway and promoting the formation of emdscs. as a result, tumor development was sped up and immune escape was demonstrated. they investigated target treatment against myeloidderived suppressor cells (emdscs), namely by blocking the stat3 signaling pathway via the mir9/socs3 and mir-181a/pias3 cerna networks, which might be a more successful therapy [31]. 5. conclusion according to prior research in the field of exosomal microrna, several exosomal micrornas have been identified as major biomarkers for the diagnosis and prognosis of cancer and other diseases. it is possible to determine the intensity and stage of sickness by screening exosomal micrornas; in this case, mir3613-3p [32], and mir-4443,[33] induce metastasis. exosomal mirnas can be used to detect cancer and track its progression by observing how they are dysregulated. this property has several advantages, the most important of which is that it may be used to identify cancer at an early stage researchers employed mir-181-5p, mir-30a-3p, mir-30e-3p, and mir-3615p to accurately diagnose non-small cell lung cancer at an early stage [34]. in addition, mirna screening can help predict cancer prognosis. elevated levels of mir-23b-3p, mir-10b3p, and mir-21-5p in exosomes were linked to poor overall survival in non-small cell lung cancer (nsclc) patients, according to researchers [32]. rather of collecting samples from blood and bodily fluids, emerging technologies such as size-based approaches, precipitation, immune-affinity capture-based techniques, and microfluidic-based techniques can make exosome extraction from clinical samples more kiyanjam et al. 6 efficient and straightforward [33, 35]. they are not affected by age, gender, bmi, or other variable traits, giving them the potential to become appropriate biomarkers for people from all walks of life [36, 37]. microrna has become increasingly frequent throughout time as technology has advanced. regardless of the advantages and practicality of exosomal micrornas, there remain still obstacles to overcome [37]. to begin with, biological bodily fluids are a rich supply of exosomes from a variety of sources, making the separation of tumor cell-derived exosomes more difficult [38]. adverbial adverbial adverbial adverbial adverbial adverbial adverbial adverbial tears were examined for biomarkers in bc in a research, and certain particular mirna were found. second, despite the fact that numerous isolation procedures have been proposed, the purity and quality of exosomes vary depending on the strategy, and there is currently no standard methodology for exosome separation and identification [37]. third, current exosome isolation techniques are frequently complicated and time-consuming, with drawbacks such as aggregation and insufficient removal of contaminating biological molecules; whereas a selective, reproducible, robust, simple, fast, and highthroughput isolation technique is critical for the field of exosomes research [39]. to summarize, exosomal mirnas can play an important role as biomarkers for cancer prediction and diagnosis; nevertheless, several issues must be addressed. authors’ contributions mk and gs developed the study concept, design, analyzing data and drafted manuscripts. fk and fr scientific editing and critical revisions of the manuscript. all authors read and approved the final manuscript. conflict of interests there is no conflict of interest. ethical declarations it is declared that this article has been completely written ethically. financial support there was no source of funding. references 1. siegel r, ward e, brawley o, jemal a. cancer statistics, 2011: the impact of eliminating socioeconomic and racial disparities on premature cancer deaths. ca cancer j clin. 2011; 61(4):212-36. 2. bartel dp. micrornas: genomics, biogenesis, mechanism, and function. cell. 2004; 116(2):281-97. 3. pogue ai, 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severe acute respiratory syndrome coronavirus 2 consideration: applied machine learning for nutrition quality, microbiome and microbial food poisoning concerns fatemeh taslimi1 , seyedeh elham hosseini ezabadi2, maed jabbari3, erfan ghanbarzadeh4, mandana ashkan5, farhad akbari afkhami6, amir rigi4 , mojtaba hedayati ch7,* 1faculty of medicine, mazandaran university of medical sciences, sari, iran 2department of nutrition, faculty of nutrition sciences and food technology, shahid beheshti university of medical sciences, tehran, iran 3school of nutritional sciences and dietetics, tehran university of medical sciences, tehran, iran 4microbial toxins physiology group, universal scientific education and research network, rasht, iran 5student research committee, guilan university of medical sciences, rasht, guilan, iran 6department of chemistry and biochemistry, university of alabama, tuscaloosa, alabama 35487, united states 7department of microbiology, virology and microbial toxins, school of medicine, guilan university of medical sciences, rasht, iran abstract although almost two years have passed since the beginning of the coronavirus disease 2019 (covid19) pandemic in the world, there is still a threat to the health of people at risk and patients. specialists in various sciences conduct various researches in order to eliminate or reduce the problems caused by this disease. nutrition is one of the sciences that plays a very important supportive role in this regard. it is important for patients to pay attention to the potential of different diets in preventing or accelerating the healing process. the relationship between nutrition and microbiome regulation or the occurrence of food microbial poisoning is one of the factors that can directly or indirectly play a key role in the body's resilience to covid-19. in this article, we introduce a link between nutrition, the microbiome, and the incidence of food microbial poisoning that may have great potential in preventing, treating covid-19, or preventing deterioration in patients. in linking the components of this network, artificial intelligence (ai), machine learning (ml) and data mining (dm) can be important strategies and lead to the creation of a conceptual model called "balance square", which we will introduce. keywords: covid-19, microbiome, microbial toxins, nutrition, machine learning 1. introduction todays, coronavirus disease 2019 (covid-19), known as significant public health challenge and is the causal agent of severe acute coronavirus respiratory syndrome. since december 2019, the covid-19 outbreak has become a significant global epidemic threat for at risk people's health and patients [1]. in this pandemic situation, we *corresponding author: mojtaba hedayati ch, ph.d department of microbiology, school of medicine, guilan university of medical sciences, rasht, guilan, iran tel/fax: +98 13 33690099 email: mhedayatich@gums.ac.ir https://orcid.org/0000-0002-8277-9841 received: august, 15, 2021 accepted: august, 28, 2021 are in constant struggle to stop virus infection or attenuate severity of disease every day [2]. specialists in various fields of sciences try to do their best, attenuating or eliminating symptoms caused by disease. recently, however, due to the more complex process of diagnosis and treatment, it seems that there is a need for multidisciplinary research and © the author(s) 2022 https://jcbior.com/ taslimi et al. 2 performance based on system biology in the face of covid-19. we consider nutrition, microbiology, microbial toxins, and computer science-based research as a comprehensive diagnostic, prophylactic, and coping strategy against covid-19. for this purpose, it is necessary to explain a series of concepts to understand the network relationship between these sciences, which we will describe below. 2. correlations of nutrition, microbiome, microbial toxins and covid-19 the determining role of nutrition, microbiome and food poisoning caused by microbial toxins in human health has long been demonstrated [3-5]. different eating habits can play a role in the development of diseases by changing human microbiome ratio [6]. eating habits, in turn, have a significant impact on the incidence of diseases such as obesity, diabetes and cardiovascular disease. each of these diseases can cause different reactions of the body in the face of covid-19 [7, 8]. therefore, diets, need to observe with high accuracy in different human population from healthy to those who suffered disease. the type of nutritional value, cooking method and hours and amount of meals are other important and influential factors in the quality of food consumed by a person [9]. consumption food groups can mainly include 9 groups of breads and cereals, fruits, vegetables, meats and meat products, legumes, nuts, fats, low-fat milk and dairy products, and high-fat milk and dairy products. different characteristics affect a person's resident or transient microbiome and the likelihood of food poisoning: age, level of education, economic status, height, weight, use of prebiotics, probiotics and synbiotic, multivitamin and mineral supplements, genetic disorders, health of drinking water, living in the city or village, cultural characteristics, lifestyle and occupation [10]. studies have shown that the gut microbiota in people with a variety of diseases, including inflammatory bowel disease, type 1 and type 2 diabetes, celiac disease, autism, multiple sclerosis or covid-19, can be different from healthy people [11]. as a result, the proper formation of the microbial population in life can, in a way, play an important role in a person's health in the future. it has even been observed that, the mother's microbiota is directly related to the baby's microbiota, and that the mother's diet during pregnancy can affect the microbial population of the baby's gut [12]. it seems that the effect of diet on many diseases can be explained by changes for example in intestinal microbiota. different eating habits can play a role in the development of diseases by changing the microbiota [13]. habits, food consumption cultures and environmental factors in different parts of the world are effective in the formation of intestinal microbiota in different forms. it is important to investigate the relationship between diet and microbiome and their toxins (such as lipopolysaccharide=lps) or food poisoning in relation to covid-19. because it can have significant direct or indirect effects on the readiness and capacity of the immune system [14]. this association between microbiome and nutrition is important, even in relation to covid-19 in pregnant mothers. in the past, it was thought that the baby's gut was sterile before birth and its microbial population formed only after birth, but now analysis of the baby's first stool, meconium, has shown that the mother's uterine environment is not sterile and contains a microbial population that was born before birth which the baby received through the placenta and amniotic fluid [15]. the origin of this microbial population can be from the mother's gastrointestinal tract, bacteria in the mother's oral cavity or from the urogenital area [16]. during pregnancy, gastrointestinal motility decreases and uterine pressure increases, especially late in this period. on the other hand, the effect of estrogen on mesenteric arteries and increasing pressure on these arteries by the fetus causes transient clogging of arteries. these factors, along with the weakening of intestinal barriers against bacterial growth, cause the transfer of bacteria from the intestine to other organs [17]. in addition, pregnancy can affect the thickness of the mucosal layer and the adhesion of enterocytes, causing bacteria to enter the blood and lymph vessels to reach other tissues, such as the placenta and amniotic fluid. this transmission is probably through dendritic cells and cd18 + [18]. surprisingly, by swallowing amniotic fluid, the germs enter the baby's gut and appear in meconium after birth [19]. the extent to which this microbiome is embedded can play a role in establishing or preventing mother-to-child reception of covid-19. taslimi et al. 3 various studies have shown that human nutrition, even before birth, can affect the future human microbiome. for example, in a study in 2018, it was shown that mothers in the normal delivery group were associated with an increase in fruit consumption during pregnancy with an increase in the streptococcus/clostridium group population in the baby's stool. interestingly, contrary to expectations, fruit consumption was inversely related to bifidobacterium spp., which is known as a beneficial bacterium, and consumption of red meat and processed foods was directly related to the amount of this bacterium. in addition, maternal dairy consumption during pregnancy was associated with an increase in clostridium in cesarean section infants. consumption of fish and seafood also showed a positive relationship with streptococcus bacteria in new born neonates [20]. also, another study in 2018 that focused on maternal food intake in the third trimester of pregnancy showed that high consumption of vegetables and reduced consumption of processed meat and fried foods during pregnancy were inversely related to the amount of bacteroides spp. and clostridium spp. in infant feces [21]. another study conducted in 2017 showed that maternal fat intake during pregnancy, including saturated fatty acid (sfa) and monounsaturated fatty acids (mufa), was associated with an increase in the order firmicutes and a decrease in some groups, such as the proteobacteria order. a number of vitamins (a, d, e, b₂, b₁, b₃, b₃, b₉, b₁₂, b₆, c, carotenoids, retinoic acid), plant protein and fiber negatively associated with coprococcus, blautia, roseburia and several bacterial family reflected ruminococcaceae and lachnospiraceae. most of these species were more abundant and positively associated with maternal fat intake, mufa, animal protein and especially significant association with sfa. total maternal fat intake during pregnancy was negatively related to escherichia and shigella genera and positively related to firmicutes genus including blautia, roseburia, rombustia and faecalibacterium genera. these groups also had a negative relationship with fiber and animal protein source. total carbohydrates, plant proteins, and fiber were inversely related to the order firmicutes. in neonates born by cesarean section, maternal fiber intake was positively associated with proteobacteria. they also reported that polyunsaturated fatty acids (pufas) levels were positively associated with proteobacteriaceae and negatively associated with firmicutes [22]. it is found that intestine microbiome changes found in covid-19 hospitalized patients. this suggests that changes in the intestinal microbiome may play a role in increasing the intensity of covid-19 [23]. as a matter of fact, gut microbiota as a dark side of life in human body set metabolic homeostasis of mankind and its imbalance could turn pathogenesis of covid-19 more complex. for this reason, various studies have been performed in which foods containing polyphenols and dietary supplements such as probiotics and vitamins such as vitamin d have been shown to be effective in preventing or reducing the severity of covid-19 [24, 25]. 3. application of computer sciences and programming: promising strategies as described before, nutritional portions and types is capable to lead to microbiome dysbiosis and eventually leads to immune imbalance which is critical in order to covid-19 management [26]. for instance, it is possible to observe simultaneous penetration of microbial toxic metabolites like bacterial lps, into intrauterine space in pregnant mothers and other human intracellular fluids and organs. lps as a potent cytokine storm inducer then might lead to immune imbalances in neonates, which are predictable. such an outcome can also be seen as a result of receiving foods that carry microbial toxins or bacteria that produce microbial toxins (staphylococcal superantigens and bacillus enterotoxins). so far, various methods have been used to increase the effectiveness of drug compounds, identify microbial toxins, different pathogens identification in different patients and make optimal use of environmental and food capacities [27]. still, there is a need for a multidisciplinary perspective to evaluate the data obtained. in linking the components of nutrition science, microbiology, microbial toxins and covid-19 network, artificial intelligence (ai), machine learning (ml) and data mining (dm) can be important strategies and lead to the creation of a conceptual model called "balance square", which we will introduce. in short, ai deals with automated methods of reasoning and inference by computers. ml is a subset of ai. this field converts data into information and taslimi et al. 4 makes decisions based on it. some of its important algorithms are: classification, clustering, feature selection and prediction. dm is about extracting information from a large amount of data or the big data. data mining is not a technical discipline but uses different algorithms related to natural language processing (nlp), ml and ai. search programs, text summaries, and question-and-answer systems are examples of data mining applications [28]. these new sciences help machines to behave like an intelligent human being and to be able to perform various tasks. ai does not have the power to learn and analyze events, but it does program the information needed to solve potential problems and provides it to the system, then uses these data and calculations to begin solving problems and issues [29]. ai is closely related to ml; in essence, a pre-written program tells the system to learn new knowledge over time from past outputs and performance to improve future performance and decision making. ml has the ability to generalize information from large amounts of data and can use algorithms to identify relationships and patterns between the results of dm, obtain useful results, and take new actions [30]. dm, ai, and ml are three sciences that, in addition to their differences, are also directly related to each other. the use of ai made it smarter [31]. modern medicine produces large volumes of data in various fields. this is clearly seen in relation to the vast amount of data obtained from covid-19. data mining has the ability to analyze large raw or multidimensional data of covid-19 that is stored in medical and clinical databases or collected from medical centers and hospitals. this knowledge has the ability to discover regular patterns involved in disease development, proposing correlations between different characteristics such as patients' personal data, disease symptoms or even predictions. one of the important aspects of data mining in accurate diagnosis of diseases and choosing the appropriate figure 1. bs-1 conceptual model. this model introduces the best model of nutrition ingredient, portion size, or diet for covid -19 patients (a) or people at high risk of getting sick (b), based on ai, ml, and dm assistance. information data of individual microbiome (c) is provided to bs-1 software through user interfaces such as a wearable gadget (d) or laboratory-recorded information (e). the best diet (f) or prescription (g) is then given to the user to create a golden ratio of microbiome (h) or to avoid microbial poisoning which can worsen the disease. the information obtained can provide health care providers with more accurate decision-making power. taslimi et al. 5 treatment for covid-19 patients can be used is the prognosis of the disease, which is of great importance in medicine. prognosis discovery improves the quality of medical decisions, minimizes medical errors, and reduces the cost and time of diagnosis, or even rescues and satisfies patients [32]. radiologists use ml image segmentation algorithms to find any emergencies in patient’s radio/x-images. they also applied mlbased software’s to assist younger radiologists for educational manner and training. ml is also helpful to prevent any misdiagnosis. radiologists usually use it in rapid lesions identification and confirm their diagnosis [33]. gender determination from hand image, automatic detection of grandiose epilepsy and recognition of normal actions in video by combining machine vision (mv) techniques and ml, selection of effective features in breast cancer diagnosis using parametric ml models, detection of mitotic cells in images breast biopsy with the help of fast learning machines, diagnostic screening of pulmonary tuberculosis using ai, heart problems and disorders prediction, helping to identify and progress neurological diseases such as ms and parkinson's and many other health problems are examples of ai, ml and dm applications in the medical sciences [34]. in the "balance square number-1 (bs-1) model (figure 1), we propose to evaluate the golden ratio of microbiome, nutrition portion and type, and microbial toxins for covid-19 patients. after analyzing the results, each person should be prescribed a golden diet related to their own systems biology. this diet, along with medication and lifestyle changes, can provide an important diagnostic, preventive, or therapeutic role for the individual. probiotics play a key role in the battle against foreign germs, causing inhibition and adhesion of the intestines in direct opposition to bacteria and viruses by inhibiting the mucosal barrier of the body. they also boost alveolar macrophage activity and are useful against lung disorders. they also enhance the immune system with their by-products [35]. another study focused on the impact of probiotics, vitamin d, and omega-3 fatty acids on intestinal issues in covid-19 illness [36]. one of the most important applications of machine models and analysis will undoubtedly be to aid in the sequencing of microbial genes and to promote microbial analysis [37]. microbial resistance and the creation of harsher toxins can also result from poor dietary habits, necessitating the development of new medications [38]. 4. comprehensive conclusion the bs-1 model is able to examine large amounts of data about individuals before or after covid-19 infection. if the laboratory information is abnormal, suggest appropriate warnings and precautions for each individual after logical mathematical reasoning. bs-1 helps physicians understand what the best diet is or diet limitations for covid-19 patients or people at high risk for the disease? what compounds are inflammatory or suppress the immune system? what behaviors lead to person's microbiome dysbiosis? so far, there has been no study conducted to test validation of such model (bs-1) according to correlation of microbiome-microbiota toxic metabolites like bacterial lps and nutrition in covid19 positive patients. we suggest to ai, ml and dm scientist to produce bs-1 software as an effective tool for better covid-19 management. arrays of different wearable gadgets could develop for each person to record and report individuals' data to bs-1. this data can ultimately help the doctor or other specialists make better decisions or help them diagnose the best time to visit and assess the patient's condition. unfortunately there are also some limitations according clear application of "balance square" concept as follows: weakness in multidisciplinary insights for scientific teams, complexity of concept‘s systems biology, poor connection between basic and medical sciences. we proposed more interdisciplinary collaboration to overcome and fill in present gaps. authors’ contributions ft, seh: collecting and summarizing nutritionrelated articles. mj, ma: collecting and summarizing both nutrition and microbiome-related articles. egh: endnote library preparation and drafting manuscript. fa: 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of patients with covid-19 in rasht, iran tofigh yaghubi kalurazi1, vahid shakoori2, sara nasiri3, ali alavi4, ezat hesni1, lida mahfoozi1, dorrin aghajani1, zahra gholamalipour1, roxana ehsani1, samaneh mirzaei dahka5,*, niloofar faraji1,* 1razi clinical research development unit, razi hospital, guilan university of medical sciences, rasht, iran 2cardiovascular diseases research center, department of cardiology, heshmat hospital, school of medicine, guilan university of medical sciences, rasht, iran 3heshmat hospital, guilan university of medical sciences, rasht, iran 4inflammatory lung diseases research center, department of internal medicine, school of medicine, razi hospital, guilan university of medical sciences, rasht, iran 5student research committee, school of nursing and midwifery, guilan university of medical sciences, rasht, iran abstract in december 2019, a pandemic of an unknown respiratory virus emerged in wuhan, china, putting the whole world in crisis; a newly emerged coronavirus called severe acute respiratory syndrome coronavirus 2 (sars-cov2). in this study, we evaluated the clinical characteristics and laboratory findings of patients with coronavirus disease 2019 (covid-19) in the north of iran. demographical data and clinical characteristics of 126 patients with covid-19 in razi hospital, rasht, iran, were recorded. the mean age of patients was 62 years old and 57.1% of cases were male. about 17.5% of patients had direct contact with a sars-cov-2-infected patient. the most common underlying diseases were lung diseases (11.9%), diabetes (11.9%), cardiovascular disease (cvd) (7.1%), and hypertension (4.8%). the mean levels of lactate dehydrogenase (ldh), creatine phosphokinase (cpk), creatine kinase mb (ck-mb), serum glutamic oxaloacetic transaminase (sgot), and erythrocyte sedimentation rate (esr) (1231.79±866.48 u/l, 766.88±2288.68 u/l, 59.2±55.18 u/l, 112.28±213.07 u/l, and 67.61±31.07 mm/hr, respectively) were remarkably higher than the normal ranges. also, the average o2 saturation (o2sat) was 61.42±26.37%. male gender, advanced age, a history of underlying diseases, higher level of cardiac enzymes, and lower level of o2sat were associated with severity in patients with covid-19. keywords: sars-cov-2, covid-19, laboratory findings, clinical characteristics 1. introduction coronaviruses, belonging to the family coronaviridae, are enveloped single‐stranded rna viruses that are widely spread in mammals including humans [1]. despite mild infections caused by *niloofar faraji, msc razi clinical research development unit, razi hospital, guilan university of medical sciences, rasht, iran tel/fax: +98 13 3355002 email: niloofarfaraji.sci@gmail.com http://orcid.org/0000-0001-5796-7157 *samaneh mirzaei dahka, msc email: samane.mirzaee@yahoo.com https://orcid.org/0000-0003-2825-9359 received: october, 05, 2021 accepted: november, 30, 2021 coronaviruses, a novel coronavirus was found as the cause of a new emerging worldwide pandemic after testing the lower respiratory tract samples, named severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [2]. the coronavirus disease 2019 © the author(s) 2022 https://jcbior.com/ https://jcbior.com/ yaghubi kalurazi et al. 2 (covid-2019) is announced as a worldwide concern by the world health organization (who). patients with covid-19 have some clinical manifestations such as fever and cough as primary clinical presentations, and shortness of breath and myalgia as the following manifestation [3]. due to the wide spectrum of sars-cov-2 symptoms, some patients may have serious and extensive complications such as acute respiratory distress syndrome (ards) and cytokine storm, which may result in organ failure and death [4, 5]. some circumstances lead to susceptibility of being fatal cases of covid-19, such as middle and upper age with a history of pre-existing underlying diseases (lung diseases, cardiovascular diseases (cvd), kidney and liver diseases, hypertension, and diabetes) [6, 7]. among underlying diseases, lung and cardiac complications are strongly related to higher inhospital mortality in patients with covid-19. the mechanisms of sars-cov-2, which causes this acute heart complication, include direct and indirect damage to cardiomyocytes and systemic inflammation [8]. a study demonstrated that the mortality was associated with an increased level of creactive protein (crp), serum glutamic oxaloacetic transaminase (sgot), potassium (k), neutrophils count, white blood cell (wbc) count, and prothrombin time (pt) in covid-19 patients with abnormal chest computed tomography (ct)-scan results. additionally, in these patients, a decrease in lymphocytopenia and o2 saturation (o2sat) levels is associated with severe conditions [9]. some laboratory findings such as lymphocytopenia, thrombocytopenia, elevated crp, and esr can also be helpful for clinicians to predict the patient’s condition [10]. the higher neutrophil to lymphocyte ratio (nlr) and upper age can be used as independent biomarkers indicators of poor clinical outcomes [11], in which nlr greater than 6.5 may reflect the progression of the disease toward an unfavorable clinical outcome; with this notion, the ratios higher than 9 may result in death [12]. in addition, in covid-19 patients with mild to severe conditions, the average level of lactate dehydrogenase (ldh), creatine phosphokinase (cpk), sgot, serum glutamic pyruvic transaminase (sgpt), and crp, are higher than the normal range [13]. since the evaluation of clinical and laboratory findings are more accessible and valuable for the diagnosis of infections such as sars-cov-2 [14]; we aimed to evaluate demographical data, clinical characteristics, and laboratory findings of patients with covid-19 in the north of iran. 2. materials and methods 2.1 data collecting this retrospective study was conducted from february to may 2020, on the 126 cases of covid-19, referring to razi hospital, rasht, iran. all cases were confirmed for sars-cov-2 infection via using the real-time polymerase chain reaction (rt-pcr) test on nasopharyngeal swabs specimens from the upper respiratory tract. demographic data and clinical characteristics of patients including age, gender, history of smoking, history of opium consumption, direct contact with covid-19 patients, history of underlying diseases, and symptoms at the time of admission were collected from the patient’s medical records. laboratory variables including complete blood count (cbc), esr, coagulation profile (pt and partial thromboplastin time (ptt)); arterial blood gases (abg); and biochemical parameters such as alkaline phosphatase (alp), sgpt, sgot, total protein, albumin, cpk, creatine kinase mb (ck-mb), calcium (ca), phosphorous, magnesium (mg), direct and indirect bilirubin, uric acid, blood urea nitrogen (bun), creatinine, amylase, blood sugar, ldh, sodium (na), and k were analyzed on blood samples of the patients with covid-19. the patients with incomplete data were excluded from the study. 2.2 statistical analysis the variables were represented by mean ± standard deviation (sd), median and interquartile range (iqr). the shapiro-wilk test was used to test the normality of data distribution. the categorical variables were presented in numbers and percentages. all statistical data were analyzed using ibm spss, version 24.0. 3. results a total number of 126 patients with covid-19 were included in this study, of which the median age of patients was 62 years, ranging from 28 to 92 years old. overall, 57.1% of cases were male and 42.9% were female. according to table 1, 17.5% of patients had direct contact with covid-19 patients. the most common underlying diseases were lung diseases (11.9%), diabetes (11.9%), cvd (7.1%), and yaghubi kalurazi et al. 3 hypertension (4.8%). fever (80.2%), cough (80.2%), myalgia (50%), and dyspnea (40.5%) were the most common symptoms, while digestive symptoms were rare. also, 81% of patients experience an o2sat level <93%, and 13.5% of patients were intubated. the mean levels of some laboratory findings represented abnormalities in esr (67.61±31.07 mm/hr), ck-mb (59.2±55.18 u/l), cpk (766.88±2288.68u/l), ldh (1231.79±866.48 u/l), sgot (112.28±213.07 u/l) and sgpt (90.33±209.47 u/l). the mean of lymphocyte, neutrophil, and o2sat were 20.03±14.05%, 76.56 ±15.5%, and 61.42±26.37%, respectively. o2sat was critically lower than the normal range, table 2. 4. discussion at the beginning of the covid-19 pandemic, the diagnosis of the disease was complicated by various symptoms, abnormal chest ct-scan results, abnormal laboratory findings, and the severity of the disease at the time of onset. due to our results, males were the majority of hospitalized patients, similar to some other reports, which consequences in severe complications [15–18]. gender differences in the frequency and outcomes of infectious diseases are reported at all ages in males [19–21]. these data represented that, while environmental factors may be affecting various aspects of the pandemic, fundamental differences in the immune response among males and females are possibly a factor behind the notably observed gender bias in the covid-19 pandemic. various studies reported the genetic differences among genders in their innate and adaptive immune system function [22–25]. the most common underlying diseases were lung diseases, diabetes, and cvd. in several studies, lung diseases and cvd were reported as frequently associated factors with sars-cov-2 infection and the severity condition in these patients [26–28]. in addition, pre-existing cvd upsurges the rate of covid-19 morbidity and mortality and is closely related to poor disease outcomes [8]. a cohort study in china illustrated that cardiac injury is frequent among hospitalized patients with covid-19, and is associated with a higher risk of in-hospital mortality [29]. along with age and cvd, one of the most common comorbidities associated with a poor prognosis in covid-19 patients is diabetes. people with diabetes and lung complications are more susceptible to get infections, and they tend to develop more severe conditions with a significantly higher mortality rate than those without diabetes [30]. fever, cough, dyspnea, and myalgia were the most reported symptoms among patients with covid-19. chen et al. reported that the majority of patients with covid-19 were males, and common symptoms were fever and cough [31]. the hematological analysis demonstrated a lower level of o2 sat, lymphocytopenia, and a higher level of esr and neutrophil count. several studies have illustrated a lower level of lymphocytes and a higher level of neutrophils in covid-19 cases with severe conditions, which were consistent with our results. the nlr can be useful in the clinical management of table 1. demographical data and clinical characteristics of patients with covid-19 demographical data n (%) gender male 72 (57.1) female 54 (42.9) contact with covid-19 cases 17 (13.5) history of smoking 2 (1.6) history of opium consumption 2 (1.6) clinical characteristics fever 101 (80.2) cough 101 (80.2) myalgia 63 (50.0) dyspnea 51 (40.5) loss of consciousness 8 (6.3) anosmia 4 (3.2) ageusia 3 (2.4) gastrointestinal manifestation 2 (1.6) nausea 1 (0.8) vomiting 1 (0.8) seizure 1 (0.8) po2 less than 93% 102 (81.0) upper than 93% 24 (19.0) intubation 17 (13.5) underlying disease lung diseases 18 (11.9) diabetes 15 (11.9) cardiovascular diseases 9 (7.1) hypertension 6 (4.8) chronic kidney disease 4 (3.2) chronic neurological disorders 4 (3.2) other chronic diseases 4 (3.2) chronic liver disease 2 (1.6) immunodeficiencies 2 (1.6) patients undergoing hemodialysis 2 (1.6) chronic hematologic diseases 2 (1.6) cancer 1 (0.8) yaghubi kalurazi et al. 4 table 2. laboratory findings of patients with covid-19 laboratory index tested median iqr1 iqr3 mean sd min max normal range wbc 122 9.5 6.2 11.6 10.04 5.86 2.5 48.1 4-10 x 103 /cumm rbc 122 4.7 4.1 5.1 4.61 0.84 82 6.69 4.5-6 x 106 miu/l hb 122 13.2 11.5 14.4 12.85 2.34 6 19.1 12-16 g/dl hct 122 41.3 36.3 44.4 40.10 7.08 8.4 61.4 35.5-48 % mch 122 28.4 27 29.5 28.23 4.94 17.9 73.2 27-32 pg mchc 122 32.1 30.9 33.4 32.28 3.98 26.8 71.4 31-37 gr/dl mcv 122 87.5 84.7 91.2 87.29 7.46 61.4 107.9 80-100 fl rdw 121 13.6 12.5 15.1 14.04 2.15 10.8 24.2 11-16 % platelet 122 203 147.5 268.5 224.16 113.19 40 812 140-450 x 103 u/l lymphocyte 36 17.5 10.25 25 20.03 14.05 5 80 25-40% neutrophil 36 80 72 85.75 76.56 15.52 15 95 40-60% eosinophil 33 2 2 2 2.58 4.192 1 26 1-4% monocyte 13 2 1 3 2.23 1.013 1 4 2-8% esr 38 70 44.25 88.25 67.61 31.07 10 138 0-22 mm/hr male 0-29 mm/hr female ptt 49 32 29 36.5 33.55 5.96 28 54 30-45 second pt 49 12 12 12.75 12.85 2.20 12 25 10-2-12 second inr 50 1 1 1.08 1.11 0.30 1 2.85 0.8-1.1 o2sat 55 68 38.5 83.3 61.42 26.37 6.8 98.8 >95% hco3 55 22.1 18.5 25.6 23.17 8.76 9.6 54.7 22-26 meq/l po2 55 36.2 24.5 48.3 43.62 30.47 8.1 148.8 80-100 mmhg pco2 55 41.1 35.7 49 43.39 14.27 20.3 96.4 35-45 mmhg ph 55 7.4 7.3 7.4 7.33 0.08 6.99 7.517 7.35-7.45 ldh 68 951 735.8 1398.3 1231.79 866.48 7 4247 230-460 u/l cpk 72 183.5 94.5 461.5 766.88 2288.68 35 18000 10-120 mcg/l cpk-mb 69 46 31 64.5 59.28 55.18 13 350 <24 u/l or 6-25% of total cpk alp 62 183.5 138.5 276.3 223.65 125.28 79 702 20-140 u/l sgpt 61 32 23 60 90.33 209.47 11 1404 7-56 u/l sgot 61 49 30 90.5 112.28 213.07 12 1377 8-45 u/l k 119 4.2 4 4.4 4.20 0.48 2.6 5.7 3.5-5 meq/l na 120 134 131 137 134.56 4.93 125 149 135-145 meq/l ca 46 8.4 8.2 8.6 8.40 0.49 6.7 9.5 8.5-10.5 mg/dl phosphorus 48 3 2.5 3.4 3.23 1.55 1.5 10.9 2.6-4.5 mg/dl mg 49 1.9 1.8 2.1 1.98 0.25 1.4 2.8 1.8-2.4 mg/dl bs 96 155 115.5 202 180.89 92.30 75 582 <140 mg/dl bun 124 21 14 29.8 27.07 22.26 8 159 7-21 mg/dl creatinine 123 1.1 0.9 1.4 1.37 1.10 0.70 10.10 0.6-1.2 mg/dl uric acid 42 6 4 8.5 6.58 2.96 2.2 15 2.6-8.2 mg/dl total protein 47 6.5 5.7 7 6.31 0.93 3.80 8 6-8.3 g/dl albumin 49 3.6 3.3 3.9 3.60 0.47 2.60 4.50 3.5-5.2 g/dl bilirubin total 59 0.7 0.5 0.9 0.78 0.57 0.30 4.40 0.3-1.2 mg/dl bilirubin direct 59 0.2 0.2 0.4 0.30 0.25 0.10 1.90 <0.2 mg/dl amylase 38 60 41.8 91.3 78.89 74.97 16 454 40-140 u/l abbreviations: cbc (complete blood count), wbc (white blood cell), rbc (red blood cell), hb (hemoglobin), hct (hematocrit), mch (mean corpuscular hemoglobin), mcv (mean corpuscular volume), mchc (mean corpuscular hemoglobin concentration), rdw (red cell distribution width), eer (erythrocyte sedimentation rate), pt (prothrombin time), ptt (partial thromboplastin time), inr (pttest/ptnormal)isi, o2sat (o2 saturation), hco3 (hydrochloric acid), po2 (arterial partial pressure of oxygen), pco2 (arterial partial pressure of carbon dioxide), ph (potential of hydrogen), ldh (lactate dehydrogenase), cpk (creatine phosphokinase), ck-mb (creatine kinase mb), k (potassium), na (sodium), ca (calcium), mg (magnesium), bs (blood sugar), alp (alkaline phosphatase), sgpt (serum glutamic pyruvic transaminase), sgot (serum glutamic oxaloacetic transaminase), sd (standard deviation), iqr (interquartile range). yaghubi kalurazi et al. 5 sars-cov-2 infection in the early stages [32–35]. besides, hypoxia and dyspnea are both reported as a sign of lung involvement. the lower level of o2sat predisposes patients with covid-19 to intensive care unit (icu) admission [36, 37]. pu et al. suggested that sars-cov-2 might trigger the changes in the form of erythrocytes or plasma characteristics through an unknown mechanism by elevating the level of esr [38]. a sustained high level of esr may lead to adverse effects on the prognosis status of patients with covid19 because a higher esr level can damage the joint and lead to joint complications such as osteoarthritis, which may be a precursor of liver and kidney damage [39–42]. the biochemical analysis reported an elevated level of sgot, sgpt, ldh, cpk, and ck-mb in patients with covid-19. due to our results, liver enzymes including, sgot and sgpt were remarkably higher than the normal range in patients with covid19 at the time of admission to the hospital, which was associated with the severity of conditions in these patients. interestingly, a study demonstrated the association between liver enzymes and severity conditions in patients with covid-19. they observed that sgot elevated first, followed by sgpt which was associated with the highest mortality [43, 44]. as sgot is found in various tissue, the elevated level of this enzyme results in multiorgan failure [45]. an investigation demonstrated that along with lymphocytopenia, elevated levels of ldh were more frequently seen in severe covid-19 illnesses [46]. furthermore, the evaluation of ck-mb in patients with covid-19 might provide specific clinical data for early risk factors in these patients [47–49]. similar to our study, it was reported that age, pre-existing underlying diseases, low o2sat, decreased lymphocyte count, and elevated levels of crp, sgot, ldh, and ck-mb can be used as predictors of the severity of covid-19 conditions [50, 51]. another study revealed that ck-mb, ldh, and sgot were higher in patients with covid-19 compared to the control group; moreover, in these patients, the levels of mentioned enzymes were higher in patients with death outcomes than in survivors [52]. according to our investigation, advanced age, male gender, a history of underlying disease, and abnormal laboratory findings predispose patients with covid-19 to severe conditions. acknowledgments we would like to thank all hospital staff and specialists for their assistance with conforming and recording cases. authors’ contributions concept and study design: ty, sm, aa. methods, data collection, and experimental work: eh, lm, da, sn, vs, zg, re, nf. results analysis and interpretation: aa, eh, lm, da, sn, vs, zg, re, sm. manuscript preparation: ty, sm, nf. all authors read and approved the final version of the manuscript. conflict of interests the authors reported no 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2department of physiology, school of medicine, guilan university of medical sciences, rasht, iran 3cellular and molecular research center, school of medicine, guilan university of medical sciences, rasht, iran abstract the nucleus locus coeruleus (lc), the main source of norepinephrine in the brain, is connected to memory processing regions such as the hippocampus and baso-lateral amygdala (bla). the lc and its mostly associated noradrenergic projections, play an important role in memory formation parallel to other neurotransmitter systems. it has been suggested that the unique response characteristics of lc to various situations strengthens different memories formation. here, we review key related findings of lc effect on memory (avoidance, spatial, cognitive) formation, memory processing regions, memory molecular mechanisms as well as its role in memory related disorders. literature review was conducted by extensive search on isi, pubmed and scopus, online databases from may 2021 to july 2021. according to the obtained results, lc noradrenergic projections to memory processing areas of the brain, can modulate the encoding, consolidation, and retrieval for different memory types. also, the lc regulates neurogenesis and neural plasticity in different areas of the brain. evidences suggested that dysfunction of the lc and its associated noradrenergic system may lead to cognitive impairment or a variety of memory-related disorders, including alzheimer's disease. finally, it can be concluded that the locus coeruleus noradrenergic system may be a suitable target for the treatment of different memory/cognitive disorders. keywords: locus coeruleus, memory, neurogenesis, neuronal plasticity, alzheimer’s disease 1. introduction memory is the dynamic, unlimited process that information is learned, stored, and retrieved through it [1]. this complex brain function has various types, such as avoidance, spatial and recognition memory and each involves different brain areas [2]. memory formation comes from interactions between different cells, neurons, neural circuits, and nuclei within the brain [3, 4]. the complicated neurobiological interplay between different parts causes molecular changes in neurons and non-neuronal cells, formation of new *corresponding author: behrooz khakpour-taleghani, ph.d department of physiology, school of medicine, guilan university of medical sciences, guilan, iran tel/fax: +98 13 33690006 email: khakpour_b@yahoo.com https://orcid.org/0000-0002-8192-0188 received: august, 31, 2021 accepted: september, 25, 2021 memories, and finally leads to individual behavioral strategies and decisions alteration [4]. although memory formation is a result of the interactions between different chemical factors and brain cells, given its central role in inducing cellular and circuit changes, norepinephrine (ne) has great importance in memory formation [5]. the nucleus locus coeruleus (lc), the primary source of brain norepinephrine, with its medium-sized dense noradrenergic neurons, is known as the locus coeruleus noradrenergic system (lc-ne) [6]. the lcne system affects the neural circuits which form the © the author(s) 2021 https://jcbior.com/ khakpour-taleghani et al. 2 basis of consciousness and cognitive processes. also, it exhibits modulatory effect on different brain related process such as arousal, sleep-wake cycle, learning, anxiety, pain, mood, and energy metabolism [7]. lc projections innervate various parts of the brain including the cerebellum, spinal cord, and hippocampus (hipp); on the other hand, it receives inputs from different brain nuclei such as the lateral raphe nucleus and nucleus of the tractus solitarius (nts) [8]. due to the substantial connectivity of the lc-ne with several parts of the hipp, the locus coeruleus plays a crucial role in synaptic amplification of neuronal circuits in this area and thereby on the cognitive functions of the brain such as mood and memory [9]. to better clarify the lc-na system critical role in different memories, we reviewed several studies and a variety of behavioral tests. moreover, alongside explaining the critical role of the lc-ne in neuronal circuits, to draw a comprehensive feasible framework to develop future researches, its effect on molecular and cellular processes of memory formation was reviewed. 2. methods a comprehensive literature search was conducted on pubmed and scopus databases looking for articles using the following key terms: "locus coeruleus", "norepinephrine", "memory", "avoidance", "spatial", "recognition", "long-term potentiation", "neurogenesis", and, "alzheimer's disease". the key terms were chosen using mesh and, boolean operators such as "and" and "or" were used to connect the key terms. from may 2021 to july 2021, three researchers conducted the search independently. in this study, papers on learning, memory and cognition associated with the nucleus locus coeruleus were selected, and eventually, 200 articles were included in the study. next, the articles about parkinson's disease, depression, as well as those written before 2000, were excluded. also, to prevent missing valuable studies, the references of the included articles were searched to extract other related studies. finally, 50 studies were extracted and included in the review. 3. lc and memory 3.1 lc and avoidance memory avoidance memory is the experience of unpleasant and emotional stimuli that subsequently causes avoidant behaviors to avoid them [10]. different parts of the brain, such as the amygdala, nts, and hipp actually play an important role in the acquisition and consolidation of this memory [11]. more specifically, various studies have shown that different amygdala nuclei have a major role in achieving an avoidance strategy or consolidation of avoidance memory [12]. it has been suggested that lc stimulation can improve avoidance learning and memory as it is the main source of the amygdala norepinephrine [13]. in line with this suggestion, llorca-torralba et al. reported, pain as an external stimulus could enhance avoidance memory through the stimulation of lc-ne and intensify its effect on the amygdala [14]. similarly, according to fan et al. obtained results of an animal study, corticosterone and stress increase the expression of adrenaline-producing enzymes in lc, boost the function of the basolateral amygdala (bla) and development of avoidance behavior [15]. also, shelkar et al. have been reported that interaction between lc-ne and no facilitates the expression of avoidance behavior [16]. moreover, khakpour-taleghani et al. revealed that while temporary lc inactivation has no influence on the acquisition of negative experiences during avoidance learning, lc plays a role in the consolidation of this memory and converting short-term into long-term memory [17]. it has also been observed that stimulation of lc noradrenergic nerves increase neuronal firing in the bla, and triggers anxiety-like responses in rodents [18]. moreover, it seems that stress can induce a reflexive response in the central region of the amygdala (cea), thereby potentiates rodents avoidance memory through enhanced bla neuronal firing following augmentation of tonic lcne activity (figure 1) [6]. 3.2 lc and spatial memory spatial memory is the ability to keep encoded and retrieved information which is related to an individual's environment and/or spatial orientation. indeed, it is a result of the interplay between different cell populations within the hipp [19], and other various areas of the brain, including the lc. given the strong mutual neuronal connections between the hipp and the lc, several studies have been conducted to assay the effect of lc stimulation on the hipp and khakpour-taleghani et al. 3 spatial memory. lemon et al. found that electrical stimulation of the lc could enhance neural encoding of spatial information in hippocampus ca1 region synapses and subsequently resulted in spatial memory improvement [20]. similarly, in another report, lc stimulation and consequent epinephrine increase enhanced the signal-to-noise ratio of sensory stimuli and thereby could improve spatial memory performance [21]. it should be emphasized that there are some discrepancies on the lc role in spatial memory processing. according to khakpourtaleghani et al., although the lc-ne activity has an effective role in spatial memory, it is not the only contributing factor in memory formation. additionally, the bilateral inactivation of lc had no major effect on the spatial memory consolidation [22]. it may be probable that other factors such as the release of norepinephrine from other parts of the brain can mitigate the negative effects of lc destruction on spatial memory consolidation [22]. in contrast to khakpour-taleghani and colleagues findings, others have mentioned impaired spatial memory after the destruction of noradrenergic neurons and the associated decrease in norepinephrine level within the brain [23, 24]. it seems, to solve such controversial findings we need further studies to clarify the role of lc-ne in spatial memory formation. generally, it appears that lc can improve spatial memory through noradrenergic projections and β-adrenergic receptordependent signaling pathway which has an effect on neurodevelopmental activity in the hipp [25]. currently, we know that dopaminergic projections which originate from the lc can improve spatial memory through targeting d1 and d5 receptors across the dorsal hippocampus (figure 2) [26]. 3.3 lc and recognition memory recognition memory, a hippocampal higher-level function, represents the ability of animals and humans to identify a stimulus or situation which previously had figure 1. path a: the amygdala is the main area for avoidance memory formation. the lc noradrenergic projections [green arrow] affect avoidance memory formation in bla. path b: stress, causes the reflective enhanced cea activity [dark arrow], which in return increases tonic activity of lc-ne and subsequently, the bla activity increases. abbreviations: bla: basolateral amygdala; cea: central nucleus of the amygdala; lc: locus coeruleus. figure 2. the brain portions involved in the spatial memory formation. the lc sends noradrenergic (green arrow) and dopaminergic (red arrow) projections to the hippocampus ca1 area and dorsal hippocampus, respectively. ultimately, these modulatory projections lead to the spatial memory formation. abbreviations: lc: locus coeruleus; hipp: hippocampus; ca1: hippocampal region ca1; ca3: hippocampal region ca3; dg: dentate gyrus. khakpour-taleghani et al. 4 encountered it [27]. given the robust association between the lc and the hipp, the hippocampal modulatory effect on recognition memory is not out of scope [28]. therefore, a large number of studies have been conducted to better understand the relationship between the lc and this type of memory. titulaer et al. reported that stimulation of lc and subsequent norepinephrine increase in the hipp ventral area plays a key role in the animals' performance improvement in recognizing new objects [29]. in addition, kempadoo et al. found that lc light stimulation increases dopamine in dorsal hipp and eventually improves animal model of recognition memory performance [26]. similarly, according to glennon et al. lc stimulation by chemical or even external stimuli such as hearing can also improve recognition memory [30]. in addition to the bilateral relationship between lc and hipp, according to mello-carpes et al. findings, the lc-hipp-ntsparagigantocellularis (pgi) pathway can significantly affect recognition memory performance [31]. furthermore, the frontal cortex-lc pathway is another modulator of this memory through α2adrenoceptor signaling [32]. based on the abovementioned reports, it can be concluded that the neurological activity of locus coeruleus and its noradrenergic projections to different brain areas, like the hipp, play a pivotal role in the development of this higher function memory (figure 3) [29]. 4. lc and cns neurological changes 4.1 lc and neurogenesis neurogenesis is a process in the central nervous system (cns) of adult vertebrates to produce more nerve cells [33]. among the various factors, the lc-ne system has a major role in the neurogenesis of the cns at different stages of life and in a variety of ways [34]. it has been observed that lc-ne induced neurogenesis begins shortly before birth and reaches its peak in infancy. it exerts significant beneficial effects on learning and memory by its trophic effect on the cns [35]. also, the lc has an improving role in the neurogenesis of other brain nuclei. according to guérin et al. study, lc removal in transgenic immature mice leads to the newborn cells death and the polysialylated neural cell adhesion molecule [psancam] decrease in the olfactory bulb and eventually, olfactory memory reduction [34]. also, bortolotto et al. found that the lc-ne intensifies hipp nerveproducing cells (npcs) neurogenesis through β2adrenoceptor stimulation [36]. furthermore, fan et al. reported that alterations of lc cells gene expression can influence neurogenesis of hipp. they revealed that in older mice the expression of a certain type of lc cells transcription factor could enhance cell division in animals’ dentate gyrus region [37]. also, it has been observed that genetic alterations such as lc cell's nucleus mirna increase, exert trophic effect on the hipp [38]. moreover, according to coradazzi and colleagues research, destruction of the lc, directly diminishes neurogenesis and reduces reproductive cells of the hipp [39]. finally, it should be noted that in addition to the lc-ne positive effect on neurogenesis, it also exerts neuroprotective effect through multiple mechanisms including decreasing the oxidative stress and inflammatory responses as well as enhancing endotrophic-neurotrophic systems (ntfs), especially brain-derived neurotrophic factor (bdnf), neurotransmitter production, cholinergic cell survival and also, modulation of cns energy metabolism alongside with regulation and maintain extracellular homeostasis [40]. figure 3. path a. the lc dopaminergic projections (red arrow) to the dorsal hippocampus have a significant role in development of recognition memory. path b. lc-ne (green arrow) promotes the recognition memory by its effect on the ventral hippocampus. path c. the lc–hipp–nts–pgi pathway (dark arrow) has an effective role in recognition memory function. path d. lc-ne (green arrow) inhibitory effect on the pre-frontal cortex modulates recognition memory performance. abbreviations: lc: locus coeruleus; hipp: hippocampus; pfc: prefrontal cortex; nts: nucleus of the solitary tract; pgi: paragigantocellularis. khakpour-taleghani et al. 5 4.2 lc and neural plasticity long-term potentiation (ltp) is a form of neuroplasticity that is thought to be a common cellular mechanism for many learning and memory processes [41]. the lc-ne system plays a pivotal role in the development of ltp in important memory processing centers such as the dentate gyrus and the ca1 region of the hippocampus. ne exerts its positive effect through different members of the adrenoreceptor family [42]. quinlan et al. found that in animal models, the lc stimulation with light can lead to ltp formation in the dentate gyrus and subsequent longterm learning and memory improvement [43]. according to reid et al. following simultaneous stimulation of the perforant pathway and the lc, norepinephrine release starts and results in hippocampal neuronal plasticity and the initiation of learning process [44]. also, lashgari et al. has reported that unlike short periods, over a long period of the time, lc noradrenergic system can induce ltp through protein synthesis enhancement [45]. moreover, according to hammerschmidt et al. results, the lc destruction can decrease the ltp occurrence in the hipp through the reduction of camkii and nr2a and finally leads to memory loss [46]. in addition, according to tamano et al. report, through βadrenoreceptors activation the lc-ne regulates zn2+ ion concentration and subsequently beta-amyloid decrement can facilitate the hippocampal dentate gyrus ltp production [47]. it should be noted that the sensitivity towards epinephrine is not the same at different parts of the hipp, and each region expresses a certain level of ltp, depending on its βadrenoreceptors expression [48]. titulaer et al. found that in addition to ne, the nucleus locus coeruleus dopaminergic projections have an effective role in the ltp production in the ventral hipp which is involved in recognition memory. they suggested that it acts through stimulation of hippocampal d1/5 receptors [29]. 5. lc and alzheimer's disease many studies have demonstrated that lc is highly susceptible to various toxins and infections, and in many cases, is the primary site where ad pathology occurs [49]. german et al. found that amyloid-beta build-up in the lc results in the destruction of its neurons [50]. similarly, ross et al. observed that excessive activity of the lc in response to hormonal stressors such as the corticotropin-releasing factor may cause production and deposition of amyloid-beta and introduced it as one of the most important pathologies of alzheimer's disease (ad) [51]. aghajanov et al. reported that in animal models of alzheimer's disease, elevated level of oxidative stress, together with increased concentration of monoamines, particularly norepinephrin, is accompanied by severe structural damage to lc monoaminergic neurons [52]. also, hopp et al. found that neuroinflammation can induce ad symptoms by disrupting the regulation of calcium ions in the lc and leads to impaired nervous pacemaker activity [53]. furthermore, it has been observed that loss of the lc noradrenergic neurons or its decreased activity causes ad symptoms in different parts of the brain [54, 55]. for example, kelly et al. demonstrated that degeneration of the lc-ne system increases the risk of early ad through impairing vascular and neurological function in the brain areas associated with memory and cognitive performances [55]. another hypothesis is that the primary signaling of aberrant forms of tau protein begins in the lc [56]. kang et al. have been reported that aberrant forms of tau protein not only cause neurological dysfunction but also are capable of spreading memory processing regions of the brain and might contribute to the ad development [57]. moreover, we know that increased lc activity by neurotrophic factors, may have beneficial effects in preventing neuropsychological signs of ad [58]. 6. conclusion the nucleus locus coeruleus through its noradrenergic projections affects various parts of memory processing areas of the brain. also, lc-ne as a potent modulatory system, as well as other important memory processing cholinergic and dopaminergic systems, play an important role in memory formation. in addition, through the stimulant noradrenergic projections to the baso-lateral amygdala, lc has an effective role in enhancing avoidance memory. of course, the effective role of lc in other brain areas such as nts should be considered in the avoidance memory formation. it develops spatial memory mainly through β-adrenergic signaling in the ca1 region of the hippocampus. furthermore, due to its stimulatory effect on the lc– hipp–nts– pgi pathway, especially the hippocampus and the modulatory effect on the frontal khakpour-taleghani et al. 6 cortex, lc has a pivotal role in the development of cognitive memory. on the other hand, lc-ne can lead to permanent ltp production and neurogenesis through the induction of cellular level changes in gene expression, protein synthesis, and ions concentration in nuclei and/or circuits involved in memory formation. moreover, we must emphasize that the role of the lc dopaminergic projections should not be overlooked in the memory 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collier tj, dorrance am, counts se. locus coeruleus degeneration induces forebrain vascular pathology in a transgenic rat model of alzheimer's disease. j alzheimers dis. 2019; 70(2):371-88. 56. kaufman sk, del tredici k, thomas tl, braak h, diamond mi. tau seeding activity begins in the transentorhinal/entorhinal regions and anticipates phospho-tau pathology in alzheimer's disease and part. acta neuropathol. 2018; 136(1):57-67. 57. kang ss, liu x, ahn eh, xiang j, manfredsson fp, yang x, et al. norepinephrine metabolite dopegal activates aep and pathological tau aggregation in locus coeruleus. j clin invest. 2020; 130(1):422-37. 58. braun d, feinstein dl. the locus coeruleus neuroprotective drug vindeburnol normalizes behavior in the 5xfad transgenic mouse model of alzheimer's disease. brain res. 2019; 1702:29-37. https://pubmed.ncbi.nlm.nih.gov/31250157/ https://pubmed.ncbi.nlm.nih.gov/31250157/ https://pubmed.ncbi.nlm.nih.gov/31250157/ https://pubmed.ncbi.nlm.nih.gov/31250157/ 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https://pubmed.ncbi.nlm.nih.gov/29752551/ https://pubmed.ncbi.nlm.nih.gov/31793911/ https://pubmed.ncbi.nlm.nih.gov/31793911/ https://pubmed.ncbi.nlm.nih.gov/31793911/ https://pubmed.ncbi.nlm.nih.gov/31793911/ https://pubmed.ncbi.nlm.nih.gov/29274883/ https://pubmed.ncbi.nlm.nih.gov/29274883/ https://pubmed.ncbi.nlm.nih.gov/29274883/ journal of current biomedical reports jcbior.com volume 2, number 4, 2021 eissn: 2717-1906 1 original research impacts of bacillus subtilis jq61816 on lipid panel and expression of genes involved in cholesterol metabolism in hypercholesterolemic rats abolfazl dehkohneh1, azam khalili1, omid ardalani2, amin mohsenzadeh3, hoda sabati4, parvaneh jafari5,* 1department of biotechnology, faculty of advanced science and technology, tehran medical sciences, islamic azad university, tehran, iran 2department of microbial biotechnology, school of biology and centre of excellence in phylogeny of living organisms, college of science, university of tehran, tehran, iran 3department of microbiology, faculty of science, ardabil branch, islamic azad university, ardabil, iran 4biotechnology and biological science research center, faculty of science, shahid chamran university of ahvaz, ahvaz, iran 5microbiology department, faculty of science, islamic azad university, arak branch, arak, iran abstract hypercholesterolemia is one of the major risk factors associated with the emergence and development of cardiovascular diseases (cvd) and atherosclerosis. the hypocholesterolemic effects of probiotics have been indicated by numerous studies. the 3-hydroxy-3-methylglutaryl-coenzyme a reductase (hmgcr) and cytochrome p450 7a1 or cholesterol 7 alpha-hydroxylase (cyp7a1) are two important genes in choletsterol metabolism. in this study, the effects of bacillus subtilis jq61816 on lipid panel, hepatic enzymes and expression levels of hmgcr and cyp7a1 were investigated. twenty-one male wistar rats were randomly allotted to 3 experimental groups; a) negative control group (nd) fed with normal diet, b) high-fat diet group (hfd) fed with high cholesterol diet, and c) probiotic group (bs) fed with high cholesterol diet supplemented with probiotic b. subtilis. serum analysis of treatment groups was performed to measure fasting blood sugar (fbs), lipid profile parameters, hepatic enzymes, urea, and uric acid. our results showed that b. subtilis could reduce the level of total cholesterol, triglycerides, and ldl and it also could increase high-density lipoprotein (hdl) level. moreover, alanine transaminase (alt), aspartate transaminase (ast), and uric acid were significantly lower in bs group compare to hfd group. furthermore, up-regulation of hmgcr and down-regulation of cyp7a1 were observed in bs group. the results of our study suggest that consumption of probiotic b. subtilis jq61816 may prevent or decline the development of hypercholesterolemia and other cardiovascular diseases. keywords: probiotics, bacillus subtilis, hypercholesterolemia, cardiovascular disease, triglyceride 1. introduction hypercholesterolemia is one of the main risk factors associated with the emergence and development of cardiovascular diseases (cvd) and *corresponding author: parvaneh jafari, ph.d microbiology department, faculty of science, islamic azad university, arak branch, arak, iran tel/fax: +98 86 3413 2451 email: p-jafari@iau-arak.ac.ir, parvaneh.jafari@gmail.com http://orcid.org/0000-0003-2771-9309 received: july, 23, 2021 accepted: september, 22, 2021 atherosclerosis, which are considered as common cause of morbidity and mortality [1]. according to world health organization (who) prediction, by 2030, cvd will be the leading global cause of deaths © the author(s) 2021 https://jcbior.com/ dehkohneh et al. 2 and will affect over 23.6 million around the world which accounts for 80% of the deaths in low-income countries [2, 3]. there are several approaches used to control or treat hypercholesterolemia, such as consumption of statins and ezetimibe, exercising and adjusting of dietary habits [4-6]. although medicinal therapy is a pioneering method in the regulation of cholesterol level, long-term use of this approach leads to various undesirable effects [7-9]. thus, there has been a growing tendency among researchers to find alternative ways, such as using biomaterials. numerous studies have indicated that probiotics, either bacteria or yeasts, have many health-promoting effects such as immunomodulatory, anti-cancer and anti-diabetic, gastrointestinal tract promotion, hypocholesterolemic and regulation of lipid metabolism [10-14]. for these reasons, probiotics are considered as a promising approach in the modulation of lipid metabolism to reduce the rate of hypercholesterolemia. probiotics are living microorganisms that exert their beneficial effects on the host if consumed in appropriate amounts. bacillus subtilis is a type of probiotics which is widely used in diets [15]. in the present study, the impact of oral administration of b. subtilis jq61816 on lipid panel parameters and the expression levels of 3-hydroxy-3methyl-glutaryl-coenzyme a reductase (hmgcr) and cholesterol 7-alpha hydroxylase (cyp7a1) genes were assessed in male wistar rats with a high-fat diet. 2. materials and methods 2.1 bacteria the b. subtilis jq61816 was purchased from takgen company as lyophilized bacterium (bactogen® contained 4×109 cfu/ml). probiotic suspension daily prepared, and to prepare probiotic suspension, lyophilized b. subtilis jq61816 mixed by phosphatebuffered saline (pbs) (sigma aldrich, usa). 2.2 animals and study design all animal experiments in this study were approved by the animal care committee of islamic azad university of medical science (tehran, iran) and were performed according to the guidelines from the nih principles for the care and use of laboratory animals. twenty-one male wistar rats aged 7-8 weeks; weighed 100-150 grams were purchased from baqiyat-allah research center (tehran, iran), and used for all experiments. rats were housed in cages maintained at 22 ± 2°c with 55% ± 5% humidity and subjected to a 12 h light/dark cycle and free access to food and water for 14 days to adapt to laboratory conditions after adaptation rats were distributed into three groups. negative control group (nd), high-fat group (hfd), and probiotic group (bs), which respectively were fed with normal diet plus 1ml pbs, high-fat diet + 1ml pbs, and high cholesterol diet + b. subtilis (4×109 cfu/ml) suspension. diet ingredients are shown in table 1. to prepare a high-fat diet, we used liquid fat and mixed it with the normal diet's ingredients. pbs and probiotic suspension were administered by daily gavage at a certain time. 2.3 blood collecting and analysis at the end of three weeks, all rats were sacrificed after 12 h fasting, and then blood was collected by cardiac puncture. the serum was collected by centrifugation at 1500×g for 15 min. an auto-analyzer t a b le 1 . c o n st it u e n t o f d ie ts u se d i n t h is r e se a rc h in g r e d ie n ts p e r c e n ta g e i n n o r m a l d ie t (% ) p e r c e n ta g e i n h ig h -f a t d ie t (% ) p ro te in 2 3 2 3 l y si n e 1 .1 5 1 .1 5 m e th io n in e 0 .3 3 0 .3 3 t h re o n in e 0 .7 2 0 .7 2 t ry p to p h a n 0 .2 5 0 .2 5 c a lc iu m 1 1 s a lt 0 .5 5 0 .5 5 f a t 0 8 dehkohneh et al. 3 (ra1000, usa) was used for the analysis of serum total cholesterol (tc), triglyceride (tg), low-density lipoprotein (ldl), high-density lipoprotein (hdl), aspartate aminotransferase (ast), alkaline phosphatase (alp), alanine aminotransferase (alt), fasting blood sugar (fbs), urea and uric acid levels. moreover, to achieve a better understanding of the impact of probiotics on serum parameters, we calculated the ratio of total cholesterol/hdl, triglycerides/hdl, and ldl/hdl. 2.4 gene expression analysis total rna was extracted from abdominal adipose tissues using rnx-plus reagent (cinaclon, iran) according to manufacturer's instruction, and 1 µg of isolated rna was converted to cdna with cdna reverse transcriptase kit (bioneer, south korea). cdna was used as a template and amplified by quantitative polymerase chain reaction (qpcr). gene expression analysis was performed by real-time pcr system (abi-stepone, usa) using sybrgreen (ampliqon, denmark). the program for pcr and relative gene expression is described in our previous study [11]. gapdh gene was considered as a housekeeping gene, and the relative quantities of each gene were analyzed using terms of 2-δδct. primers were designed for hmgcr, and cyp7a1 genes are obtained from our previous research [11]. the primers are designed using primer3plus software and primer analysis was performed by oligoanalyzer 3.1. resultant primers are shown in table 2. the whole experiments were carried out in duplicate. 2.5 statistical analysis all data are reported as the mean ± sem. statistical significance was analyzed by the student ttest method using graphpad prism 8.2.1 software (graphpad software, inc., us). a p-value < 0.05 was considered statistically significant. 3. results 3.1 the effect of b. subtilis jq61816 on fbs hfd group had the highest level of fbs among the experimental group. although b. subtilis jq61816 could reduce the fbs level, there was not a significant difference compared to control groups (figure 1a). 3.2 the effect of b. subtilis jq61816 on lipid profile 3.2.1 total cholesterol the level of total cholesterol was significantly increased in hfd group by 22.9% (p< 0.05), while b. subtilis jq61816 caused decreasing of total cholesterol level by 9.9% compared to hfd. however, the difference was not significant (figure 1b). 3.2.2 triglycerides the serum analysis indicated that b. subtilis jq61816 consumption could significantly decrease the triglycerides level by 30.4 % in bs group compared to the hfd group (p<0.05). the results are shown in figure 1c. 3.2.3 hdl bs group revealed the highest level of hdl among experimental groups. the level of hdl in bs was climbed by 11.76% compared to hfd group. moreover, nd group had the lowest amount of hdl unexpectedly (figure 1d). 3.2.4 ldl as depicted in figure 1e, hfd group had the highest level among all groups. the serum ldl was significantly decreased after 21 days of consumption of t a b le 2 . t h e l is t o f p ri m e r u se d i n r t -p c r g e n e p r im e r s e q u e n c e ( 5 '3 ') p r o d u c t si z e ( b p ) h m g c r f : a a g c t g t c a t t c c a g c c a a g r : g g c c a c a t g c a a t g t a g a t g 1 7 1 c y p 7 a 1 f : c t g c g t g a c g a a a t t g a c a g r : c t t g c a c t t c a c g g a t g a t g 1 6 9 g a p d h f : t t g t g a t g g g t g t g a a c c a c r : a g t c t t c t g a g t g g c a g t g a t g 1 7 0 dehkohneh et al. 4 probiotics by 22.2% compared to hfd group (p<0.05). 3.2.5 total cholesterol/hdl ratio as expected, the measurement of total cholesterol to hdl ratio indicated that bs group was lower than other experimental groups (p<0.05). the result of the analysis is shown in figure 2a. 3.2.6 triglycerides/hdl ratio the ratio of triglycerides number to hdl number displayed a 16.9% reduction in hfd group compared to nd group, while a statistical significance was figure1. serum analysis results of fbs and lipid profile parameters. a) serum fbs level: bacillus subtilis jq61816 could decline the level of serum fbs compared to hfd group, but it was not statistically significant; b) total cholesterol level: the quantity of total serum cholesterol was elevated significantly by 22.9 % in hfd group due to having a high-fat diet while the level of total cholesterol was reduced by 9.9% due to consumption of b. subtilis jq61816; c) the level of serum triglycerides: consumption of b. subtilis jq61816 could significantly decrease the serum triglyceride compared to hfd group; d) serum hdl level: bs group showed the highest level of hdl, which was elevated by 11.76% compared to hfd. unexpectedly, the nd group had the lowest level of hdl among all experimental groups; e) the serum level of ldl: bs group was significantly declined by 22.2% compared to hfd group due to consumption of probiotics. figure 2. ratio analysis of lipid profile parameters. a) total cholesterol to hdl ratio: bs group is declined compared to hfd group due to consumption of b. subtilis jq61816; b) triglyceride to hdl ratio: bs group was shown a significant difference compared to hfd group by 36.8%; c) ldl / hdl ratio: the highest number of ldl/hdl ratio was belonged to hfd group, while bs group had the lowest level due to probiotics consumption. dehkohneh et al. 5 observed in bs group compared to hfd group by 36.8% (p<0.05) (figure 2b). 3.2.7 ldl/hdl ratio the analysis of ldl/ hdl ratio revealed that the consumption of b. subtilis jq61816 was caused a significant decline compared to hfd group. the results of the analysis are shown in figure 2c. 3.3 the effect of b. subtilis jq61816 on hepatic enzymes 3.3.1 ast and alt the measurement of serum ast and alt levels revealed that the administration of probiotics could reduce these hepatic enzymes significantly compared to hfd group by 41.4% and 23.8%, respectively (p<0.05). in contrast, the serum level of ast and alt were climbed due to a high-fat diet (figure 3a, 3b). 3.3.2 alp the level of alp in bs group was decreased compared to hfd. although the amount of alp was increased in both groups of hfd and bs compared to nd group, a significant difference was observed between hfd and nd groups (p<0.05). results are shown in figure 3c. 3.4 uric acid the measurement of serum uric acid showed that orally administration of b. subtilis jq61816 could reduce uric acid level by 35.4% compared to uric acid level in hfd group (p<0.05). the results are shown in figure 3d. 3.5 urea the analysis of urea level demonstrated that there was no significant difference between all experimental figure 3. the level of hepatic enzymes, uric acid and urea. a) the serum levels of ast: consumption of bacillus subtilis jq61816 could significantly decline the amount of ast enzyme while the hfd group had the highest level of this hepatic enzyme among other experimental groups; b) the serum level of alt: bs group had the lowest level of alt among experimental groups and b. subtilis reduced the alt level significantly compared to hfd group; c) the serum level of alp: the amount of alp was raised in hfd and bs groups compared to nd group. although the bs group had a lower amount of serum alp, there was no significant difference between hfd and bs groups; d) the serum level of uric acid: administration of probiotics was caused to reduce the uric acid level in bs group by 35.4% compared to hfd group; e) the serum level of urea: although the level of urea in bs group is lower than hfd group, but the difference was not statistically significant. dehkohneh et al. 6 groups. however, the serum level of uric acid in bs group was lower than hfd group (figure 3e). 3.6 the effect of b. subtilis jq61816 on key genes in cholesterol metabolism the expression of hmgcr gene was significantly climbed in bs group compared to hfd group (p<0.05). in contrast, the down regulation of cyp7a1 was observed in bs group. results are shown in figure 4a, 4b. 4. discussion hypercholesterolemia is a complex metabolic disorder caused by an elevated level of blood cholesterol and affected by lipid metabolism factors and diet. numerous clinical, epidemiological, animal and genetic studies have indicated that a high amount of blood cholesterol is associated with an elevated risk of cardiovascular diseases [16, 17]. using statins (e.g., rosuvastatin and atorvastatin), as lipid-lowering medication, is the most common approach to reduce serum cholesterol, triglycerides, and ldl levels. although this method might be effective, some undesirable effects have been reported, including myopathy and albuminuria [18, 19]. hence, using alternative approaches are crucial, which are safer and more cost-effective. for a long time, probiotics have been widely used, and the hypocholesterolemic effect of them has been wellstudied in vivo and in vitro [12, 20]. in this study, we used b. subtilis jq61816 as a probiotic. a high level of cholesterol in hfd group indicated that high-cholesterol diet had led to hypercholesterolemic rats. our results indicated that b. subtilis jq61816 could decrease the serum levels of total cholesterol, triglycerides, and ldl, while it can elevate hdl level. although b. subtilis jq61816 could reduce total cholesterol level, it was not significant statistically. the hypocholesterolemic effects of probiotics have been studied in humans and animals over the years. similarities between humans and some animals (e.g., rats, hamsters, and pigs) in bile acid and cholesterol metabolism and plasma lipoprotein distribution make these animals as useful experimental models [21-23]. therefore, the acquired results from animal studies can be reliable and transferable to humans. in this study, b. subtilis jq61816 has been chosen to make a comparison with other probiotics with hypocholesterolemic effects. according to previous reports, various probiotic bacteria may have hypercholesterolemic impacts by various levels [24-26]. there are several plausible mechanisms for hypocholesterolemic effects of probiotics, including enzymatic deconjugation of bile acids by bile salt hydrolase, assimilation of cholesterol by growing cell, production of short-chain fatty acids from oligosaccharides and cholesterol binding to cellular surface of probiotics [24-26]. also, b. subtilis bacterium can produce extracellular cholesterol oxidase, which led to cholesterol level reduction [27, 28]. heo et al. indicated that lactobacillus plantarum lrcc 5273 could reduce total cholesterol and ldl figure 4. the effect of bacillus subtilis jq61816 on key genes related to cholesterol metabolism: the level of hmgcr expression was increased in bs group compared to hfd group. in contrast, unexpectedly, the level of cyp7a1 gene expression was significantly decreased in bs group due to using probiotics. dehkohneh et al. 7 cholesterol levels significantly, while the significant reduction in triglycerides and hdl-cholesterol were not observed [29]. in another study, briand et al. showed that the administration of saccharomyces boulardii led to a significant reduction in total plasma cholesterol and hdl at day 21. however, in the liver, the significant reduction was not observed in total cholesterol level, while triglycerides level significantly decreased by 25% at day 39 [30]. moreover, according to some reports the consumption of l. plantarum ky1032 and lactobacillus curvatus at 5×109 cfu/day can significantly reduce the triglycerides level by 22% in rats with high-fat diet while consumption of these bacteria at 1010 cfu/day can decrease the level of triglycerides by 46% in highfructose-fed rats [31, 32]. there are several human studies that have indicated that the administration of probiotics has led to a decrease in serum total cholesterol, triglyceride, and ldl [33-35]. on the other hand, some clinical trial reports have shown that the consumption of some probiotics might have no significant effects on lipid profile [36, 37]. numerous studies have shown probiotics may affect cholesterol metabolism through the regulation of genes involved in cholesterol biosynthesis pathway [29, 38, 39]. in contrast to the findings of some studies, we observed a significant decrease in the expression of hmgcr gene in bs group compared to the hfd group. however, some studies have indicated that the administration of some probiotics such as l. plantarum, and lactobacillus rhamnosus, can lead to up-regulation of hmgcr in caco-2 cells and liver, respectively [38, 40, 41]. interestingly, in vivo experiments have shown that taking cholesterollowering medicines (e.g., ezetimibe and simvastatin) elevates intestinal expression of hmgcr [42, 43]. besides, our findings showed that b. subtilis jq61816 cause a significant decrease in cyp7a1 expression in bs group. our result is in agreement with yadav et al. study, which has shown that consumption of milk fermented with two strains of l. rhamnosus can cause down-regulation of cyp7a1 in hypercholesterolemic rats [44]. however, our findings are in contrast to some studies which have shown probiotics consumption leads to overexpression of cyp7a1 [39, 45]. cyp7a1 gene is a part of bile acid-producing pathway which utilize cholesterol as its substrate, downregulation of this gene might occur for two reasons, in the first hypothesis, due to consumption of diet cholesterol by b. subtilis cholesterol oxidase [46], plasma cholesterol level could decrease, therefore due to depletion of cyp7a1 substrate this gene could be downregulated. in the second hypothesis, b. subtilis squalene synthase-like enzyme (yisp) can catalyze the formation of farnesol (foh) from farnesyl diphosphate (fpp) [47]. farnesol subsequently can activate farnesoid x receptor (fxr) [48]. activation of this receptor would lead to downregulation of cyp7a1 [49] and also reported to has a triglyceride-lowering effect. in the current study, these effects were observed in bs group. on the other hand, fxr activation is shown to be effective in intestinal cholesterol excretion and hyperlipidemia attenuation [50]. it seems that the excretion of intestinal cholesterol may lead to a decrease in blood cholesterol levels. elevation of the hmgcr gene expression in bs group might occur to compensate for this reduction by increasing cellular biosynthesis of cholesterol. the proposed hypotheses are depicted in supplementary figure 1. here, we are reporting that b. subtilis jq61816 exerts cholesterol-lowering and triglycerides-lowering effects on the host through regulation of expression of genes involved in lipid metabolism. according to numerous studies, it seems probiotics exert their effects in various mechanisms and several factors are associated with the mechanisms, including dosage of probiotics, type of probiotics bacteria, gut microbiota composition, etc. we suggest that administration of different doses of b. subtilis jq61816 in a long period of time for further studies. among the limitations of the present study we make a suggestion according to the results as the mechanisms of hypocholesterolemic effects of probiotics are not clear yet. the period of time for using probiotics may have roles in its hypocholesterolemic effects. moreover, it is possible that the administration of this bacterium with other probiotics and even prebiotics may elevate its hypocholesterolemic effects. our findings showed that the administration of b. subtilis jq61816 can reduce total serum cholesterol, triglycerides, and ldl, while increasing hdl levels. moreover, b. subtilis jq61816 can reduce hepatic enzymes that are associated with liver disorders such as fatty liver. also, the level of urea and uric acid were decreased due to administration 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https://pubmed.ncbi.nlm.nih.gov/32170842/ https://pubmed.ncbi.nlm.nih.gov/32170842/ https://pubmed.ncbi.nlm.nih.gov/32170842/ https://pubmed.ncbi.nlm.nih.gov/32170842/ journal of current biomedical reports jcbior.com volume 2, number 4, 2021 eissn: 2717-1906 1 review the current advances of crispr/cas-based systems for the detection of covid-19 sasan ghaffari1,2,3, hanif kazerooni1,*, amir salehi-najafabadi3,4,* 1department of science and technology, supreme national defense university, tehran, iran 2department of hematology, school of allied medical sciences, tehran university of medical sciences, tehran, iran 3research center for new technologies in life science engineering, university of tehran, tehran, iran 4department of microbiology, school of biology, college of science, university of tehran, tehran, iran abstract the novel coronavirus disease 2019 (covid-19) still poses a serious threat to every human on the planet nearly 1.5 years after its beginning. unfortunately, the current diagnostic methods, although highly sensitive and specific, still suffer from many shortcomings. faster and easy-to-operate diagnostic systems are undoubtedly sorely needed. the crispr/cas platform has gained much attention in recent years in a wide range of biomedical sciences. besides its treatment potential and drug and vaccine development, it can be used for disease diagnosis. thus, the crispr/cas-based system holds great promise for providing a rapid and easily deployable covid-19 detection and is on par with the conventional diagnostic approaches in terms of sensitivity and specificity. in this review article, we discuss the latest advances of crispr/cas technology as a fast and reliable severe acute respiratory syndrome coronavirus 2 (sars-cov-2) detection method. keywords: covid-19, sars-cov-2, diagnosis, crispr 1. introduction the emergence of the new severe acute respiratory syndrome coronavirus-2 (sars-cov-2) in december 2019 in wuhan, china marks a major turning point for mankind. the health-related and economic issues associated with the devastating coronavirus disease 2019 (covid-19) quickly swept across the globe while threatening the lives of billions with a global pandemic. the disease has taken the lives of millions and shows no signs of slowing down even with the *corresponding author: hanif kazerooni, ph.d department of science and technology, supreme national defense university, tehran, iran tel/fax: +98 910 2118860 email: kazeroonihanif1970@gmail.com https://orcid.org/0000-0002-7159-1237 amir salehi-najafabadi, ph.d department of microbiology, university of tehran, p.o. box 11155-4563, tehran, iran tel/fax: +98 912 2984877 email: amirslehi@ut.ac.ir https://orcid.org/0000-0002-3661-4915 received: august, 08, 2021 accepted: september, 20, 2021 advent of vaccines. the alpha (229e and nl63) and beta (oc43 and hku1) groups of the coronavirinae subfamily can infect humans; two other beta group viruses, middle east respiratory syndrome (merscov) and severe acute respiratory syndrome (sarscov), were previously endemic to humans, none of which have caused such destructive effects [1, 2]. these viruses primarily infect the respiratory tract cells that carry angiotensin-converting enzyme ii (ace2) and subsequently other cells that express ace2. © the author(s) 2021 https://jcbior.com/ https://orcid.org/0000-0002-7159-1237 ghaffari et al. 2 ultimately, the unhinged virus replication and the uncontrolled inflammatory response of the immune system result in acute respiratory distress syndrome (ards), multiple organ failure, and death [2]. besides finding a potential treatment for covid19, early and specific detection of the virus are crucial in breaking the transmission cycle and stopping the disease from spreading. the positive-sense rna of sars-cov-2 is quite similar to that of sars-cov and is comprised of up to 14 open-reading frames (orfs). orf1a and orf1b encode 16 non-structural proteins and constitute the vast majority of the genome. at the 3' end of the genome, the structural proteins include spike (s), membrane (m), envelope (e), and nucleocapsid (n) [3]. quantitative real-time reverse transcriptase-polymerase chain reaction (qrt-pcr) is currently the standard method recommended by the world health organization (who) for specific detection of the mentioned sars-cov-2 genome sequences. however, due to various reasons such as being time-consuming, requiring rigid sample preparations and trained operators, and having stationary equipment, wide-range use of rt-pcr is somewhat difficult. also, in some cases, the sensitivity of the method is reported to be as low as 42.1% [4, 5], which calls for the introduction of more sensitive and versatile detection systems. clustered regularly interspaced short palindromic repeats (crispr) and its associated protein (cas) have been the focal point of advanced gene-editing since this platform is much more flexible and precise than its predecessors. considering the emergency of covid-19, scientists have focused on repurposing crispr/cas system toward a diagnostic and even therapeutic approach [6]. in this article, we review the current sars-cov-2 detection methods and introduce various crispr/cas-based approaches to virus detection. 2. sars-cov-2 routine detection methods currently, the most widely used method of detecting sars-cov-2 involves nucleic acid (na) amplification and subsequent detection. na detection methods have proven successful in the past with mers and sars-cov outbreaks [7], and constitute the current standard sars-cov-2 detection method as rt-pcr, a variant of the pcr method. in rt-pcr, the sample is collected, lysed, and its rna extracted. the purified rna is reversely transcribed to complementary dna (cdna) and amplified. the detection of certain regions of the cdna, such as n, s, and orf1 genes, equals virus detection and sars-cov2 infection. the sensitivity of rt-pcr greatly depends on the chosen primer/probes [8]. using unspecific primer/probe sets results in no detection of virus in low load (cycle threshold>30), or unspecific detection of other coronaviruses (hcov-oc43, hcov-229e, and mers-cov) due to cross-reactivity [9]. unfortunately, the rt-pcr method is dependent on expensive instruments and trained laboratory scientists and is a lengthy process that is in contrast to create rapid, highly sensitive, and point-of-care (poc) diagnostic systems. over the years, other prominent pcr-based methods have emerged as alternatives to rt-pcr. digital pcr (dpcr) and reverse-transcription loopmediated isothermal amplification (rt-lamp) are to name a few. in dpcr, the sample is separated into discrete reactions and each is carried out separately. dpcr does not rely on a standard curve. it can detect much less viral load and it is less susceptible to amplification inhibitors [10]. lamp is a step forward toward more rapid and poc testing which is crucial in the current pandemic. the method amplifies na using 6 specific primers and a dna polymerase enzyme with chain displacement activity. such an activity circumvents the need for dna denaturation via heat. thus, the reaction may be carried out at one constant temperature (isothermal) making the process simpler and the device less intricate. if the starting na is rna, reverse transcriptase is added to create cdna (rtlamp) (table 1) [11]. reverse transcription recombinase polymerase amplification (rt-rpa) is another isothermal amplification method that works on the same basis as rt-lamp and eliminates the need for thermal cycling machines. all in all, accurate diagnosis of covid-19 in suspected individuals is imperative to hinder the spread of the virus. the sensitivity and specificity of the current diagnostic methods are crucial in drawing an accurate comparison between each platform. the specificity and sensitivity of na amplification methods depend on various conditions. while targeting the orf1, e, and rdrp genes produce accurate results, n gene detection decreases sensitivity [12]. the contamination of the specimen has shown a decrease in the sensitivity of rt-pcr [13]. a study on 194 samples compared dpcr and rt-pcr and reported higher overall specificity and sensitivity of dpcr ghaffari et al. 3 compared to rt-pcr [14]. these results were further confirmed by another study on 77 clinical samples, especially on low viral loads [15]. rt-lamp provides the same level of specificity and sensitivity as rt-pcr and also has the added benefit of not requiring a thermal cycler. serological methods, such as enzymelinked immunosorbent assay (elisa) have the advantage of being rapid and high throughput; however, they cannot be designated as a gold-standard method since antibody response and its clinical significance in covid-19 is not yet fully understood [16]. they are not as sensitive as nucleic acid-based techniques. in crispr/cas platforms, the specificity is guaranteed by cas enzyme activation after crispr rna binds to the target sequence. signal emission after the cleavage of the labels, on the other hand, ensures sensitivity. in a study by hou et al. on 52 samples, the crispr platform showed 100% specificity and sensitivity compared to the sensitivity (90.4%) and specificity (89.2%) of rt-pcr [17]. depending on the detected gene, the crispr method had the same or higher sensitivity and specificity compared to rt-pcr in another study [18]. considering its numerous advantages discussed below, crispr could potentially revolutionize the way we fight invading pathogens, such as sars-cov-2. 3. a primer on crispr/cas in response to the disastrous outcome of the covid-19 pandemic, it is crucial to push forward the detection capabilities of laboratories worldwide. early detection of the virus is crucial since it allows for rapid clinical intervention and stops the unsuspecting infected carrier from spreading the virus. crispr was first discovered in the 1980s as a part of the immune system of prokaryotes against the invasion of viruses and plasmids [19]. the system essentially works on the rna-guided catalytic activity of cas proteins. apart from crispr/cas sensational reception in the field of gene editing, it has gained much attention as an na detection platform due to its efficacy and simplicity. the two major constituents of crispr/cas are a single guide rna (grna) that recognizes the target sequence and a cas endonuclease that proceeds to cleave the target sequence. the grna itself is made up of trans-activating rna (tracrrna) and crispr prerna (crrna) which recognizes a small 3-8 bp dna sequence called proto space adjacent motif (pam). crispr/cas essentially works like molecular scissors and based on the cas protein, it is divided into two classes and each of them consists of three types. class 1 is type i, iii, and iv, while class 2 includes type ii, v, and vi [20]. class 2 crisprs are widely used in preclinical and clinical studies. crispr/cas9 of class 2, type ii is used to cleave double-stranded dna (dsdna) to delete or insert certain genes in somatic cells (table 2). this method has shown great promise in clinical trials of t cell therapy for cancer treatment [21]. other types of class 2 crispr systems are also demonstrating the potential for genetic manipulation table 1. the most widely used sars-cov-2 diagnostic tools method detection time feature disadvantage rt-pcr na >2h the most common method/simple time-consuming and low sensitivity dpcr na <2h simpler and more sensitive than rt-pcr expensive and less prevalent equipment rt-lamp na 40-60 min highly sensitive, rapid, and good for poc testing susceptible to false positive elisa antibody/antigen >2h fast, easy to operate cross-reactivity with other proteins/antibodies are produced after 7 days crisprbased na 30-60 min high sensitivity and specificity/rapid and good for poc testing not widespread ct scan radiological detection <2h excellent as confirmation and checking disease progression unspecific pathogen detection/expensive equipment/heavy radiation na: nucleic acid; ct; chest tomography; elisa: enzyme-linked immunosorbent assay ghaffari et al. 4 and diagnosis of infectious diseases that will be discussed later. 4. crispr-based detection methods rt-pcr is currently the most prevalent mode of virus detection, including sars-cov-2. still, other methods of virus detection are being explored because of some challenges associated with rt-pcr. difficulties of costs, availability, time, the need for trained laboratory scientists, and false negative with low viral load or emerging sars-cov-2 variants are some instances of rt-pcr shortcomings [22]. regardless of its recent introduction as a gene-editing platform, the novel crispr/cas system is capable of detecting virus-containing samples. the platform has been used for the detection of viruses, such as the dengue virus (denv) and zika virus (zikv). crispr platform's potentials in virology were first introduced in 2018 by myhrvold et al. reporting the capacity of the platform to detect 1 copy of the virus in samples of zikv and denv [23]. this prompted the scientific community to manipulate different aspects of the platform to develop approaches for virus detection and eradication. various crispr-based diagnostic (crispr-dx) systems can be used as a stand-alone platform or combined with other established methods to provide a rapid and precise sars-cov-2 detection system. 5. crispr/cas12-based sars-cov-2 detection a novel technique called sars-cov-2 dna endonuclease targeted crispr trans reporter (detectr), and it was recently reported to detect sars-cov-2 rna in samples as low as 10 virus copies/µl in less than 40 minutes. the technique utilizes crispr/cas12a in tandem with rt-lamp [24]. the rt-lamp performs the reverse transcription of the virus rna into cdna. then, the crrna of crispr which is designated to recognize certain virus sequences binds to target dna allowing for cas12a to cleave the single-stranded target dna (ssdna). cas12a subsequently cleaves the reporter molecule, and the resultant fluorescent signal is captured via lateral flow strips to indicate the presence of the virus (figure 1). ding et al. reported the development of the all-in-one dual crispr/cas12a (aiod-crispr) system for rapid, ultrasensitive, visualized detection of sars-cov-2 and hiv viruses [25]. aiod-crispr uses two crrnas for a more specific dna recognition, and all the required components for na amplification are mixed into a single reaction. based on the color change in the reaction mix, the results can be interpreted with the naked eye. crispr/cas12a-based assay with a naked eye readout (crispr/cas12a-ner) is another similar model of rapid sars-cov-2 detection. the system can detect 10 copies/µl in a sample under 40 minutes and uses crrna as a recognition tool and ssdna as a reporter. in the presence of the virus, cas12a also cleaves the reporter molecule which emits a green fluorescent color in 458 nm and it is conceivable by the naked eye [26]. further increases in the sensitivity of crispr/cas12 systems, huang and colleagues developed the crispr-fluorescence detection system (crispr-fds) method that detects viral genome of two copies in a sample in under 50 minutes after the amplicons are produced using rt-pcr or rpa. although crispr-fds is unable to quantitatively determine virus titers, the system is much more sensitive than the conventional rt-pcr [27]. by coupling rt-lamp with crispr/cas12, another team created iscan (in vitro specific crispr-based assay for nucleic acids detection) to detect sarscov-2 infection in 60 minutes. the system is aimed at low-resource areas and it has claimed that the reagents can be produced locally [28]. cas12b-mediated dna t a b le 2 . d if fe re n t c la ss e s a n d t y p e s o f c a s p ro te in s c la ss t y p e n u m b e r o f su b ty p e s e x a m p le o f c a s t a r g e t n a 1 i 7 c a s3 d n a ii i 4 c a s1 0 d n a /r n a iv 1 c a s7 d n a 2 ii 3 c a s9 d n a v 3 c a s1 2 d n a v i 3 c a s1 3 r n a ghaffari et al. 5 detection (cdetection) uses cas12b for dna cleavage and it is reported to be more sensitive than detectr (cas12a) and sherlock (cas13) [29]. other crispr systems of this category include one hour low-cost multipurpose highly efficient system (holmes), crispr-assisted detection (casdetec), various nucleotide guard (vanguard), (stopcovid), isotachophoresis crispr (itp-crispr) [30]. one important aspect of any diagnostic method is its ability to be translated into clinical settings. dtectr system, for instance, has shown such potential by being a convenient, rapid, and sensitive alternative to the traditional rt-pcr. a study on 378 covid-19 patients compared the sensitivity of rtpcr and dtectr and found both to be equally sensitive which is a positive point for dtectr [31]. another important aspect of the crispr-based method illustrated in this study is the capacity of the system to detect multiple sars-cov-2 genes, such as n or orf1. hence, if one gene is mutated and undetectable, the presence of the other gene will correctly diagnose the sample as positive. to further decrease the test time (to 30 min) and increase the automation, microfluidic systems are being developed to automatically extract the rna of sars-cov-2 samples, and control and accelerate the process using electric field gradient [32]. 6. crispr/cas13-based sars-cov-2 detection scrispr/cas13 like crispr/cas12 systems depends on collateral cleavage, meaning that after the protein binds to its designated na sequence, it starts cleaving its surrounding single-stranded na indiscriminately. thus, the probes which consist of quencher and fluorophore are cut, and a fluorescence signal is emitted because of the distance between them. specific high-sensitivity enzymatic reporter unlocking (sherlock) is an ultrasensitive system of detecting sars-cov-2 that similarly to detectr depends on the rt-rpa for reverse transcription and amplification of viral rna into dsdna. then, after t7 transcription into single-stranded rna (ssrna), crispr/cas13a enzymology is responsible for the cleavage of the rna. to minimize the need for extraction equipment for na on-site, one team added heating unextracted diagnostic samples to obliterate nucleases (hudson) to the sherlock system. the system uses heat and chemical reduction to lyse viral particles and destroy interfering ribonucleases found in bodily fluids, allowing a field-deployable virus detection method [23]. however, the system is weighed down due to the need for multiple reagent mixtures. shine (sherlock and hudson integration to navigate epidemics) combines the two previous systems to detect sars-cov-2 in samples figure 1. a schematic of the protocols for sars-cov-2 detection using crispr/cas. the viral rna is first extracted, purified, and amplified into dsdna by rt-rpa or rt-lamp. for sherlock, the dna is first transcribed to ssrna followed by recognition of crispr/cas13a and cleavage of reporters. in the case of detectr, the amplified dna is recognized by crispr/cas1, and the cleavage of reporters produces signals. the emitted signals are picked up and visualized either by lateral flow readouts or fluorescence devices (figure illustrated with the help of https://smart.servier.com/). ghaffari et al. 6 without the need for na extraction. the device was reported to have a specificity of 100% and sensitivity of 90% in 50 patients which is much higher than rt-pcr [33]. the results of the 50 nasopharyngeal samples were visualized by either an in-tube fluorescent or a paper-based colorimetric readout. crest (cas13based, rugged, equitable, scalable testing) was developed to further increase the scale and availability of crispr-based methods. the system uses accessible reagents and enzymes, cheap thermal cyclers, and easy-to-operate fluorescent detectors. the device is highly sensitive, and while it takes approximately 2 hours to deliver results, it requires no ac power to operate [34]. it was briefly mentioned that mutations in viruses are not only pivotal in their pathogenesis and escape from the immune system but also in their diagnosis. viruses are well known to have various mutations due to faulty na replication. although most mutations are neutral or deleterious and result in virus death, a few table 3. the most notable crispr/cas-based sars-cov-2 detection platforms crispr system cas time advantages disadvantages detectr cas12a <40 min accurate, easy to use, no thermocyclers, rapid results needs na extraction; thus, limited by the quality of extraction kits and reagents aiod-crispr cas12a 40 min rapid and highly sensitive and specific, one-pot reaction; thus, no need for product transferring and less contamination, results visible with the naked eye needs na extraction; thus, limited by the quality and access to extraction kits and reagents crispr/cas12aner cas12a <40 min rapid and highly sensitive and specific, results visible with the naked eye, no need for sophisticated equipment needs na extraction; thus, limited by the quality of extraction kits and reagents crispr-fds cas12a <50 min rapid and highly sensitive and specific, no need for sophisticated equipment needs na extraction; thus, limited by the quality of extraction kits and reagents iscan cas12a 1 h rapid and sensitive, easy-to-make reagents; thus, field-deployable and proper for large-scale detections needs na extraction; thus, limited by the quality of extraction kits and reagents vanguard cas12a 30 min rapid and sensitive, robust and affordable, can tolerate single nucleotide mutations casdetec cas12b 1 h accurate with no cross-reactivity; thus, less false positive needs na extraction; thus, limited by the quality of extraction kits and reagents itp-crispr cas12a 30 min easy to use, automated, uses little reagents stopcovid cas12b 1 h easy to use, available reagents; thus, low-cost, no need for na extraction crest cas13a 2 h sensitive, low-cost, adaptable to largescale detection settings, no need for sophisticated equipment needs na extraction; thus, limited by the quality of extraction kits and reagents shine cas13a 50 min simple, sensitive, one-step reaction sherlock cas13a <1 h sensitive, rapid, no need for sophisticated equipment feluda fncas9 1 h specific and sensitive, able to detect single nucleotide mutations conan cas3 40 min rapid, sensitive, low-cost, able to detect single nucleotide mutations needs na extraction; thus, limited by the quality of extraction kits and reagents ghaffari et al. 7 of them alter the properties of the virus, such as infectivity, host immunity interaction, and severity of the disease [35]. in the context of virus detection, if the mutations occur in important areas of the virus diagnostic test, they will negatively impact virus detection using rt-pcr or other methods in the sense that crispr is unable to bind to the target sequence and false-negative results are reported. it was recently shown that enhanced ascas12a (enascas12a) as a variant of ascas12a was able to tolerate single nucleotide variations in the target sequence and was particularly potent in differentiating sars-cov-2 from sars-cov and mers-cov [36]. this vanguard system was able to tolerate point mutations in samples. 7. miscellaneous crispr/cas-based detection systems although most crispr systems utilize cas12 and cas13 as their basis of na recognition and cleavage, there are other crispr/cas systems currently under investigation. in a preprint, a cas9-based system termed fncas9 editor linked uniform detection assay (feluda) was stated to accurately detect sars-cov2 within one hour [37]. belonging to the class 2 type ii, fncas9 is an ortholog of cas9 derived from francisella novicida (fncas9) which can cut dna. it has been reported that the protein was also highly sensitive in the case of single nucleotide mismatches [38]. it has also been reported that the feluda system was not only able to sensitively detect sars-cov-2 na but also differentiate between the target sequences of sarscov1 and sars-cov-2 that differed in only a single nucleotide [37]. crispr/cas3 is also documented to be used in sars-cov-2 detection. cas3 belongs to class 1 type i category of cas proteins and it is derived from escherichia coli and cleaves ssdna. yoshimi et al. introduced a cas3-centered sars-cov-2 detection system called cas3-operated nucleic acid detection n (conan) [39]. conan is described to be fast (40 minutes) and low-cost similar to its peers. however, cas3 capacity to cleave target na and reporters compared to cas12 (detectr) and cas13 (sherlock) was unknown until recently. yoshimi and colleagues compared the specificity of conan with detectr in 10 positive and 15 negative samples (confirmed via pcr) [39]. the detection rate of both methods was higher than 94%, and the difference was not significant. table 3 summarizes the most prominent crispr/cas-based modes of sars-cov-2 detection. 8. conclusion the outbreak of covid-19 has a caused major disruption of our way of life. specific and sensitive detection of the virus in carriers and asymptomatic patients is pivotal in breaking the transmission cycle of the virus and reducing the infection rates. the advent of gene-editing techniques has brought along the promise of revolutionizing the detection and treatments of our ailments. crispr/cas is the latest and most versatile version of gene-editing platforms, and it can be easily redirected to target a desired genetic sequence. recently, crispr/cas-based methods have been introduced as rapid, low-cost, sensitive, and specific modes of sars-cov-2 detection. different groups have utilized different cas proteins, such as cas3, cas9, cas12, and cas13 to develop a unique system of detection which are on average faster than the conventional rt-pcr with comparable sensitivity. by obviating the need for a thermal cycler machine, crispr/cas provides rapid results by making it desirable in low-income countries without the need for sophisticated equipment. on the other hand, there are some challenges associated with crispr/cas. one downfall of the crispr/cas-based methods is their inability to quantitatively measure the viral load in samples. this is crucial since the level of virus infection is a determinant of disease progression. furthermore, the limited number of targetable sequences is a hindrance for crispr/cas which allows a restricted number of grnas to be engineered. in addition, the target sequence must bear a pam sequence for the grna to complement the target sequence which is not always present. the tolerance for the number and location of mismatches is another aspect of virus detection; the more the mismatches or the closer to the pam sequence, the less the mismatches are tolerated. crispr/cas13 circumvents this by detecting the target sequence independent of pam. also, the inherent capacity of crispr/cas to detect multiple genes by using multiple grnas is another benefit of the platform. overall, crispr/cas systems have many advantages that justify them as poc detection methods of sarscov-2 that can be used in the field to lessen the burden ghaffari et al. 8 of the covid-19 pandemic by providing a rapid and reliable detection platform. acknowledgments the authors appreciate the efforts of the tehran university of medical sciences in helping with the data gathering. author contributions sg and hk wrote the first draft of the manuscript. hk and asn conceived the idea of the manuscript and gathered the data. asn and hk finalized the draft. all the authors read and approved the finalized manuscript. conflict of interests none declared. ethical declarations not applicable. financial support no funding was received for this study. references 1. chan jf, lau sk, to kk, cheng vc, woo pc, yuen ky. middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing 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https://www.biorxiv.org/content/10.1101/2020.04.07.028167v2 https://www.biorxiv.org/content/10.1101/2020.04.07.028167v2 https://pubmed.ncbi.nlm.nih.gov/31570623/ https://pubmed.ncbi.nlm.nih.gov/31570623/ https://pubmed.ncbi.nlm.nih.gov/31570623/ https://pubmed.ncbi.nlm.nih.gov/31570623/ https://www.medrxiv.org/content/10.1101/2020.06.02.20119875v1.full https://www.medrxiv.org/content/10.1101/2020.06.02.20119875v1.full https://www.medrxiv.org/content/10.1101/2020.06.02.20119875v1.full journal of current biomedical reports jcbior.com volume 2, number 4, 2021 eissn: 2717-1906 1 review fungi associated oral cavity hygiene and denture stomatitis: a review on diagnostic and treatment ali farzanegan1,*, halala khalandi2, mehraban falahati2, parisa kardouni khoozestani3 1department of microbiology, guilan university of medical science, anzali international campus, guilan, iran 2department of parasitology and mycology, iran university of medical science, tehran, iran 3department of oral and maxillofacial pathology, dental school, guilan university of medical science, anzali international campus, guilan, iran abstract oral mucosal diseases, communications of opportunistic fungi with oral bacteria, host defense, and predisposing factors can affect oral hygiene. denture stomatitis is a common inflammatory reaction with multifactorial etiology that is affected by oral hygiene. one of the most important factors that affect this condition is fungi, particularly candida albicans. accurate identification of etiologic agents of stomatitis at the species level is essential for selecting an effective drug and definitive treatment. the purpose of this article is to investigate the factors that are affected by oral hygiene with priority on the effect of candida spp. on denture wearers. keywords: candida albicans, denture stomatitis, oral hygiene 1. introduction a complex public of bacteria, viruses, fungi, and archaea, which live human bodies are very important to humans life [1]. the human microbes at various body sites are affected by nutrients, environmental exposure, and the immunological system [2]. differences in properties of microbial related to the use of different dietary regimens, and medicine exposures, such as antibiotics and corticosteroids use [3, 4]. in addition, fungi like bacteria have played major roles in human health and disease [5, 6]. culture of the fungi populating, presentation that fungi belonging to four main mycological phyla, ascomycota, basidiomycota, chytridiomycota, and zygomycota, (about 2–3%). many culture-dependent examinations on human niches, from the mouth, nails, and rectum of healthy humans have isolated yeasts, such as candida spp. [7]. most studies on oral fungi have focused on candida and the candida albicans are the most common yeasts that causing oral disease. *corresponding author: ali farzanegan, msc thirty metery blvd, anzali, guilan, iran. zip code: 4314637758 tel/fax: +98 911 982 9233 alifarzanegan1349@gmail.com https://orcid.org/0000-0002-2425-8634 received: june, 28, 2021 accepted: september, 05, 2021 oral candida infection usually occurs by predisposing factors (local or systemic host factors). decreased salivation, poor oral hygiene, wearing dentures is local factors, and diabetes mellitus, nutritional deficiency, hiv/aids are systemic factors. if do not pay attention to oral hygiene, wearing dentures increases the incidence of candida to 60-100%. denture stomatitis is a pathological symptom as inflammation and erythema of the oral mucosal zones exposed by the denture, which is typically linked with candida species, predominantly candida albicans, by reason of production biofilms on oral hole soft tissue and denture shells [8]. diagnosis of genus and species of oral pathogenic fungi is useful for definitive therapy because fungal has a wide variety of treatments. for example, nystatin and amphotericin b, fluconazole oral suspension are used for oral candidiasis and itraconazole or ketoconazole are used for candida strains resist to fluconazole. many antifungal drugs have side effects and we need new drugs and new © the author(s) 2021 https://jcbior.com/ farzanegan et al. 2 therapeutic strategies [9]. 2. factors affecting oral cavity hygiene 2.1 interactions of fungi with oral illnesses some of the oral mucosal diseases present with symptoms that are pathognomonic for the condition, whereas others oral mucosal diseases present with similar features can make identification difficult to do based on clinical examination only. these disorders are categorized based on different clinical structures, such as acute or chronic conditions, single or multiple lesions, primary or recurrent flora, and local or general disease [10, 11] (figure 1). oral mucosal diseases etiology causes as result infectious and non-infectious. oral mucosal infection diseases are less diagnosed and microbiological examinations must be done frequently. one of the most common microbiotas that reasons contamination in the oral mucosal is candida spp. although noncandidal fungal infections like aspergillosis, cryptococcosis, histoplasmosis, blastomycosis, paracoccidioidomycosis, zygomycosis (mucormycosis), oral geotrichosis, rhodotorula infection, and fusariosis are considerably less common than oral candidiasis, they can cause oral infections. more than 60% of candida spp. may habitat in the human’s oropharynx. candida can exist in dimorphism and producing toxins for invading tissue. several factors such as variations to saliva, dentures, drug, nutritional deficiencies, and immunosuppressive diseases, make an individual candida infection. the most common kinds of clinical conditions in the human oropharynx are pseudomembranous candidiasis (angular cheilitis), epithelial cell desquamation, and necrotic tissue. erythematous (atrophic) candidiasis, which is frequently related with the use of corticosteroids, broad-spectrum antibiotics, and chronic hyperplastic candidiasis are other kinds of clinical conditions [11]. 2.2 interactions of opportunistic fungi with oral bacteria a healthy oral cavity has dissimilar bacterial, viral, and fungal species. maximum are also commensal species, but they can become pathogenic in response to variations in the environment and personal hygiene. bacteria have synergistic communications on biofilms production through their secretory molecules. these secretory molecules are acylhomoserine lactones, peptide autoinducers, autoinducers-2 (ai-2), which have been identified in bacteria and fungi [12]. the peptide autoinducers are the most predominant in the oral cavity. human oral microbiome genetics are an influence on oral viruses, bacteria, and fungi. metagenomics technologies can help to identify strains causing biofilm in the oral cavity [13]. the fungi’s role in the oral cavity is mainly indefinite and interactions with bacteria can affect oral health. about 600 bacterial species and many of fungal species live the oral cavity of humans. most figure 1. infectious and noninfectious factors affecting oral cavity hygiene farzanegan et al. 3 study on oral molds has focused on candida species. study on the oral mycobiome by molecular characterizations showing the great variety of fungi inhabit in the oral cavity sequencing of 16s rrna and internal transcribed spacer 1 (its1)-based amplicon library can let rapid identification of bacteria and fungi species in oral samples [14]. oral fungus is an significant part of the oral microbiota that has received increased consideration. the variety of the fungal community in the oral cavity is significantly higher than any other body site. any difference of local mold groups in immunosuppressed people could be produced oropharyngeal fungal infections. variations in the human location are essential in developing fungal diseases and communication between fungi, bacteria and host can affect the formation of oral cavity infection [15-17]. entering ecological fungi via food intake and mouth breathing can increase the diversity of fungal in the oral cavity. among fungi, candida species, and among bacteria, the streptococcus assortment (including streptococcus gordonii, streptococcus oralis, and streptococcus sanguinis) have identified more than another microbiota [18]. signaling, metabiotic and physical adhesion molecules between those can influence fungal growth, gene expression, and pathogenicity. fungal o-mannosyl and bacterial and fungal soluble αand β-glucans cause co-aggregation interactions [19, 20]. this interaction also augmented the harshness and incidence of oral candidiasis lesions and mucosal inflammatory responses, resulting in amplification of toll-like receptor 2 (tlr2) and neutrophilic response [21]. 2.3 the commonest fungi in oral infections among the mycobiota in the healthy human oral cavity, 74 cultivable and 11 no cultivable fungal genera are identification, which comprises candida species (c. albicans, c. parapsilosis, c. tropicalis, c. khmerensis, and c. metapsilosis), cladosporium, aureobasidium, saccharomycetales, aspergillus, fusarium and cryptococcus [22]. aspergillus, fusarium, cryptococcus, and cladosporium, are human pathogens. the 60 nonpathogenic molds noticed in the oral rinse samples in the form of spores from the air or food. recently, malassezia spp. in the saliva of healthy detected with sequencing analysis of its1 amplicons [23]. conventional and molecular methods identified 144 isolates of candida species. among the c. albicans complex, neither c. stellatoidea nor c. africana was identified by hwp1 gene amplification. subsequently, other isolates were identified and confirmed with maldi-tof at the species level [24]. geotrichum spp. is a commensal fungus in humans and no studies have reported that this fungus is the normal oral. this fungus has affected the respiratory tract (bronchi, lungs), superficial skin infections, invasive infection in immunosuppressed patients, and cause oral geotrichosis associated with diabetes mellitus [25]. mucormycosis is an invasive fungal infection that can develop in immunocompromised patients. it is caused by periorbital infection and meningoencephalitis in patients with uncontrolled diabetes [26]. paracoccidioidomycosis (pcm) is one of the most frequent systemic fungal infections that cause oral lesions and may affect the gingiva, buccal mucosa, hard palate, lips, and tongue [27]. 2.4 host defense against oral fungal infection the antifungal intrinsic immune cells are mainly phagocytes, including neutrophils and macrophages, and epithelial and endothelial cells, which eliminate mycobiota through phagocytosis and direct pathogen killing [28, 29]. neutrophils, migrating from the blood vessels to the affected tissue, are the first cells that killing mycobiota by the oxidative burst and proteases [27]. therefore, the oral epithelium is able to secrete a variation of protection effector molecules. in early and later life, mucosal and cutaneous fungal infections are increasing worldwide, especially with oral candidiasis. tlrs are receptors that recognize conserved pathogen-associated molecular patterns (pamps), like bacterial, viral, or fungal antigens. c-type lectins are other receptors (clrs) that recognize carbohydrates found in the fungal cell wall, including mannose (the mannose receptor-mr), β-glucan (dectin-1), mannans (dectin-2, -3), and chitin (receptor unknown). most tlrs communicated in oral epithelial cells, healthy epithelial tissue, and oral mucosa from patients with oral candidiasis [28]. saliva in the oral cavity is immune modulators host defense against oral fungal infections. secretory immunoglobulin a (siga) is an abundant antibody produced by salivary glands and secreted in saliva, and it has multiple roles in mucosal immunity. salivary histatin 5 (hst 5) is the main antifungal protein synthesized by human salivary glands. farzanegan et al. 4 another antifungal protein in the oral cavity are ll37. it is a gene encoding for the only member of the human cathelicidin family, created by neutrophils or mucosal tissues. calprotectin is another antifungal protein, produced by mucosal keratinocytes that have synergistic activity in the presence of lactoferrin. both of these proteins decrease fungal growth by withholding important metal nutrients from c. albicans. lysozyme with the potential to candidacidal activities in saliva produces by oral neutrophil leukocytes [21]. activation of tlrs is an antiinflammatory that helps to homeostasis. il-6, il-1β, tgfβ, and il-23 cause arise in th17 cells. these cells express il17 (il-17a) in addition il-17f, il-21 il-22 and gm-csf [30]. dendritic cells (dcs) can uptake fungi and stimulates the diversity of innocent t cells into effector th-cell subtypes like th17 [7]. c. albicans express primes th17 cells that produce il-17 and ifnγ, but not il-10. antimicrobial peptide (amp) production in the oral cavity can help to fungi elimination. il-17 is very important in reaction to candida at mucosal surfaces. this mediator protective neutrophil influx and amps via collaboration to control overgrowth and switching of candida to invasive form [30]. 3. predisposing factors for fungal oral infection 3.1 systemic host factors one of the main risk factors for mycotic infections that reason morbidity or mortality among hemodialysis and post-transplant patients is diabetes mellitus (endocrine disorders). a high level of glycemic can lessen salivary flow and ph, increase salivary glucose level, and facilitate oral candidiasis like middle glossitis, atrophic glossitis, denture stomatitis, and angular cheilitis. non albicans species like pichia, trichosporon, geotrichum can recognize in poorly controlled diabetes patients. removable prostheses and cigarette smoking in diabetic patients can cause a higher rate of fungal infections [31].oral candidiasis is associated with autoimmune disorders like sjögren’s syndrome (exocrine disorders). in this syndrome, the salivary gland has dysfunction (hyposalivation based on unstimulated and stimulated flow rates) that causes oral complications [32, 33]. autoimmune disease focused on t cells that increasing chronic fungal infection [34]. innate and adaptive t-helper cells are essential to the protection and prevent the expansion of fungal infection in the human body. a mutation in the autoimmune regulator (aire) gene in the thymus, a transcriptional factor, regulates the expression of cytokines and chemokines in medullary thymic epithelial cells (mtecs), that essential for selecting proper t-cell (helper (cd4) and cytotoxic (cd8) t cells). lack of mtecs permits the autoreactive t cells, stimulates tissue injury, and make systemic inflammation [17]. when the host immune system agonizes, like immunodeficiency, c. albicans can become invasive and involving the oral cavity and other locations in the body [35]. over 60% of hiv-ill patients and more than 80% of patients detected with aids, infected by candida spp. among the disease in which candidiasis occurs can be mention immunodeficiency syndrome, digeorge syndrome, hereditary myeloperoxidase deficiency, and chediakhigashi [36-38]. one of the predisposing factors in elderly people that cases of oral infection is kidney’s disease that occurs by treatments with antibiotics and sulphonamides. alcoholism, high caffeine use, chronic hepatitis c virus, hiv infection, diabetes mellitus, and other metabolic illnesses can reason xerostomia that reduces the salivary flow in elderly patients. these disorders are predisposing factors to the virulence of the candida species and affect oral hygiene and denture wearing [39, 40]. cytotoxic chemotherapy and radiotherapy in solid organ or hematological malignancies are the leading cause of 30 to 94% oral candidiasis. prophylaxis with antifungal medicines is very important to prevent oral mycosis. however, nystatin appears ineffective in these patients [41]. iron and folate, vitamin c, b12, and a deficiencies, cause to reduced host defenses, and on the other hand, carbohydrate-rich diets are a risk factor that to increases the adherence of candida species to epithelial cells [42]. broad-spectrum antibiotics, immunomodulatory and xerogenic drugs, and drugs cause xerostomia (dry mouth) side effects, like corticosteroids, antidepressants, antipsychotics, anticholinergics, antihypertensives, and antiadrenergic can predispose to oral candidiasis [43]. cell-mediated immunity, which is defense against candida infection, declines with advancing age. oral environmental factors and age are associated with microbes in the saliva because aging makes a proliferation in the concentration of microorganisms in saliva [44]. farzanegan et al. 5 3.2 local host factors mechanical trauma like a unwell fitting denture and saliva increases the risk of tissue diffusion and colonization by candida and candida infection is responsible for the pathogenesis of the denture stomatitis [45]. saliva is very important in innate immunity because including lysozyme, hystatin, lactoferrin, calprotectin, and iga. on the other hand, salivary protein as the mucinous and the catherine is like adhesion receptors that detected the mannoproteins present in the candida species. saliva disorder with xerostomia induces the variation of the normal microbial and level of ph [39]. the flow of saliva eliminates the carbohydrates in the food that help the biofilm creation of candida yeasts on denture acrylic materials and neutralized ph levels that are decreased by bacteria, via the saliva buffering activity thus wasted nourishing source to the candida spp. denture age is an important factor for the accumulation of colonization candida in acrylic materials. only 25% of dentures for less than a year were detected with denture stomatitis and over 84% more than five years had the disease [39, 46]. stimulants in the denture that playing an important role in causing allergic with continuous denture wearing at night are two other predisposing factors that cause disease in the oral cavity [38]. 3.3 other factors several studies have been shown tobacco smoking, alone and with other systemic or local factors, can increase oral c. albicans and streptococcus mutans adhesion that resulting biofilm in the denture. studies have established that the h antigen in the blood group is a receptor for candida albicans. thus, people with blood group o that have more h antigen on their cell surfaces may be at higher risk of increasing oral candidiasis. moreover, candidiasis is associated with gender and has been reported higher in women compared to men, and this connection would be increasing during pregnancy [41, 47]. 4. diagnostic of oral fungal infection the identification of oral candidiasis is based on clinical diagnosis with a medical history. oral fungal infection presents in one of two forms: erythematous or white. erythematous is characterized by lesions that are red, including acute atrophic candidiasis, chronic atrophic candidiasis, median glossitis, angular cheilitis, and linear gingival erythema. white is characterized by lesions that are white, including pseudomembranous candidiasis and hyperplastic candidiasis [48]. laboratory tests of clinical specimens include: direct microscopy test (using 10% potassium hydr oxide (koh)), rapid staining (giemsa or gram staining), gomori-grocott methenamine silver (gms) staining (the main staining for tissue samples in mycology), culture on the selective medium (especially sabouraud dextrose agar (sc) containing chloramphenicol or gentamycin and sabouraud dextrose agar containing chloramphenicol and cycloheximide (scc)), the germ tube test, the enzyme techniques (chromogenic media), the immunological diagnostic techniques and the collection of saliva and incisional biopsy [49]. the genetic methods offer high sensitivity and specificity, and allow identification of the pathogen without having to perform cultures. additionally, samples from patients getting antifungal treatment can be used. however, these methods are more expensive and are not available in most hospital centers. molecular methods include: polymerase chain reaction (pcr), nucleic acid sequence-based amplification (nasba), amplified fragment length polymorphism (aflp), randomly amplified polymorphic dna (rapd) analysis, and malditof ms-based. these identifications may be useful for definitive antifungal therapy [8, 24]. according to the american academy of oral and maxillofacial pathology, any atypical tissue must be biopsy for a microscopic examination that is the gold standard for the diagnosis of most lesions. any lesion from the oral cavity, if continuing for more than 2 weeks should be sent for the histopathological exam. oropharyngeal candidiasis like acute pseudomembranous, acute atrophic, chronic atrophic, chronic hyperplastic, and angular cheilitis need used to swabs and oral rinse samples for smear preparation and culture for diagnosing candidiasis [50]. for chronic hyperplastic candidiasis (chc), histopathologic examination shows parakeratosis, neutrophilic micro-abscesses, a thickened spinous layer, and chronic inflammation of the underlying connective tissue associated with longstanding candida infection of the mucosa. the candida hyphae are embedding in the parakeratin into the viable cell layers of the epithelium. a biopsy of abnormal tissue is generally advisable (pas staining is https://www.sciencedirect.com/topics/medicine-and-dentistry/glossitis https://www.sciencedirect.com/topics/medicine-and-dentistry/angular-cheilitis https://www.sciencedirect.com/topics/medicine-and-dentistry/angular-cheilitis https://www.sciencedirect.com/topics/medicine-and-dentistry/linear-gingival-erythema https://www.sciencedirect.com/topics/medicine-and-dentistry/candidiasis farzanegan et al. 6 used). about 35% of chc cases show a mainly "mucosal" reaction pattern, 25% show features of squamous epithelial dysplasia, mild or moderate grade, and in some cases, dyskeratosis affecting all the keratinocyte cell layers. re-biopsy after antifungal therapy is suitable because this is possible that the candida infection is superimposed on an innate dysplastic lesion [51]. 5. prevention and treatment the treatment of candidiasis is difficult due to the recurrence, the limitation of antifungal drugs, and their side effects. the common antifungal mediators for the management of oral candidiasis are topical (nystatin, amphotericins, miconazole, clotrimazole) and systemic (ketoconazole, fluconazole, itraconazole) management [8, 52]. posaconazole, ravuconazole, and echinocandins such as caspofungin, micafungin, and anidulafungin are newer antifungals in the prophylaxis [53]. the patient’s therapeutic history, oral signs, and laboratory testing of clinical specimens include direct microscopy tests, culture and biopsy can help to the diagnosis of oral microbiota and drug choice [41]. identification of species is essential for rapid treatment [45]. nystatin is the primary typical treatment for denture stomatitis [43, 54], but ineffective for candidal lesions in cancer patients [53].for the treatment of fungal angular cheilitis (with candida priority) must be used to nystatin cream, or miconazole cream (2%), or clotrimazole cream (1%) [55]. if these treatments are unsuccessful, systemic antifungal agents should be used. on the other hand, the prolonged or frequent use of antifungal medicines is a major problem for the development of resistant species. moreover, therapeutic drug levels are important, especially in patients with reduced saliva production [56]. regardless expend the systemic antifungal medicine; the use of a contaminated denture can be recurrence oral candidiasis [8, 44, 57]. denture base material, age of denture, age of patients, gender, and dietary factors (high carbohydrate diet, vegetarian diet consisting of vegetables, fruits, cereals, and pulses) are very important for denture hygiene. one of the important methods for denture hygiene is chemical (antimicrobial cleansing agent) and mechanical (brushing, sonic vibrators, and ultrasonic) methods. the use of toothbrushing, mouthwashes, and cleaning tablets for acrylic denture overnight storage can be alone effective in reduces denture biofilm mass and treating denture stomatitis [39, 42, 58]. use of precoating biomaterials (silicone rubber or denture acrylic) with chemicals resources (silanes, chlorhexidine, histatins, metal nanoparticles (tio2, fe2o3, agnps)) is the strategy to decrease the adhesion and constrain the growth of candida [58, 59]. alkaline peroxides, alkaline hypochlorite acids, and chlorhexidine gluconate are disinfectant agents, that have inhabited bacteria, viruses, and fungi infection and denture biofilm formation [52], but used the combination with nystatin, reduced efficacy of nystatin [39, 58]. sodium hypochlorite (naocl) is a gold standard solution but not be used always. citric acid, immediately after treatments, can be active in decreasing c. albicans cell viability in a biofilm [60, 61]. however, these disinfectant agents do not completely take out the biofilm and do not prevent its formation after 48h [62]. another technique that can be used not only to sterilize but also for the cure of denture stomatitis is microwave irradiation that demonstrated to be safe, simple, easy to use, effective, and low-cost [46]. this method does not induce resistance for fungi and it seems not to alter the color or smell of the dentures [63] and prevents the unwanted effects due to antifungal drugs, such as nausea, vomiting, and hepatotoxic and nephrotoxic effects [46]. this method is effective against microorganisms via 650 w of microwave irradiation for 3 minutes [42, 63]. essential oils of plants are a new trend in treatment. eugenol, linalool, menthol, farnesol, menthone, geraniol, terpine-4-ol, αterpineol, and tyrosol, carvacrol have durable antifungal activity in vitro [55]. moreover, garlic has biological effects with a deficiency of side effects so it could be a replacement for nystatin in oral candidiasis [8, 43]. probiotic bacteria such as lactobacillus rhamnosus gg, lactobacillus rhamnosus lc705, propionibacterium freudenreichii, and shermanii js can reduce the growth of pathogenic microbes like candida spp. in the oral cavity [53]. one of the preventive treatments for denture stomatitis is combination antifungals (nystatin, amphotericin, fluconazole, clotrimazole) with thin-film polymer materials that could be inhibiting c. albicans biofilm growth in denture materials [56]. furthermore, conjugation nanoparticles (like silver-zinc zeolite) with thin-film polymer materials exhibited that the farzanegan et al. 7 biofilm formation will be lesser [59]. 6. conclusions denture stomatitis is the inflammatory response that is generally seen in oral cavity hygiene and dental studies. systemic and local host factors are predisposing factors for fungi infections. c. albicans is the most common microbe species capable of producing infection at several anatomical locations and its diagnosis is significant to the choice of antifungal therapy. direct microscopy tests and culture with metagenomics technologies are the best methods that can help to identify strains causing biofilm in the oral cavity. on the other, the limitation of antifungal medicines, and their side effects are the most common problems for the treatment of candidiasis. use of essential oils and probiotic bacteria in the oral cavity, and use of combination antifungals or nanoparticles with thin-film polymer materials could be inhibited c. albicans biofilm growth on denture materials that recommended. author contributions all authors contributed equally to this manuscript and approved the final version of the manuscript. conflict of interests the authors declare that they have no conflicts of interest. ethical declarations not applicable. financial support none to 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https://pubmed.ncbi.nlm.nih.gov/24957210/ https://pubmed.ncbi.nlm.nih.gov/24957210/ https://pubmed.ncbi.nlm.nih.gov/24957210/ https://pubmed.ncbi.nlm.nih.gov/24957210/ https://pubmed.ncbi.nlm.nih.gov/24957210/ https://pubmed.ncbi.nlm.nih.gov/21083738/ https://pubmed.ncbi.nlm.nih.gov/21083738/ https://pubmed.ncbi.nlm.nih.gov/21083738/ journal of current biomedical reports jcbior.com volume 2, number 3, 2021 eissn: 2717-1906 1 research article investigating the prevalence of antibiotics prescription in the toxicology ward of a referral hospital in the north of iran hamid mohammadi kojidi1, tofigh yaghubi kalurazi2, morteza rahbar taramsari1, fatemeh daneshsefatdoost1, alireza badsar1, paria okhovat2, mirsaeed attarchi1,2,* 1department of forensic medicine, school of medicine, guilan university of medical sciences, rasht, iran 2razi clinical research development unit, razi hospital, guilan university of medical sciences, rasht, iran abstract antibiotic resistance of microorganisms is a serious problem in medicine. one of the most common reasons for this issue is the improper prescription of antibiotics by physicians. the purpose of the study was to investigate the prevalence of using antibiotics in the toxicology ward of razi hospital. in this retrospective descriptive cross-sectional study, 338 patients admitted to the toxicology ward of razi hospital were enrolled from march 2018 until march 2019. a checklist of investigated factors including age, gender, fever, paraclinical findings, type of poisoning, and type of antibiotic prescribed was completed for all patients. about 52.1% of the participants were males and 47.9% were females. the mean age of participants was 37.07 ± 17.36 years. antibiotics had been prescribed for 49 patients. the most common form of poisoning was benzodiazepines. also, ceftriaxone was the most common antibiotic prescribed to the patients. fever existed in 69.4% of the patients receiving antibiotics. the most common paraclinical finding was abnormal complete blood count (cbc) in patients (leukocytosis) which was seen in 83.7% of them. the antibiotics mostly had been prescribed in the first two days of hospitalization. in the present study, antibiotics were prescribed in the toxicology ward for only 14.5% of the poisoned patients. considering that ceftriaxone has been the most commonly prescribed antibiotic, its use has to be based on clinical evidence based on the necessary indications according to scientifically substantiated sources. keywords: drug use, antibiotics, toxicology ward 1. introduction the introduction and use of antibiotics have led to a great revolution in the treatment of infectious diseases such that the epidemiological pattern of diseases changed from infectious to non-infectious and accidents [1, 2]. after analgesics, antibiotics are the second most widely used drugs and their use has shown an upward trend over the years [3, 4]. concerns about using antibiotics became known shortly after their introduction to the medical world [5]. using new antibiotics in the following decades was accompanied *corresponding author: mirsaeed attarchi, md department of forensic medicine, school of medicine, guilan university of medical sciences, rasht, iran tel/fax: +98 13 33690006 email: : msattarchi@yahoo.com https://orcid.org/0000-0002-7730-688x received: may, 07, 2021 accepted: july, 19, 2021 by an irrational increase in their use, leading to the emergence of nosocomial infections and emerging infections [6, 7]. antibiotic resistance in microorganisms is one of the serious problems in infectious medicine, the main reason is the improper prescription of antibiotics by physicians. the research has shown that at least onethird of the hospitalized patients receive a course of antibiotics, and almost half of these antibiotics are prescribed unnecessarily or incorrectly [8, 9]. even though antibiotics are required in most bacterial © the author(s) 2021 https://jcbior.com/ mohammadi kojidi et al. 2 infections and do not pose a threat to the patient’s life, most studies have shown that 30 to 60% of cases are inappropriate [10]. the global cost of antibiotics in 2005 was estimated at $40 billion, of which developing countries accounted for about one-third [11]. according to a world health organization (who) report based on data collected from 65 countries in 2015, amoxicillin and amoxicillin-clavulanate were the most widely used antibiotics [12]. as there are few studies on the pattern of antibiotic administration in iran, the present study was conducted to investigate the frequency of antibiotic prescribing in the toxicology ward of a referral hospital in the north of iran. 2. materials and methods 2.1 design and the population the population of this study included the patients admitted to the toxicology ward of razi hospital in 2018. in this cross-sectional descriptive study, the files of 338 patients in the toxicology ward of razi hospital were selected using regular systematic random sampling. then, they were investigated over one year from april 2018 to march 2019 and the number of patients being prescribed at least one antibiotic during this time was recorded. razi hospital in rasht is the main referral center for patients suffering from toxication, in gilan province, north of iran. for each patient, demographic information and the type of antibiotic prescribed were collected from the file. in this study, fever was reported to be above 37.2°c in the morning and above 37.7°c in the evening by the oral method. abnormal complete blood count (cbc) was considered leukocytosis (wbc> 11000) in the study. the study design was approved by the regional ethic committee of guilan university of medical sciences with the approval code of ir.gums.rec.1399.051. information was collected and recorded from patients’ files anonymously and in complete confidentiality. the researchers of the project adhered to the code of ethics in all stages of the study. 2.2 statistical analysis the collected data were entered into spss ver.22 and were analyzed. the results were presented as frequency and percentage. chi–square or fisher's exact tests was used to estimate any statistical association for quantitative variables, and paired ttests were used to compare means. a p <0.05 was regarded as significant relevance. 3. results in this study, the files of 338 patients were examined from april 2018 until march 2019. among these patients, 176 patients (52.1%) were males and 162 patients (47.9%) were females. the mean age of the entire patient was 37.07 ± 17.36 years. the mean age of the patients prescribed antibiotics (44.83 ± 18.97 years) was significantly higher than patients not prescribed antibiotics (35.93 ± 16.73 years) (p < 0.05). moreover, 170 (50.3%) patients were in the age group 20-40 years, 67 (19.8%) and 55 (16.3%) in 40-60 years and less than 20 years, and 46 (13.6%) were in the group over 60 years. of the 338 patients, antibiotics were prescribed for 49 patients (14.5%). table 1 presents the distribution of the frequency of poisoning agents and the frequency of antibiotic administration according to the toxicology ward. as can be seen from the table, the highest frequency of poisoning is related to benzodiazepines. the highest frequency of antibiotic use was in multidrug poisoning (24.5%), followed by methadone (22.4%). of the 49 patients to whom antibiotics were prescribed, 63.3% (31 people) were males and 36.7% (18 people) were females. overall, the frequency of antibiotics was higher for men compared to women so that 17.6% of male patients and 11.1% of female patients were prescribed antibiotics. as specified in table 2, the highest prevalence of antibiotics was in people over 60 years of age so that 46 of the poisoned people were over 60 years of age. table 2 shows the frequency of antibiotic prescribing by age group and hospitalization days. of the 49 patients to whom antibiotics were prescribed, 21 were in the age group of 20-40 years and the highest frequency of antibiotic administration was with 21 cases on the first day of hospitalization (table 2). table 3 presents the frequency distribution of different types of prescribed antibiotics. as can be seen, ceftriaxone was the most commonly used antibiotic with 41 cases. moreover, it is the most common antibiotic used in both genders and all age groups. table 4 presents the clinical and paraclinical findings regarding the cases in which antibiotics were prescribed. abnormal fever and whole blood test (leukocytosis) were seen in 69.4% and 57.1% of patients receiving antibiotics, respectively. in 9 cases mohammadi kojidi et al. 3 for whom antibiotics were prescribed, fever and abnormal paraclinical findings did not exist and antibiotics were used based on the patient’s complaints and symptoms or for prophylaxis. 4. discussion irrational use of antibiotics in hospitalized patients is a critical issue in medical and environmental studies. unnecessary use of antibiotics, inadequate monitoring, inappropriate dosing, and administration of multiple antibiotics are examples of cases of inappropriate use of antibiotics in hospital settings. these cases can increase the prevalence of antibiotic-resistant strains and lead to prolonged hospitalization, side effects, and loss of medical budget [13-15]. in this cross-sectional study, the prevalence of antibiotics in patients was investigated by studying the files of patients admitted to the toxicology ward of an educational and medical center. in the current study, of 338 patients investigated, the highest number of poisoned patients was in the age range of 20-40 years. the most common type of poisoning was benzodiazepines. overall, antibiotics were prescribed in 14.5% of patients admitted to the toxicology ward, which is less common compared to similar internal and external studies performed in other hospital wards. the history of chronic disease has a key role in the physician’s decision for prescribing antibiotics and most of the patients hospitalized in the toxicology ward were young and in the age range 20-40 years with no underlying disease. therefore, this factor can be considered as the hypothesis of the reasons for the table 1. the frequency of poisoning agent and antibiotic prescription according to poisoning factor cause of poisoning poisoning antibiotic prescription frequency percentage frequency percentage benzodiazepine 74 21.9 5 10.2 aluminum phosphide 28 8.3 2 4.1 methadone 29 8.6 11 22.4 metformin and glibenclamide 3 0.9 0 0 opium 23 6.8 4 8.2 snake and spider bites 5 1.5 3 6.1 rat poison 3 0.9 0 0 alcohol 17 5 0 0 cannabis and amphetamines 5 1.5 1 2 tramadol 16 4.7 2 4.1 propranolol 1 0.3 0 0 acetaminophen and nsaids 12 3.6 0 0 hydrogen chloride 2 0.6 0 0 diesel and hydrocarbons 2 0.6 1 2 expectorant and rasperidone 3 0.9 1 2 carbon monoxide 2 0.6 1 2 pepsinogen inhibitor 1 0.3 0 0 barbiturates and antiepileptics 2 0.6 1 2 mushrooms 6 1.8 2 4.1 lead and arsenic 2 0.6 1 2 insecticides and herbicides 19 5.6 0 0 ssri and tca 4 1.2 1 2 bleach 6 1.8 0 0 botulism 8 2.4 0 0 multi-drug 47 13.9 12 24.5 unknown 18 5.3 1 2 total 338 100 49 100 abbreviations: nsaids, nonsteroidal anti-inflammatory drugs; ssris, selective serotonin reuptake inhibitors; tca, tricyclic antidepressants mohammadi kojidi et al. 4 low prevalence of antibiotics in this ward. ceftriaxone was the most commonly prescribed antibiotic in both genders. in a study by gholami et al., 190 patients receiving antibiotics in the emergency ward of a teaching hospital in tehran were examined for the accuracy of the indication, the type of antibiotic, dose, and method of administration. in their study, ceftriaxone was the most commonly prescribed antibiotic with 72.1%, while 36.3% of the prescribed antibiotics being inappropriate, and in 15.8% of the cases, antibiotics prescription was unnecessary. overall, considering mistakes in the diagnosis, indication, type of antibiotic, dose, and method of drug administration, 77 mistakes were made in the antibiotics prescription [16]. ebrahimzadeh et al. examined antibiotic use patterns in several hospital wards in sari from 2000 to 2005 using atc/ddd method. the use of antibiotics from april 2000 for six months was 95.4 units and from april 2005 for six months was 124 units. this was obtained by dividing the specified daily value by the number of occupied beds, multiplied by the number of days, multiplied by 100 (ddd/100bed-day). cefazolin was the most widely used drug of all time. intensive care unit (icu) and obstetrics wards had the highest dose of antibiotics from march to august 2005. over time, the largest increases were related to vancomycin and clindamycin [17]. in a study by robert et al., the spot prevalence of antibiotic use was evaluated in a volunteer sample from french hospitals. among 3,964 patients in 38 hospitals, 343 (8.7%) patients used antimicrobial prophylaxis and 1276 (32.2%) antimicrobial therapy. among patients receiving antimicrobial therapy, 959 (75.2%) patients received beta-lactam, 34.8% penicillin beta-lactam inhibitor, 22.1% third-generation cephalosporins, and 7.8% carbapenems. in aggregate, 518 (40.6%) patients received more than one antibiotic [18]. in the present study, the most prevalent paraclinical result in patients receiving antibiotics was leukocytosis. blood culture was carried out in only 2 cases that were reported negatively. fever existed in 69.4%. of the 49 patients receiving antibiotics, 9 had neither abnormal paraclinical findings nor fever, and antibiotics were administered just according to the patient’s symptoms and complaints or for prophylaxis. in our study, in line with other studies from iran, cephalosporins were the most commonly used antibiotic. out of the 49 patients to whom antibiotics were prescribed, 83.7% were prescribed antibiotics in the first two days of hospitalization. therefore, antibiotics are unlikely in most cases due to table 2. frequency of antibiotic prescription by the time of administration and age group variable frequency percentage antibiotic time (day of hospitalization) first 21 42.9 second 20 40.8 third 6 12.2 fourth 0 0 fifth 2 4.1 age groups (years) under 20 4 8.2 20 to 40 21 42.8 41 to 60 14 28.6 over 60 10 20.4 total 49 100 table 3. the frequency of various types of prescribed antibiotics types of antibiotic frequency percentage ceftriaxone 41 12.1 clindamycin 17 5 meropenem 5 1.5 vancomycin 5 1.5 cefazolin 3 0.9 metronidazole 2 0.6 levofloxacin 2 0.6 penicillin 2 0.6 ciprofloxacin 2 0.6 imipenem 1 0.3 cephalexin 1 0.3 piperacillin 1 0.3 azithromycin 1 0.3 ceftazidime 1 0.3 table 4. frequency of abnormal clinical and paraclinical finding in patients receiving antibiotics abnormal finding frequency percentage complete blood test 28 57.1 chest x-ray 11 22.4 complete urine test or urine culture 4 8.2 fever 34 69.4 mohammadi kojidi et al. 5 nosocomial infections. one of the limitations of this study was that it is retrospective, leading to some problems in obtaining information. therefore, it is recommended to conduct a more comprehensive study with a larger sample size. moreover, a prospective study with a more detailed checklist is needed to conduct in other teaching hospitals in this province. improper prescription of antibiotics can affect the whole society by creating resistant strains. moreover, one of the reasons for over-prescribing antibiotics is doctors’ lack of knowledge about the indications for prescribing antibiotics. hence, it seems that codified teaching and emphasis on how to prescribe properly these drugs can be very effective. currently, in the modern hospitals of the world, different measures like training programs, the use of antibiotic prescription forms, clinical guides of prescribing antibiotics, and direct or telephone consultation with infectious disease specialists are performed to limit the prescription of antibiotics to prevent the overprescription of antibiotics, thereby reducing the hospital costs and the emergence of resistant species [19]. in the present study, antibiotics were prescribed in the toxicology ward for only 14.5% of the poisoned patients. considering that ceftriaxone has been the most commonly prescribed antibiotic, its use has to be based on clinical evidence based on the necessary indications according to scientifically substantiated sources. author contributions conception or design of the work: hm, mr, ma; data collection: ty, fd, ab, po; data analysis and interpretation: hm, fd, po, ma; drafting and critical revision of the manuscript: hm, ty, mr, ab, ma. all authors read and approved the final version of the manuscript. conflict of interests none. ethical declarations the study design was approved by the local ethics committee of guilan university of medical sciences (approval no. ir.gums.rec.1399.051). financial support none. references 1. sepehri gr, haj akbari n, mousavi a. prescribing 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https://pubmed.ncbi.nlm.nih.gov/12766839/ mohammadi kojidi et al. 6 16. gholami a, barati m, vahdani m, vahdani h, karimi ma. pattern of empirical antibiotic administration in emergency department of an educational hospital in tehran. razi j med sci. 2011; 18(82):17-23. 17. ebrahimzadeh ma, ansari f, ramezani a, shokrzadeh m, shabankhani b, saeedi ss, et al. utilization pattern of antibiotics in different wards of sari imam khomeini teaching hospital. j mazandaran univ med sci. 2007; 17(61):166-9. 18. robert j, péan y, varon e, bru j-p, bedos j-p, bertrand x, et al. point prevalence survey of antibiotic use in french hospitals in 2009. j antimicrob chemother. 2012; 67(4):1020-6. 19. radyowijati a, haak h. improving antibiotic use in low-income countries: an overview of evidence on determinants. soc sci med. 2003; 57(4):733-44. http://rjms.iums.ac.ir/article-1-1629-en.html http://rjms.iums.ac.ir/article-1-1629-en.html http://rjms.iums.ac.ir/article-1-1629-en.html http://rjms.iums.ac.ir/article-1-1629-en.html http://jmums.mazums.ac.ir/article-1-278-en.html http://jmums.mazums.ac.ir/article-1-278-en.html http://jmums.mazums.ac.ir/article-1-278-en.html http://jmums.mazums.ac.ir/article-1-278-en.html https://pubmed.ncbi.nlm.nih.gov/22258928/ https://pubmed.ncbi.nlm.nih.gov/22258928/ https://pubmed.ncbi.nlm.nih.gov/22258928/ https://pubmed.ncbi.nlm.nih.gov/12821020/ https://pubmed.ncbi.nlm.nih.gov/12821020/ https://pubmed.ncbi.nlm.nih.gov/12821020/ journal of current biomedical reports jcbior.com volume 2, number 2, 2021 eissn: 2717-1906 1 review the role of immunotherapy in eliminating high-risk treatments for pediatric cancer zahra farhoud1,*, sepehr bozorgchenani2 1department of veterinary medicine, rasht branch, islamic azad university, rasht, iran 2department of bioinformatics, tehran branch, university of science and culture, tehran, iran abstract chemotherapy, radiation, and surgery are often ineffective in treating refractory or chronic tumors in children. recent advances in cancer immunotherapy have also improved the outcome of many human tumors, resulting in significant responses in patients who have previously refused to respond to other treatments. the immune system is made up of cells and proteins that work together to maintain immunity against pathogens. cancer cells can adapt and metastasize over time, express different neoantigens, or express more immune-suppressing mechanisms, allowing them to be identified and eradicated. in this study, we focus on immunotherapy for pediatric cancer and its progression using natural killer (nk) cells, chimeric antigen receptor t cells (car-t), and oncolytic virus cells in these patients. for all intents and purposes, toxicity is a concern. although immunotherapy is believed to have fewer long-term side effects than chemotherapy and radiation therapy, they may have a high rate of short-term side effects, depending on the cause and purpose. these complications can lead to lifethreatening illnesses, from mild illnesses such as fever and headaches to more severe illnesses such as autoimmune, neurotoxicity, and opportunistic infections. any of the problems listed, for example, cytokine release syndrome, can be fatal. different intensities require multiple tests and trials. keywords: children cancer, natural killer cells, car-t 1. introduction chemotherapy, radiation, and surgery are often ineffective in treating resistant or chronic tumors in children. for many cancer patients, immunotherapies have been a promising treatment choice [1]. despite advances in chemotherapy and radiotherapy that have increased average survival rates for children with cancer in recent decades, childhood cancer remains the leading risk factor for mortality under the age of 20 [2]. recent advances in cancer immunotherapy have improved the outcome of several human cancers and in some cases led to promising responses in patients * corresponding author: zahra farhoud, bsc department of veterinary medicine, rasht branch, islamic azad university, rasht, iran tel/fax: +98 937 7811335 email: z.farhoudkhomeirani@gmail.com https://orcid.org/0000-0001-6598-2832 received: may, 13, 2021 accepted: may, 27, 2021 who were previously unable to respond to other types of care [3-6]. immunotherapy has been used to treat cancer since the late 1800s. wilhelm bush observed tumor regression in 1866 after a rose infection in a patient with sarcoma. shortly afterward, in 1891, streptococcus and its toxins were injected into the bloodstream by gypsies, and some patients with nonsurgical sarcoma recovered [7-10]. the immune system is a diverse and intertwined collection of cells and proteins that function together to kill pathogens while preserving resistance to autoimmunity [11]. other functions of the immune system include focusing on immune monitoring and © the author(s) 2021 https://jcbior.com/ farhoud et al. 2 penetration of immune cells and tumor cytolysis in cancer patients. burnet and thomas first described immunosurveillance in 1957, which occurs when a tumor is recognized as 'foreign' in the body [12, 13]. the evolution, metastasis, and expression of neoantigens, as well as the use of different pathways to inhibit the immune system, are all influential characteristics of cancer cells. as a result, they are resistant to identification and deletion. thus, the safety version should focus on 3 steps, which include deletion, balance, and escape [14]. during the removal of cancer cells, immune and innate cells detect new antigens, followed by the formation of tumor-reacting t cells and the removal of the tumor. any cancer cells that exist in the removal process make it to the equilibrium level. during the equilibrium time, the adaptive immune system keeps the tumor inactive. finally, cancer cells evolve to avoid recognition by the immune system, resulting in an escape pathway marked by tumor growth and/or t-cell exhaustion [14, 15]. in this study, we intend to examine immunotherapy methods to reduce the adverse effects of previous traditional therapies, considering the differences between pediatric and adult cancers. 2. the difference between cancer in children and adults in pediatric cancer, most embryonic cells are involved in the development of the disease, and thus the accumulation of genetic mutations and transcriptional abnormalities is thought to cause cancer in children [16]. other research on the causes of childhood cancer has shown that children with fanconi anemia are more likely to grow cancer at a median age of 16 years [17]. brca2 mutations have been identified in fanconi anemia, along with 15 other potential mutations, resulting in congenital abnormalities, bone marrow dysfunction, and an elevated risk of developing myeloid and solid malignancies [18, 19]. 3. chimeric antigen receptors t cells in pediatric cancer immunotherapy with chimeric antigen receptors t cells (car-t), first recorded in the mid-1980s, increased hope for cancer care and ignited global interest in immuno-oncology [20]. the formation of car-t cells and natural killer (nk) cells resulted from combining the anti-cancer action of immune system cells such as t and nk cells with the idea of antibody specificity to redirect these cells' cytotoxic activity to target tumor cells expressing a specific antigen [21]. tumors' ability to escape the immune system is due to the tumor's composition, which modulates the function of human leukocyte antigen (hla) molecules or tumor antigens, as previously mentioned. car is an acceptable therapy choice since it is designed to contain a specific antigen without the need for hla neoantigens of tumor genes to overcome the difficulties. a chimeric antigen receptor is composed of an extracellular domain with an antigen-binding domain derived from a monoclonal antibody specific for a tumor surface antigen, a spacer domain, a transmembrane domain, and an intracellular signaltransducing chain of the t-cell receptor [22, 23]. a specific vector could be used to diagnose most patients with cancers that express the target antigen [24]. 4. natural killer cells human nk cells are formed from multipotent cd34+ hematopoietic progenitors in the bone marrow. nk cell maturation occurs in both the bone marrow and the lymphoid glands and, unlike t cells, does not include the thymus [25-27]. nk cells are intrinsic lymphocytes that can suppress cancer. unlike t cells, nk cells are activated without any previous contact with cancer cells. the function of nk cells is determined by a combination of inhibitory activation signals by receptor-ligand interaction. activating receptors such as nkp46, nkp44, nkp30, dnam1, and nkg2d identify tumor antigens and activate nk cells. these nk cells secrete cytotoxic granules containing perforin and granzymes, which directly kill cancer cells by inducing apoptosis through various mechanisms including the fas and trail pathways. nk cell stimulation in the tumor microenvironment can also play a role in dendritic cell and t cell recruitment to the tumor site. nk cells may also be activated by monoclonal antibodies that target cd16, such as daratumumab for multiple myeloma and tall or rituximab for b cell non-hodgkin lymphoma [28, 29]. peripheral tissue, cord blood, and induced pluripotent stem cells (ipsc) from haploidentical, allogeneic, or autologous donors can all be used to separate nk cells [30]. nk cells play an important role in the transplantation of allogeneic and autologous hematopoietic stem cells. a few genes, including kir, farhoud et al. 3 nkg2d, and their ligands, regulate cytotoxicity in leukemia. these cells can invade several leukemic cell types, including acute myeloid leukemia (aml), chronic myeloid leukemia (cml), and chronic lymphocytic leukemia (cll), with little risk of acute graft against host disease [31-33]. 5. pediatric brain tumor immunotherapy the immune system finds the central nervous system (cns) to be a privileged location, in addition to complementing immune cells and signals that act on the rest of the body. the blood-brain barrier (bbb), a highly specialized bond between blood and the cns parenchyma caused by capillary endothelium, nursing cells, and astrocytes, determines this safe score [34]. pediatric brain tumors are made up of a variety of histologic subtypes that occur with varying degrees of aggressiveness in the central nervous system [35]. cns tumors are the most prevalent solid tumors in children, as well as the high incidence of cancer-related mortality [35]. although overall childhood cancer survival rates have risen significantly in recent decades, improvement in pediatric cns tumors has lagged behind that of hematological cancers [36]. william coley introduced bacteria into various tumors in the late 1800s to stimulate an immune response, and hence the idea of stimulating the immune system to combat cancer (also known as rapid immunotherapy) was born [8]. researchers have since discovered how the immune system communicates with tumor cells in the hopes of channeling these abilities to develop effective immunotherapies. antigen presentation on major histocompatibility complex (mhc) molecules, as well as antigen presentation that is not mhc-dependent, are used to activate the immune system. by digesting intracellular and extracellular proteins into small peptides, extracellular display on class i or class ii mhc molecules for mhc-dependent activation is accomplished [37].t cells use their t-cell receptor (tcr) to recognize peptides on the mhc molecule as antigens, eliciting an immune reaction [38]. peptide binding to the mhc is strongly influenced by a patient's hla genotype, which must be taken into account when developing and implementing antigenbased therapies. antigen epitopes can also stimulate the immune system in non-mhc-dependent ways, such as antibody-dependent b-cell activation [39]. 6. the role of oncolytic viruses in cancer immunotherapy another way to stimulate the immune system to fight tumors is to use oncolytic viruses, so viruses that are widely used in this field can be called herpes simplex virus, adenoviruses, measles virus, and poliovirus to attack and infect cells noted cancer. this specificity can be achieved by inserting tissue-specific promoters that ensure viral replication occurs only in target organs, or by removing specific genes that ensure viral replication occurs only in actively individual cells [40]. an oncolytic virus infection not only kills tumor cells directly but also stimulates pamps and damps, resulting in immunogenic cell death and adaptive immune responses [41]. 6. conclusion given all of the benefits, toxicity is a cause for concern. although immunotherapies are believed to have fewer long-term side effects than chemotherapy and radiation, they may have a high rate of short-term adverse effects that range in magnitude based on the agent and the target. this can vary from mild inconveniences such as fevers, headaches, and chills to more severe conditions such as myalgia, autoimmunity, neurotoxicity, and opportunistic infections; any of these incidents for example, cytokine release syndrome can be fatal. the immunosuppressive tumor microenvironment is also a recurring issue in solid cancers, since it may reduce or eliminate all immunotherapeutic gain [2]. as a result, various intensities necessitate several measurements and trials. author contributions all authors are equally involved in the preparation of this manuscript and endorse the manuscript. conflict of interests the authors declare that they have no conflicts of interest. ethical declarations not applicable. financial support none to be declared. farhoud et al. 4 references 1. wedekind mf, denton nl, chen cy, cripe tp. pediatric cancer immunotherapy: opportunities and challenges. paediatr drugs. 2018; 20(5):395-408. 2. hutzen b, paudel sn, naeimi kararoudi m, cassady ka, lee da, cripe tp. 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https://pubmed.ncbi.nlm.nih.gov/28712033/ https://pubmed.ncbi.nlm.nih.gov/28712033/ https://pubmed.ncbi.nlm.nih.gov/28712033/ https://pubmed.ncbi.nlm.nih.gov/28695111/ https://pubmed.ncbi.nlm.nih.gov/28695111/ https://pubmed.ncbi.nlm.nih.gov/28695111/ [arshid yousefi avarvand] [department of laboratory sciences, school of allied medical sciences, ahvaz jundishapur university of medical sciences, ahvaz, iran.] [arshid.yousefi5@gmail.com] dear editor i wish to submit a new manuscript entitled “[frequency and antibiotic susceptibility of streptococcus pneumonia isolated from hospitalized and outpatient's children in educational hospitals ahvaz, 2020]” for consideration by the [journal of current biomedical reports]. i confirm that this work is original and has not been published elsewhere nor is it currently under consideration for publication elsewhere. in this paper, we report on the prevalence of pneumococcus and its antimicrobial resistance. this is significant because of its high rate of morbidity and mortality in children and the unstoppable growth of resistance to a number of antibiotics. the paper should be of interest to readers in the areas of microbiology, bacteriology, pharmacology and pediatrics. please address all correspondence concerning this manuscript to me at [maryammoti75@gmail.com]. thank you for your consideration of this manuscript. sincerely, [arshid yousefi avarvand, phd of bacteriology] 1. javad fathi , department of bacteriology and virology, school of medicine, shiraz university of medical sciences, shiraz.email: javadfathi70@yahoo.com 2. mansoor khaledi, department of microbiology, faculty of medicine, shahed university, tehran, iran. email: mansoor.khaledi@yahoo.com journal of current biomedical reports jcbior.com volume 3, number 2, 2022 eissn: 2717-1906 erratum erratum: a survey of medical students' attitudes and practices towards narcotics and psychotropic drugs shahab dastmardi1, alireza kohansal vajargah1, morteza rahbar taromsari2, hamid mohammadi kojidi2, ardalan majidinia3, fariba asgari bozayeh4, kaveh ghaderi1, kourosh delpasand5,* 1student research committee, school of pharmacy, guilan university of medical sciences, rasht, iran 2department of forensic medicine and toxicology, school of medicine, guilan university of medical sciences, rasht, iran 3student research committee, school of medicine, guilan university of medical sciences, rasht, iran 4department of nursing, school of nursing and midwifery, medical education research center (merc), guilan university of medical sciences, rasht, iran 5department of medical ethics, school of medicine, guilan university of medical sciences, rasht, iran there was an error in the author list of the published article. two authors (a kohansal vajargah, k ghaderi) requested to add to the authors lists. after obtaining the agreement of the authors and the corresponding author, and describing author contributions, editor-in-chief accept the corrections as listed below. the correct author list is: shahab dastmardi, alireza kohansal vajargah, morteza rahbar taromsari, hamid mohammadi kojidi, ardalan majidinia, fariba asgari bozayeh, kaveh ghaderi, kourosh delpasand we apologize for any inconvenience this may have caused. *corresponding author: kourosh delpasand, md, ph.d department of medical ethics, school of medicine, guilan university of medical sciences, rasht, iran tel/fax: +98 13 33690006 email: kd388@yahoo.com http://orcid.org/0000-0001-6842-3374 received: november, 02, 2022 accepted: november, 19, 2022 https://jcbior.com/ https://jcbior.com/ journal of current biomedical reports jcbior.com volume 3, number 1, 2022 eissn: 2717-1906 1 brief report cost analysis of educational courses of medical students in iran shima nikjoo1,*, aziz rezapour1, torandokht baluchnejad mojarad2, reza jahangiri1, asma rashki kemak1, sajad vahedi3, hiro farabi1 1health management and economics research center, school of health management and information sciences, iran university of medical sciences, tehran, iran 2department of physiology, faculty of medicine, iran university of medical science, tehran, iran 3department of health services management, school of health, ahvaz jundishapur university of medical sciences, ahvaz, iran abstract estimating the cost of graduate courses can play an important role in deciding on the allocation of required resources. this study aimed to determine the cost of educational activities of graduate students in hospitals of medical universities in iran. this study was performed in eight autonomous educational medical centers of iran university of medical sciences, tehran, iran. after identifying the cost topics, the required information was collected through a semi-structured interview with the officials and students. then, the amount of use of the graduate students from the cost headings was determined. the results identified that doctor of philosophy (phd) students of microbiology with more than 950 united states dollar (usd) and phd students of medical education with about 7 usd have created the highest and lowest costs, respectively. this study, for the first time, examined the educational costs of iranian postgraduate students in the hospital. in conclusion, the results of this study indicated that the amount of the costs of different fields of graduate education in medical sciences is very different and it should be considered in the allocation of the resources. keywords: medical course, economics of education, postgraduate student 1. introduction to plan and achieve the educational goals, organizations must have information about the cost of each educational course. analysis of this type of information causes policy makers to make more appropriate decisions and move towards achieving the goals, increasing the efficiency and excellence of the organization [1]. identifying the actual cost of various courses can help to better allocate of scarce resources [2]. calculating the student's per capita cost can also help to determine the ratio of different educational departments in the total cost of education [3]. in addition, knowledge of financial information and the *corresponding author: shima nikjoo, ph.d health management and economics research center, school of health management and information sciences, iran university of medical sciences, tehran, iran tel/fax: +98 21 88794301 email: shimanikjoo@gmail.com https://orcid.org/0000-0001-6048-691x received: may, 22, 2021 accepted: july, 12, 2021 cost of training can play an important role in the appropriate decision of educational managers and increase the economic efficiency of the educational systems [1]. this study aimed to estimate the educational costs of graduate students of medical sciences in autonomous hospitals of the iran university of medical sciences, tehran, iran. 2. materials and methods the cross-sectional was performed on all graduate departments of iran university of medical sciences in 2018 who had educational activities in autonomous © the author(s) 2022 https://jcbior.com/ nikjoo et al. 2 educational hospitals of iran university of medical sciences, tehran, iran. the calculation of direct costs of educational services provided to graduate students of medical schools in independent educational centers by academic years was examined as follows: in the first stage, the cost headings of postgraduate medical students in the hospital were identified. this step was performed qualitatively using a semi-structured interview. in order to identify cost topics, interview with the vice chancellor of the faculty, heads of departments, educational assistants of independent medical centers, heads of clinical and financial departments, and the professors and students (n=20). after identifying the cost headings, in the next step, the amount of educational activities and services, as well as the general and specific requirements used for training in educational and medical centers were measured. since the activities and services performed in educational environments are not completely consistent with the topics and it is possible to use different and diverse educational processes, so the basis for identifying the activities in this study was the real educational services that took place in educational centers. this was done through observation and interviews with the internship officials of the students in the hospital, the heads of the departments and the resident professors in the hospitals. in the third stage, the cost of resources used to educate students were calculated. in the last step, the cost of education of each student in each course was calculated. the costs calculated in this study were a combination of direct and indirect training costs in medical training centers. direct educational costs included the costs of the hospital's resources for education and the costs of general and specific necessities for education in medical centers. indirect educational expenses also included the cost of renting classrooms and other educational spaces, maintenance of office and educational equipment related to education, and the cost of welfare services (e.g. accommodation, food, and transportation). finally, from the total direct and indirect costs, the student per capita was calculated. 3. results in total, 24 graduate courses (master of science; msc and doctor of philosophy; phd) of the postgraduate departments in the medical school of iran university of medical sciences in the academic year of 2018-2019 were included. among the available courses, 7 internship courses are spent in the affiliated hospitals of the university, and the students of other courses study their practical units in the laboratories located in the medical school. the total current cost spent on graduate education services in the medical school was 1173 united states dollar (usd). the cost of consumables was the largest cost category accounted for 63% of the total cost. in terms of the cost of training of graduate students of the medical school was reported in, all costs incurred in educating students in educational and medical centers were calculated. the phd in microbiology had the highest per capita cost. phd students in microbiology with more than 950 usd and phd students in medical education with about 7 usd had the highest and lowest costs, respectively. 4. discussion the purpose of this study was to obtain the costs incurred for educational activities of post-graduate students of medical school in independent hospitals of iran university of medical sciences. internship is a tool for turning theory into practice [4]. postgraduate education costs of medical school students in the academic year of 2017-2018 in the educational hospitals of iran university of medical sciences showed that some of these courses do not incur significant costs for the university in the process of doing their internship. these students carry out their practical units in a rotating manner in the teachingmedical hospitals under the supervision of the relevant educational instructors. in most studies conducted in iran, the activity-based costing method has been used to calculate the student cost [5-9]. due to the low overhead costs which do not support all related costs and considering that the present study was conducted to identify and estimate the costs of internships of students in hospitals, a semi-qualitative method of assessment and timing was used to estimate the cost. the great difference between the findings of the previous studies and the present study is that all of these studies have included the total cost of student education, including the costs of educational research, internship, support, etc., while this study only focused on the cost of internship. a large part of the cost of educating students is educational expenses. on the other hand, in post-graduate courses, including msc nikjoo et al. 3 and phd degrees, a significant part of the costs is allocated to research costs. the most important limitations of this study were the inefficiency of systems for reporting, failure to record a lot of cost information in hospitals, and failure to measure cost headings related to post-graduate students in the wards. also, this study was hospitalbased and some cost topics such as human resources salaries, doctoral students' tuition fees, transportation costs and building depreciation were not included in the calculations because they were related to the university. this study, for the first time, examined the educational costs of students in the hospital during the internship. the results of this study showed that postgraduate students do not incur significant costs for the university in their internship process. according to the results of this study, it seems that the major costs allocated to the education of this groups of students are related to theoretical and practical training courses within the university. it is also suggested that overhead costs be calculated in addition to direct and obvious costs in order to accurately estimate of training costs. also, training needs should be collected from different departments and the existing differences should be considered in budgeting. authors’ contributions shn conducted the study. shn, ar, tbm, rj, ark, sv, and hf collaborated in designing the study, analyzing the data, and writing the article. all authors read and approved the final version of article. conflict of interests there is no conflict of interest. ethical declarations the paper does not contain clinical studies or patient data. the project was confirmed by research ethics committees of iran university of medical sciences and was found to be in accordance to the ethical principles and the national norms and standards for conducting medical research in iran (ethical code: ir.iums.rec.1397.923). financial support this study was part of a research project that is supported by the health management and economics research center affiliated to iran university of medical sciences (grant no. 12865). references 1. ghaffari s, mohamadzadeh a, akbari s, salem safi p, yousefi m. costing in hospital services: economists and accountants' approaches. j hospital. 2013; 11(4):75-84. 2. franzini l, low md, proll ma. using a cost-construction model to assess the cost of educating undergraduate medical students at the university of texas--houston medical school. acad med. 1997; 72(3):228-37. 3. moradi s, hedayatizadeh-omran a, janbabaei g, alizadehnavaei r, panbehchi m, geraili b, et al. activity based costing of educational services in faculty of medicine in mazandaran university of medical sciences, iran, 2015. j mazandaran univ med sci. 2018; 28(163):86-92. 4. daugherty sr, baldwin dc, jr., rowley bd. learning, satisfaction, and mistreatment during medical internship: a national survey of working conditions. jama. 1998; 279(15):11949. 5. ebadi azar f, a.gorj h, hadian m, m.ahari a. 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http://jmums.mazums.ac.ir/browse.php?a_id=11606&sid=1&slc_lang=en http://jmums.mazums.ac.ir/browse.php?a_id=11606&sid=1&slc_lang=en http://jmums.mazums.ac.ir/browse.php?a_id=11606&sid=1&slc_lang=en http://jmums.mazums.ac.ir/browse.php?a_id=11606&sid=1&slc_lang=en https://pubmed.ncbi.nlm.nih.gov/9555759/ https://pubmed.ncbi.nlm.nih.gov/9555759/ https://pubmed.ncbi.nlm.nih.gov/9555759/ https://pubmed.ncbi.nlm.nih.gov/9555759/ https://jha.iums.ac.ir/article-1-309-en.html https://jha.iums.ac.ir/article-1-309-en.html https://jha.iums.ac.ir/article-1-309-en.html https://jha.iums.ac.ir/article-1-309-en.html https://jha.iums.ac.ir/article-1-309-en.html https://ijvlms.sums.ac.ir/article_46104.html https://ijvlms.sums.ac.ir/article_46104.html https://ijvlms.sums.ac.ir/article_46104.html https://ijvlms.sums.ac.ir/article_46104.html https://ijvlms.sums.ac.ir/article_46104.html https://ijvlms.sums.ac.ir/article_46104.html https://sdme.kmu.ac.ir/article_90306.html https://sdme.kmu.ac.ir/article_90306.html https://sdme.kmu.ac.ir/article_90306.html https://sdme.kmu.ac.ir/article_90306.html https://sdme.kmu.ac.ir/article_90306.html https://payavard.tums.ac.ir/browse.php?a_id=5973&sid=1&slc_lang=en https://payavard.tums.ac.ir/browse.php?a_id=5973&sid=1&slc_lang=en https://payavard.tums.ac.ir/browse.php?a_id=5973&sid=1&slc_lang=en https://payavard.tums.ac.ir/browse.php?a_id=5973&sid=1&slc_lang=en https://journal.irphe.ac.ir/article-1-80-en.html https://journal.irphe.ac.ir/article-1-80-en.html https://journal.irphe.ac.ir/article-1-80-en.html the role of microrna-2911 in control of viral infections running title: microrna-2911 in viral infection zahra iman dust 1, nastaran saadat 2, iman owliaee 1, shahab mahmudvand 1, somayeh shokri 1, hossein vakilimofrad 3, farid azizi jalilian 1 * 1 department of virology, school of medicine, hamadan university of medical sciences, hamadan, iran. 2 department of virology, faculty of medicine, kerman university of medical sciences, kerman, iran. 3 department of medical library and information sciences, school of paramedicine, hamadan university of medical sciences, hamadan, iran. * corresponding author: farid azizi jalilian, department of virology, faculty of medicine, hamadan university of medical sciences, hamadan, iran. phone number: +989180189029, email: azizifarid@gmail.com orcid: https://orcid.org/0000-0003-3134-1441 journal of current biomedical reports jcbior.com volume 2, number 3, 2021 eissn: 2717-1906 1 original research a survey of medical students' attitudes and practices towards narcotics and psychotropic drugs shahab dastmardi1, morteza rahbar taromsari2, hamid mohammadi kojidi2, ardalan majidinia3, fariba asgari bozayeh4, kourosh delpasand5,* 1student research committee, school of pharmacy, guilan university of medical sciences, rasht, iran 2department of forensic medicine and toxicology, school of medicine, guilan university of medical sciences, rasht, iran 3student research committee, school of medicine, guilan university of medical sciences, rasht, iran 4department of nursing, school of nursing and midwifery, medical education research center (merc), guilan university of medical sciences, rasht, iran 5department of medical ethics, school of medicine, guilan university of medical sciences, rasht, iran abstract substance abuse is one of the major behavioral problems in today's human society. one of the medical uses of drugs is to relieve the pain and suffering of patients. today, due to the widespread use of narcotics and psychotropic drugs in the control and treatment of the disease and also its use among medical students, the present study aimed to determine the attitude and practice of medical students towards the use of narcotics and psychotropic drugs. the present study was a cross-sectional analytical study that was performed on 102 medical interns of guilan university of medical sciences who were selected by available methods. to collect information, a researcher-made questionnaire was used which has three sections including a checklist of demographic information, attitude assessment questionnaire of medical students and performance assessment questionnaire. the mean age of the interns was 23.8±0.21 years. in the analysis of the results, it was found that there is a significant relationship between marital status and residence status with performance score (p <0.05). based on the results of the present study, the level of students' knowledge about narcotics and psychotropic drugs among them is not at the desired level. therefore, due to the lack of knowledge of medical students about the dangerous side effects of these drugs, education on the nature, symptoms, and side effects of psychotropic substances is recommended. keywords: narcotics, psychotropic, medical students, drugs 1. introduction substance abuse is one of the major behavioral problems in today's human society, which the world health organization has described as a worrying event in the world [1]. narcotics (a substance used to relieve pain such as morphine and codeine, but are not made from opium) and psychotropic (a drug or chemical substance such as alcohol, caffeine, nicotine, marijuana, and certain pain medicines that affects *corresponding author: kourosh delpasand, md, ph.d department of medical ethics, school of medicine, guilan university of medical sciences, rasht, iran tel/fax: +98 13 33690006 email: kd388@yahoo.com http://orcid.org/0000-0001-6842-3374 received: may, 16, 2021 accepted: july, 17, 2021 how the brain works and results in alterations in mood) drugs use in young people is an important issue and is considered a public health problem. substance use is associated with an increased risk of death, psychological and interpersonal problems, academic failure, difficulties in establishing relationships and having unwanted or unprotected sex, crime, accidents, and injuries [2]. in this regard, educational institutions © the author(s) 2021 https://jcbior.com/ dastmardi et al. 2 should be an organizing element in a system for preventing and confronting addictive substances [3]. drug addiction is one of the major problems in developing countries. since these countries have a young population, they are naturally more at risk and therefore these young people as the main and most vulnerable group are more at risk [4]. high school and college students, like other young people, are not immune to this problem [5, 6]. people may turn to addictive substances to reduce anxiety and avoid problems such as financial poverty, family problems, marital discord, inappropriate patterns of assimilation, and ultimately unemployment or euphoria. substance abuse is a maladaptive pattern of using drugs that leads to recurring problems and adverse outcomes and involves a set of cognitive, behavioral, and psychological symptoms [7, 8]. drug use sometimes increases physical or psychological dependence. physical dependence increases drug tolerance, i.e., reduced drug sensitivity and withdrawal symptoms when the drug is not available [9-11]. there are many types of drugs and their range is increasing every day and new compounds are produced. the criterion of being addictive and not addictive is not a good criterion for distinguishing illegal drugs from other substances because some substances, although addictive, are not banned, such as benzodiazepines. addictive substances are classified in several ways. in medicine and pharmacology, an addictive substance can be classified based on its chemical structure or on the effects, side effects, and conditions it causes as a result of consumption [12]. according to the controlled substances act (csa), addictive substances are often divided into five categories, including narcotics, sedatives, stimulants, hallucinogens, and anabolic steroids [13]. due to the widespread use of narcotics and psychotropic drugs in society and also its use among medical interns, the present study was designed and conducted to determine the attitude of medical interns towards the use and prescription of narcotics and psychotropic drugs and their performance. 2. materials and methods the present study is a cross-sectional analytical study that was performed on 102 medical interns of guilan university of medical sciences, rasht, iran. sampling was done by the available method in 2020. the instrument used includes a researcher-made questionnaire that has three parts. the first part includes a checklist of demographic information that includes questions about gender, age, marital status, residence status, income, and education of parents. the second part of the questionnaire was to evaluate the attitude of medical interns in the use of narcotics and psychotropic drugs and the third part is a questionnaire to evaluate the performance of medical interns in the use of narcotics and psychotropic drugs. the attitude questionnaire had 16 items and the answer method was 5-point likert. to determine the validity of the content, the content validation method (survey of 5 clinical faculty members of guilan university of medical sciences) and content validity index (cvi) and content validity ratio (cvr) were used, which were 0.88 and 0.92, respectively. to evaluate the interns' performance, a researcher-made checklist with 8 questions was used and the answers were in the form of "yes" and "no" options, which were awarded 1 and 0 points, respectively. to assess the reliability, the questionnaires were distributed twice but one week apart among 25 medical interns, and the retest test method was used and the cronbach's alpha value was 0.84. data were collected in spss software version 22 and descriptive (mean, frequency, and standard deviation) and inferential (mann-whitney and kruskal-wallis) tests were used. the p-value of less than 0.05 was considered statistically significant. 3. results out of 102 distributed questionnaires, four were excluded due to incomplete responding and 98 questionnaires were reviewed, of which 50 (51%) were male and 48 (49%) were female and their mean age was 23.8±0.21 years. other demographic characteristics are presented in table 1. according to mann-whitney and kruskal-wallis tests, no significant relationship was found between the attitude score of medical interns and their place of residence, income and education of parents, and marital status (p > 0.05). on the other hand, marriage status had a significant relationship with the performance score of narcotics and psychotropic drugs use among medical interns (p = 0.031). the average performance score of married people is higher than the average performance score of single people (table 2). dastmardi et al. 3 residence status had a significant relationship with performance score on narcotics and psychotropic drugs use among medical interns (p = 0.022). the average performance score of medical interns who lived with their friends was lower than the average score of other interns (table 2). also, the performance score on the use of narcotics and psychotropic drugs among medical interns was not significantly related to the relationship between parents 'education and parents' income and gender and age (p > 0.05). 4. discussion substance abuse is one of the scourges that has befallen communities and, unfortunately, it is most prevalent among young people and adolescents. since these materials are more emerging, the knowledge of the society is not enough about it [14]; therefore, it is necessary to conduct extensive research in this area. the present study showed that medical interns do not have the desired knowledge and attitude towards psychotropic substances. in this regard, in a study conducted on medical students in shiraz (south of iran), it was found that the rate of drug use was higher among single people than married people [6]. in our study, marriage status was significantly associated with performance scores on the use of narcotics and psychotropic drugs among medical interns. so that the average performance score of married people was higher than the average performance score of single table 1. demographic characteristics of medical interns variable name frequency percentage gender male 50 51 female 48 49 marital status single 70 71.4 married 28 28.6 place of residence with parents 45 45.9 with friends 19 19.4 dorm 18 18.4 private house 16 16.4 parents' education high school 6 6.1 diploma 13 13.3 bachelor and associate degree 48 49 masters and phd 12 12.2 md 19 19.4 parents' income (irr) under 5 million 21 21.4 between 5 and 10 million 27 27.6 between 10 and 15 million 30 30.6 over 15 million 20 20.4 abbreviations: md: medical doctor; irr: iranian rial table 2. distribution of performance scores on the use of narcotics and psychotropic drugs among medical interns status performance score p-value x̅ ± s. e min max marital status single 4.0 ± 44.21 2 8 0.031 married 5.0 ± 43.32 2 8 total 4.0 ± 72.18 2 8 residence status parents 4.0 ± 56.28 2 8 0.022 dorm 5.0 ± 17.41 2 8 friends 3.0 ± 89.40 2 7 private house 5.0 ± 69.35 3 8 total 4.0 ± 72.18 2 8 dastmardi et al. 4 people, which can indicate the responsibility of married people compared to single people regarding the performance of these people. the results of our study were consistent with a study by masibo et al. in tanzania, which was conducted in both qualitative and quantitative ways [14]. in the present study, there was no significant relationship between gender and attitudes towards narcotics and psychotropic substances, which is consistent with the previous iranian study [6]. one of the most important factors in the spread of drug use is the influence of peers and friends. young people who have drug-addicted friends are more likely to use drugs. therefore, if they are close friends of drug users, these young people are more likely to be driven to use drugs [2]. in this regard, in our study, residence status had a significant relationship with performance scores on narcotics and psychotropic drug use among medical interns, so that the average performance score of medical interns living with friends is lower than the average score of other interns. peer pressure can have both positive and negative effects, depending on the quality of the peer group. in this study, living with friends may in some ways lead to poor performance. in a study by masibo et al., which examined high school students' knowledge and awareness of psychotropic substances, they found that students were sufficiently aware of drugs, their effects, and the problems associated with their use. the reason for reporting sufficient knowledge about psychotropic substances and their effects was to have information about the dangers of using psychotropic substances that they received from parents, siblings, media, and teachers. in the masibo study, students were unaware of the long-term effects of psychotropic substance use, but more than 50% of our students were sufficiently aware [14]. factors associated with narcotics and psychotropic drugs abuses are many and vary depending on individual contexts, family characteristics, complex social and environmental factors. the results of the masibo study showed that the majority of participants had never used psychotropic substances, and few reported that they intended to use psychotropic substances [14]. in our study, 63% of students even refused to be friends with these people. in our study, it was shown that parents 'education and parents' income had no significant relationship with performance scores on the use of narcotics and psychotropic drugs, which was consistent with other studies [14, 15]. finally, it should be acknowledged that our sample size and studied population were limited, so our findings can not be generalized and further studies are demanded. the results of the present study showed that the level of knowledge and practice of medical students about narcotics and psychotropic drugs is not at the desired level; therefore, due to the lack of knowledge of medical students about the dangerous side effects of psychotropic substances, education on the nature, symptoms and side effects of psychotropic substances is recommended. also, because an unprincipled prescription of narcotics can be a violation of ethical codes, it is very important and necessary to inform the staff of the medical department about the legal and ethical standards in prescribing narcotics. acknowledgments the present study was extracted from the result of an md thesis in general medicine. this project has been done with the support of guilan university of medical sciences, and therefore the researchers consider it necessary to express their gratitude to the esteemed deputy. author contributions all authors contributed equally to this manuscript, and approved the final version of manuscripts. conflict of interests the authors declare that they have no conflicts of interest. ethical declarations it should be noted that this research design has been approved by the ethics committee of guilan university of medical sciences and has an ethics code of ir.gums.rec.1399.46. consent to publish a written informed consent was taken from the participants to publish their data. financial support none. dastmardi et al. 5 references 1. hockenberry mj, wilson d. wong's nursing care of infants and children-e-book: elsevier health sciences; 2018. 2. abbasi-ghahramanloo a, fotouhi a, zeraati h, rahimimovaghar a. prescription drugs, alcohol, and illicit substance use and their correlations among medical sciences students in iran. int j high risk behav addict. 2015; 4(1):e21945. 3. pishchulin vi, rogacheva li, fokina lv, fadeeva o, novikov r, kolupaev rv. drug addiction prevention: experience of high education institute. life sci j. 2014; 11(12):566-9. 4. rahimi pordanjani s, fallah zadeh h, mousavi m, khazaei s, sohrabivafa m, momenabadi v, et al. prevalence and reasons for psychoactive drugs use among university students of medical sciences in yazd, iran. iran j psychiatry behav sci. 2018; 12(1):e9384. 5. cox rg, zhang l, johnson wd, bender dr. academic performance and substance use: findings from a state survey of public high school students. j sch health. 2007; 77(3):109-15. 6. ashrafi hafez a, fakor ziba m, babaee haidar abadi a, hosaini f, razmposh e, gharlipour z, et al. assessment of psychoactive substances use and their associated factors among students of shiraz university and shiraz university of medical sciences. j ilam univ med sci. 2013; 21(4):58-66. 7. bastani p, marshall bdl, rahimi-movaghar a, noroozi a. the risk environments of people who use drugs accessing two harm reduction centers in tehran, iran: a qualitative study. int j drug policy. 2019; 63:90-6. 8. mchugh rk, hearon ba, otto mw. cognitive behavioral therapy for substance use disorders. psychiatr clin north am. 2010; 33(3):511-25. 9. grella ce, lovinger k. gender differences in physical and mental health outcomes among an aging cohort of individuals with a history of heroin dependence. addict behav. 2012; 37(3):306-12. 10. sinha r. chronic stress, drug use, and vulnerability to addiction. ann n y acad sci. 2008; 1141:105-30. 11. mclellan at. substance misuse and substance use disorders: why do they matter in healthcare? trans am clin climatol assoc. 2017; 128:112-30. 12. nattel s. antiarrhythmic drug classifications. a critical appraisal of their history, present status, and clinical relevance. drugs. 1991; 41(5):672-701. 13. sajjadi m, shariatifar n, matlabi m, abbasnezhad a, basiri k, nazemi h. the rate of knowledge and attitude toward psychoactive drugs and its abuse prevalence in gonabad university students. horizon med sci. 2009; 15(2):58-64. 14. masibo rm, mndeme e, nsimba se. an assessment of knowledge, attitudes and practices of psychoactive substance use among secondary school students in dodoma municipality, tanzania. american j res commun. 2013; 1(4):200-40. 15. ahmadi-nejad m, jadidi f, dehghani mr, divsalar k. studying prevalence and pattern of taking narcotic and ecstasy drugs by patients admitted to special care centers of shahid bahonar hospital, kerman, iran. addict health. 2012; 4(1-2):57-64. https://pubmed.ncbi.nlm.nih.gov/25821750/ https://pubmed.ncbi.nlm.nih.gov/25821750/ https://pubmed.ncbi.nlm.nih.gov/25821750/ https://pubmed.ncbi.nlm.nih.gov/25821750/ http://www.dx.doi.org/10.7537/marslsj111214.110 http://www.dx.doi.org/10.7537/marslsj111214.110 http://www.dx.doi.org/10.7537/marslsj111214.110 http://www.dx.doi.org/10.7537/marslsj111214.110 https://dx.doi.org/10.5812/ijpbs.9384 https://dx.doi.org/10.5812/ijpbs.9384 https://dx.doi.org/10.5812/ijpbs.9384 https://dx.doi.org/10.5812/ijpbs.9384 https://dx.doi.org/10.5812/ijpbs.9384 https://pubmed.ncbi.nlm.nih.gov/17302852/ https://pubmed.ncbi.nlm.nih.gov/17302852/ https://pubmed.ncbi.nlm.nih.gov/17302852/ http://sjimu.medilam.ac.ir/article-1-1238-en.html http://sjimu.medilam.ac.ir/article-1-1238-en.html http://sjimu.medilam.ac.ir/article-1-1238-en.html http://sjimu.medilam.ac.ir/article-1-1238-en.html http://sjimu.medilam.ac.ir/article-1-1238-en.html https://pubmed.ncbi.nlm.nih.gov/30513474/ https://pubmed.ncbi.nlm.nih.gov/30513474/ https://pubmed.ncbi.nlm.nih.gov/30513474/ https://pubmed.ncbi.nlm.nih.gov/30513474/ https://pubmed.ncbi.nlm.nih.gov/20599130/ https://pubmed.ncbi.nlm.nih.gov/20599130/ https://pubmed.ncbi.nlm.nih.gov/20599130/ https://pubmed.ncbi.nlm.nih.gov/22154506/ https://pubmed.ncbi.nlm.nih.gov/22154506/ https://pubmed.ncbi.nlm.nih.gov/22154506/ https://pubmed.ncbi.nlm.nih.gov/18991954/ https://pubmed.ncbi.nlm.nih.gov/18991954/ https://pubmed.ncbi.nlm.nih.gov/28790493/ https://pubmed.ncbi.nlm.nih.gov/28790493/ https://pubmed.ncbi.nlm.nih.gov/28790493/ https://pubmed.ncbi.nlm.nih.gov/1712704/ https://pubmed.ncbi.nlm.nih.gov/1712704/ https://pubmed.ncbi.nlm.nih.gov/1712704/ http://hms.gmu.ac.ir/article-1-599-en.html http://hms.gmu.ac.ir/article-1-599-en.html http://hms.gmu.ac.ir/article-1-599-en.html http://hms.gmu.ac.ir/article-1-599-en.html http://www.usa-journals.com/wp-content/uploads/2013/03/masibo_vol14.pdf http://www.usa-journals.com/wp-content/uploads/2013/03/masibo_vol14.pdf http://www.usa-journals.com/wp-content/uploads/2013/03/masibo_vol14.pdf http://www.usa-journals.com/wp-content/uploads/2013/03/masibo_vol14.pdf https://pubmed.ncbi.nlm.nih.gov/24494137/ https://pubmed.ncbi.nlm.nih.gov/24494137/ https://pubmed.ncbi.nlm.nih.gov/24494137/ https://pubmed.ncbi.nlm.nih.gov/24494137/ type of article: letter to the editor title: can the microbiome affect human reproductive function? authors: mohammad akhoondian a, shervin yavari b, moslem karampoor c, amin fouladpour d, alireza samadnia e, samad karkhah f* affiliations: a. department of physiology, school of medicine, cellular and the molecular research center, guilan university of medical science, rasht, iran. b. department of parasitology, school of medicine, guilan university of medical science, rasht, iran. c. department of microbiology, school of medicine, guilan university of medical sciences, rasht, iran. d. department of orthopedics, poorsina hospital, faculty of medicine, guilan university of medical sciences, rasht, iran. e. student research committee, school of medicine, guilan university of medical sciences, rasht, iran. f. department of medical-surgical nursing, school of nursing and midwifery, guilan university of medical sciences, rasht, iran. *corresponding author: samad karkhah (msc) address: nursing and midwifery school of shahid dr beheshti, hamidyan shahrak, shahid dr beheshti ave., rasht, guilan, iran fax: +98-13-33550097; postal code: 41469-39841 tel: +98-9032598167; +98-13-33552088; email: sami.karkhah@yahoo.com emails and degree: samad karkhah (msc), email: sami.karkhah@yahoo.com (orcid: 0000-0001-9193-9176). short title: can the microbiome affect human reproductive function? number of tables: 0 number of figures: 0 words count: main text: 497. 23/11/2022 dear prof. seyed asghar havaei editor in chief of journal of current biomedical reports journal enclosed please find our manuscript entitled: “emergence of amoebic dysentery mimicking covid-19: a human case report from iran”. to be considered for publication in the journal of current biomedical reports as a case report. we hope this manuscript meets the requirement for publication in the journal. meanwhile, we would like to declare we are not in any financial or personal relationship with other people or organizations that could inappropriately influence this investigation and we have no conflict of interest in this study. also, we have obtained a written consent from the patient for publication of her personal and clinical details. please let me know if there is any further information you may require regarding this manuscript. i can be contacted at e-mail: mj.afshar1990@gmail.com sincerely yours, corresponding author dr. mohammad javad abbaszadeh afshar department of medical parasitology, school of medicine, jiroft university of medical sciences, jiroft, iran type of article: letter to the editor title: upregulated shared genes of il1rn and socs3: identification of a genetic connection key and proinflammatory cytokine regulator between burns and crohn's disease authors: mohammad reza zabihi 1, mohammad akhoondian 2, babak pourgholamali 3, narges norouzkhani 4, pooyan ghorbani vajargah 5, 6, amirabbas mollaei 5, 6, samad karkhah 5, 6* affiliations: 1. department of immunology, school of medicine, tehran university of medical sciences, tehran, iran. 2. department of physiology, school of medicine, cellular and the molecular research center, guilan university of medical science, rasht, iran. 3. cellular and molecular research center, faculty of medicine, guilan university of medical sciences, rasht, iran. 4. department of medical informatics, faculty of medicine, mashhad university of medical sciences, mashhad, iran. 5. burn and regenerative medicine research center, guilan university of medical sciences, rasht, iran. 6. department of medical-surgical nursing, school of nursing and midwifery, guilan university of medical sciences, rasht, iran. *corresponding author: samad karkhah (msc) address: nursing and midwifery school of shahid dr beheshti, hamidyan shahrak, shahid dr beheshti ave., rasht, guilan, iran fax: +98-13-33550097; postal code: 41469-39841 tel: +98-9032598167; +98-13-33552088; email: sami.karkhah@yahoo.com emails and degree: samad karkhah (msc), email: sami.karkhah@yahoo.com (orcid: 0000-0001-9193-9176). short title: identification of upregulated shared genes of il1rn and socs3 between burns and crohn's disease number of tables: 0 number of figures: 1 journal of current biomedical reports jcbior.com volume 1, number 2, 2020 eissn: 2717-1906 1 editorial letter the effect of sars-cov-2 on hiv-positive individuals kimia sharifian1, razieh dowran1,* 1 department of virology, school of public health, tehran university of medical sciences, tehran, iran keywords: sars-cov-2, covid-19, hiv severe acute respiratory syndrome coronavirus 2 (sars-cov-2) which was first identified in december 2019 has been announced as a pandemic by the world health organization (who) in march 2020 after infecting millions of people all over the world [1]. according to the centers for disease control and prevention (cdc), some people are at higher risk for severe illness from covid-19 including people over 65 years old, people who live in nursing home or long term care facility, people with chronic lung disease, people who have serious heart conditions, people with severe obesity, diabetes, chronic kidney disease undergoing dialysis, liver disease, sickle cell anemia and immunocompromised like hiv positive individuals [2]. previously it was believed that low count of cd4 might protect hiv infected individuals from progress to the cytokine storm observed in covid-19 patients [3], but later studies suggested low count of cd4 (lower than 200 cells per microliter) may be associated with disease severity [4, 5]. according to a prospective cohort which investigates covid-19 in hiv-infected individuals, it was demonstrated that hiv-infected individuals should be considered to have equal (hiv positive patients who are on regular antiretroviral treatment with normal cd4 count and low viral load) or even more risk of severe disease (especially *corresponding author: dr. razieh dowran, ph.d department of virology, school of public health, tehran university of medical sciences, tehran, iran tel/fax: +98 917 1201832 email: raziehdowran@yahoo.com https://orcid.org/0000-0002-2318-4397 received: august, 7, 2020 accepted: august, 15, 2020 individuals with comorbidities, lower cd4 cell counts, or high hiv viral load) [5]. the genome of sars-cov2 was detected after approximately 40 days from the onset of symptoms in six (32%) individuals, it was remarkable that four of them had the severe disease or aids [5]. moreover, they suggest that the sars-cov2 presentation in hiv positive patients may have similar or even worse outcomes to general population. it was also mentioned that these patients should be treated same as hiv negative individuals [5]. although there are numerous mysterious about sars-cov-2 and its effects on hiv positive individuals, the prevalence of covid-19 in hiv positive patients and severity of the symptoms and illness in these people depend on different factors such as age, gender, cd4 count, viral load, body mass index (bmi), comorbidity, current antiretroviral therapy, smoking and even socioeconomic factors [3-6]. according to existing studies, symptoms of disease in hiv positive patients may be similar to hiv negative ones and the severity of symptoms in hiv positive individuals with covid-19 were almost similar to hiv negative covid-19 patients [4-7]. however, it should be noted that because of the small sample size of available studies the results may subject to bias and other studies in future may get it more sense. https://jcbior.com/ https://orcid.org/0000-0002-2318-4397 sharifian et al. 2 author contributions all authors contributed equally to this manuscript, and approved the final version of manuscripts. conflict of interests the authors declare that they have no conflicts of interest. ethical declarations not applicable. financial support none to be declared. references 1. valencia dn. brief review on covid-19: the 2020 pandemic caused by sars-cov-2. cureus. 2020; 12(3):e7386. 2. people at increased risk and other people who need to take extra precautions. available at: https://www.cdc.gov/coronavirus/2019-ncov/need-extraprecautions/index.html. 3. guo w, ming f, dong y, zhang q, zhang x ,mo p, et al. a survey for covid-19 among hiv/aids patients in two districts of wuhan, china (3/4/2020). available at ssrn: https://ssrn.com/abstract=3550029 or http://dx.doi.org/10.2139/ssrn.3550029. 4. härter g, spinner cd, roider j, bickel m, krznaric i, grunwald s, et al. covid-19 in people living with human immunodeficiency virus: a case series of 33 patients. infection. 2020:1-6. [in press]. doi: 10.1007/s15010-020-01438-z. 5. vizcarra p, perez-elias mj, quereda c, moreno a, vivancos mj, dronda f, et al. description of covid-19 in hiv-infected individuals: a single-centre, prospective cohort. lancet hiv. 2020. doi: https://doi.org/10.1016/s2352-3018(20)30164-8. 6. shalev n, scherer m, lasota ed, antoniou p, yin mt, zucker j, et al. clinical characteristics and outcomes in people living with hiv hospitalized for covid-19. clin infect dis. 2020 [in press]. doi: 10.1093/cid/ciaa635. 7. xu z, zhang c, wang f-s. covid-19 in people with hiv. lancet hiv. 2020;7(8): e524-6. doi: https://doi.org/10.1016/s23523018(20)30163-6 https://pubmed.ncbi.nlm.nih.gov/32337113/ https://pubmed.ncbi.nlm.nih.gov/32337113/ https://www.cdc.gov/coronavirus/2019-ncov/need-extra-precautions/index.html https://www.cdc.gov/coronavirus/2019-ncov/need-extra-precautions/index.html https://pubmed.ncbi.nlm.nih.gov/32394344/ https://pubmed.ncbi.nlm.nih.gov/32394344/ https://pubmed.ncbi.nlm.nih.gov/32394344/ journal of current biomedical reports jcbior.com volume 2, number 3, 2021 eissn: 2717-1906 1 review a review on the epidemiology and characteristics of covid-19 ranjit barua1,*, sudipto datta1, amit roychowdhury1,2, pallab datta3 1centre for healthcare science and technology, indian institute of engineering science and technology, shibpur, howrah 711103, west bengal, india 2department of aerospace engineering and applied mechanics, indian institute of engineering science and technology, shibpur, howrah 711103, west bengal, india 3national institute of pharmaceutical education and research, kolkata, kolkata, india abstract in december 2019, there was a health emergency worldwide named novel coronavirus or covid-19 by the world health organization (who). it originated from the wuhan seafood market, hubei province, china. till now severe acute respiratory syndrome coronavirus-2 or sars-cov-2 spread over 216 countries with 177,108,695 confirmed cases and 3,840,223 confirmed death cases has been reported (5:31 pm cest, 18 june 2021; who). analyzing the risk factor of this pandemic situation, different government health organizations of all the countries including who are taking several preventive measures with ongoing research works, even the vaccination process started. in this study, we tried to analyze all the available information on pandemic covid-19, which includes the origin of covid-19, pathogenic mechanism, transmission, diagnosis, treatment, and control-preventive measures, also the additional treatment and prevention taken by the indian government is being studied here. keywords: sars-cov-2, who, outbreak, pathogenic mechanism, india 1. introduction in december 2019, world health organization (who) was reported unknown symptoms of pneumonia cases by the chinese health authority in wuhan city in hubei province, china. it was reported that most of the patients with positive cases were connected to the wuhan seafood wholesale market [1]. who named it the novel coronavirus or 2019-ncov on 7th january 2020 [2]. it was identified from the swab test sample of patients. later, the coronavirus study group and who renamed it as severe acute respiratory syndrome coronavirus-2 (sars-cov-2) [3]. on 30th january 2020, china reported 79,394 confirmed cases and 12,167 suspected cases; more than 82 confirmed cases were reported from eighteen other countries [4]. on the same day, who declared it as a worldwide pandemic emergency [5]. initially, *corresponding author: ranjit barua, ph.d centre for healthcare science and technology, indian institute of engineering science and technology, shibpur, howrah 711103, west bengal, india tel/fax: +91 8910642686 email: ranjitjgec007@gmail.com https://orcid.org/0000-0003-2236-3876 received: may, 29, 2021 accepted: june, 21, 2021 national health commission (china) reported to who that inside of china the mortality rate was 2.1%, whereas the outside of china was reported with a 0.2% mortality rate (4th feb 2020) [6]. among the admitted patients, the mortality rate was between 11%-15% [7]. coronavirus disease 2019 (covid-19) was reported as a highly infectious disease with a higher spreading rate than the mortality rate [8]. this study aimed to analyze the available information of pandemic covid-19 including pathogenic mechanism, transmission, diagnosis, treatment, and control-preventive measures. 1.1 origin and cataloging of covid-19 the analysis of the genome sequence of sarscov-2 reveals the whole genome sequence recognition rates of bat sars coronavirus (sarsr-cov-ratg13) © the author(s) 2021 https://jcbior.com/ barua et al. 2 was 96% whereas sars-cov was 79.5% [9]. this shows that covid-19 might have their origination from bats. gradually the rate of active cases is increasing [10]. sars-cov-2 is a family member of coronaviridae and the order nidovirales. the family is subdivided into torovirinae and coronaviridae, furthermore, coronaviridae is divided into four categories: (i) α-coronavirus consists of human coronavirus (hcov-229e and hcov-nl63); (ii) βcoronavirus consists of sars-hcov (severe acute respiratory syndrome), hcov-oc43, mers-cov (middle-eastern respiratory syndrome coronavirus); (iii) γ-coronavirus consists of the viruses for birds and whales; (iv) δ-coronavirus [11]. sars-cov-2 originated from β-coronavirus combined with two highly pathogenic viruses middleeastern respiratory syndrome coronavirus (merscov) and severe acute respiratory syndrome (sarshcov), respectively [12]. the phylogenetic analysis of novel coronavirus or sars-cov-2 shows that the connection of the virus is linked with two different viruses (88% related) which derive from the bat, batsl-covzxc21, and bat-sl-covzc45, respectively which genetically differs from mers-cov and sarscov [13]. further research work with the help of genome sequences of ratg13, sars-cov, and sarscov-2 shows that the virus is associated with bat cov ratg13 which was previously found in rhinolophusaffinis from wuhan province (overall 96% genome sequence) [14]. no evidence of recombination was found in the genome of a novel coronavirus from other viruses which originated from bats liked bat cov ratg13, sars-cov. further studies suggest that the origin of this virus is from the bat family [13]. although, a related study is going on if there was any mediator via which the virus was transmitted to human bodies. reasons which shows that bats are not solely responsible for the human transmission of coronavirus are as follows: (i) availability of several non-aquatic animals which also consists of mammals for selling in wuhan seafood market but there was no sign of bats; (ii) novel coronavirus and its neighbors bat-sl-covzxc21 and bat-sl-covzc45 which have a comparatively long branch (less than 90% sequence identity), recommends that those viruses have no direct connection with sars-cov-2; (iii) other animals (civets, most probably camels respectively) acted as a mediator where the bat is the host in the coronavirus cases like mers-cov and sars-cov [14]. basically, coronavirus consists of single-stranded rna viruses with a diameter of 80–120 nm. it can be categorized into four types(i) α-coronavirus; (ii) βcoronavirus; (iii) γcoronavirus; and (iv) δcoronavirus [15]. figure 1 shows the various types of coronavirus. other than sars-cov-2, more than six coronaviruses including mers-cov and sars-cov have been identified before. the homology of sarscov and sars-cov-2 in the genome sequence is roughly 79%; which is similar to sars-like bat coronavirus (mg772933) [16, 17]. 1.2 covid-19 pathogenic mechanism several studies show that novel coronavirus uses the ace2 which is known as the “angiotension converting enzyme-2” receptor, which is similar to sars-cov [18]. this virus mostly knows it’s consistent that targets the upper layer of the cells by the by s proteins (figure 2a); which results in infection. a study indicates that novel coronavirus fixes to ace2 with more than ten folds higher similarity than sars-cov, the stage beyond the inception needed for virus contagion [19]. the complete process through which covid-19 affects humans by fixing spike protein to ace2 shows in figure 2b, the strong point of the contact for the threat of human transmission, and how this virus causes organ failure is still unknown. these outcomes describe the quicker transmission ability of novel coronavirus in humans as related to sars-cov, and figure 1. various categories of coronavirus barua et al. 3 the most number of confirmed cases of sars-cov-2 as associated with the infection of sars-cov. since the most similarity of novel coronavirus fixes to ace2, this receptor can be a possible applicant for the cure of novel coronavirus [20]. 1.3 transmission of sars-cov-2 the spreading of coronavirus or sars-cov-2 by the wuhan seafood market is still not clear. initially, it was said that the novel corona virus-positive incidents were associated with this marketplace (animal to human transmission) [21]. genomic research has delivered such evidence which shows the virus was originated from an unidentified place in the market, where it has spread nearer, however it was noticed that the transmission from human to human has happened previously [22]. the human-to-human transmission has been confirmed by the infected family members and health workers [23]. it was reported after 1st january that only 10% of the positive cases were related to the wuhan seafood market and more than 70% of the patients were not related to that market [21]. it was predicted that human transmission happens in close contact especially through respiratory droplets originate from an infected persons’ sneezes and coughs. it was found that sarscov and other coronaviruses can stay on the surfaces for up to 96 hrs and 9 days, respectively [24, 25]. on 30th january 2020, an asymptomatic transmission was reported [26], but by the researchers, no direct interview of the patient was reported, who had symptoms of transmission of disease [27]. an asymptomatic transmission was reported on 21st february 2020 [28]. the mean incubation period was reported to be 5.2 (95% ci: 4.1– 7.0) days [16]. this period can be elongated to 19 or 24 days [29, 30], however, the case descriptions usually depend on a fourteen days window [31]. also, reproductive number (r0) is predicted with fluctuating outcomes and analyses. it also measures the typical number of infected cases which results in one infected case to the whole population [32]. figure 3 shows the transmission of coronavirus. previous studies found reproductive number (r0) is to be 2.7 and 2.4 for sars [33], and 2009 pandemic h1n1 influenza [34]. another study predicted that the reproductive number (95% ci: 1.4–3.9) was 2.2 [13]. although, further studies suggested the reproductive number (r0) to be 3.28 [35]. normally reproductive number (r0) represents the typical value which is considered to be the sole transmitter, which is most responsible for the spreading of epidemics in large groups [36]. in a severe case like epidemic or pandemic, reproductive number (r0) is unstable [32]. there is a risk of transmission if a pregnant woman is admitted to the hospital but no such maternal and fetal casualty, and vertical utero-transmission were noticed [37]. currently, 177,108,695 confirmed cases and 3,840,223 confirmed death cases have been reported [5:31 pm cest, 18 june 2021; who] (supplementary figure 1). the confirmed cases of the individual countries affected by covid-19 are given in descending (high to low) order in supplementary table 1. figure 2. a. genome structure of sars-cov-2; b. sars-cov-2 infects the host cell barua et al. 4 1.4 the epidemiology of novel coronavirus on 3rd march 2020, a total of 87,317 positive cases has been confirmed by who. among all the definite cases, about 2,977 cases have yielded to the virus. the common symptoms and demises are informed by china. from the entire figure of positive cases, 79,968 infected patients have been recognized in china [38]. the common symptoms and demises are informed by china. from the entire figure of positive cases, 79,968 infected patients have been recognized in china [39]. it has been found that the clinical characteristics of 1,099 numbers of covid19 patients, where the median incubation period time was approximately 3 days, basically the range is 0–24 days, the average period from the development of the symptoms to death was approximately 14 days [40]. for sars-cov septicity, the median expectancy was four days, the typical intermission from indication beginning to the taken to hospital was three days, and the usual interim from hospital entry time to death was seventeen days [41]. the median expectancy of the infection of mers-cov was seven days. the average development period for novel coronavirus is smaller than that for mers and sars, respectively. conversely, the maximum expectancy of novel coronavirus currently detected is as high as twentyfour days, which may raise the threat of virus transmission. furthermore, persons aged more than seventy years had a shorter medium interval approximately eleven days from the indication start to death associated with patients aged less than seventy years, signifying that infection progress is speedier in aged persons related with elder persons [38, 42]. 1.5 risk factors of covid-19 the cases of sars-cov-2 septicity are realized most frequently in mature man patients with the middle age of the peoples was between thirty-four and fifty-nine years old [43, 44]. the novel coronavirus is also more likely to contaminate people with lifelong diseases like cerebrovascular and diabetes diseases, respectively [45]. the maximum amount of severe problems happens in mature ages more than sixty years, and with assured essential circumstances, for example, diabetes, cerebrovascular and cardiovascular diseases, respectively [46]. severe appearances may be also connected with infected bacteria and fungi respectively. also, below 15 years of age lower number of covid-19 cases are reported [47]. figure 4 shows the effect of coronavirus on the human body. a recent study published on jan 29th stated that among 425 patients of covid-19 cases in wuhan, china no cases were reported below 15 years of age [48]. the medical features of the pediatric infected patients were different, and the majority of patients had mild symptoms, having no low-grade fever and or pneumonia along with a good prognosis. another report stated that though a kid had ground-glass radiological lung denseness, while the patient showed no symptoms [49]. from this, the conclusion that can be made is that children are less likely to get an infection than adults. also, if infection occurs in children mild manifestations are shown than adults which can lead to no treatment of their parents because of underestimate covid-19 symptoms in that age range. figure 3. transmission routes of coronavirus barua et al. 5 1.6 clinical features novel coronavirus generates an acute viral infection in patients with a middle incubation period of 3 days [48]; which is related to sars, with two to ten days of incubation period [49]. the main signs of novel coronavirus are cough, fever, fatigue, and diarrhea, respectively [50], like other coronavirus symptoms. many positive patients had an appearance of some degree of dyspnea; the intermission from indication start to the appearance of severe respiratory distress syndrome which was only nine days within the initial cases [51]. moreover, severe cases are prone to a variety of complications, including secondary infection, and acute respiratory distress syndrome [52]. based on the report in wuhan's first 425 detected cases, the mutual indications contain fever, dry cough, myalgia, and fatigue with fewer normal are headache, hemoptysis, sputum production, diarrhea, and abdominal [21]. nearly 75% of patients had bilateral pneumonia [53]. unalike from mers-cov and sarscov symptoms, sars-cov-2 less likely indicate projecting upper respiratory tract marks and sore throat, sneezing, signifying that the virus might have a better tendency for contaminating the lower respiratory tract [54]. it is reported that pregnant women may have fewer covid-19 symptoms than non-pregnant women but should take intensive care if severely infected [55]. some reports also state that novel coronavirus can be harmful to organs and tissues other than the lungs. another study states that out of 214 positive patients 78 patients had neurological symptoms [56]. additionally, there is evidence sars-cov-2 rna was detected in eye secretions of patients [57]. computerized tomography (ct) scan is an essential tool to identify this pneumonia. numerous usual imaging topographies are normally detected in novel coronavirus pneumonia, as well as 50% of leading ground-glass consolidations, 65% of opacity, 35% of a plane or unequal interlobular septal thickening, 47% of lower lobe involvement air bronchograms, predominantly peripheral and 32% of condensing of the adjacent pleura [58]. a current study described that 90% of patients had bilateral chest ct scan report results, and the understanding of chest ct scan report to suggest 97% of novel coronavirus [59]. merging ct scan reports with clinical indication and laboratory results could allow initialing medicare treatment of pneumonia of novel coronavirus. 1.7 diagnosis the who endorses gathering samples from lower as well as upper respiratory tracts. this can be attained by endotracheal aspirate, expectorated sputum, and bronchoalveolar lavage [59]. these models are then evaluated for viral rna by polymerase chain reaction (pcr). when a positive result comes retest on the figure 4. effects of covid-19 on human figure 5. different diagnoses process of covid-19 barua et al. 6 sample should be done and if negative comes a retest is also done for confirmation. the recognition of viral nucleic acid is normal for the non-invasive analysis of the novel covid-19. figure 5 shows the various diagnosis process of covid-19. 1.8 treatment currently like the previous coronaviruses sarscov and mers-cov there is no specific treatment for covid-19. supportive care like fluid management, oxygen therapy, antibiotic treatment of the bacterial secondary infection, and isolation are suggested [60]. few cases of covid-19 patients advance quickly to septic shock and acute respiratory distress syndrome (ards) which leads to multiple organ failure [61]. so it is very much important to stop the spread of the disease by infection control measures and isolating the patient and providing necessary treatments for recovering from the disease [62]. currently, covid-19 patient treatments are commonly symptomatic. for this kind of rna coronaviruses remdesivir an antiviral drug showed positive results in most cases. in a report, holshue et al. stated that remdesivir shower good results on many patients of covid-19 [63]. a drug for treating malaria, chloroquine has been found to show the activity of immune-modulatory and has shown an antiviral effect on sars-cov-2 in vitro. in the clinic also it is seen that the drug chloroquine showed positive results [64]. currently, the drug remdesivir is also being used on the covid-19 patients till now the drug efficacy is unknown [65]. influenza viruses fusion were been blocked by a molecule umifenovir (trade name arbido) which is a small indole-derivative molecule. recently it has been suggested that umifenovir can help fight against covid-19 [66]. if sepsis is recognized, antibiotics of empiric type should be directed depending on clinical diagnosis and susceptibility information, and local epidemiology. daily glucocorticoid direction is not suggested if there are additional signs [67]. usage of intravenous immunoglobulin could be helpful for extremely ill patients. drug evaluation in line with past inquiries into the treatments for therapeutic in mers and sars [68]. though there is no proof that these drugs can improve the patient's condition clinically. an empirical antibiotic treatment combined with antiviral drug oseltamivir was employed for treating covid-19 cases [71]. in the usa ebola antiviral drug, remdesivir is employed for the treatment of covid19 patients. for 3 to 4 infected covid-19 patients combination of drugs like shufeng jiedu capsule (sfjdc), lopinavir/ritonavir a traditional chinese medicine, arbidol showed promising results [65, 6971]. various other antiviral drugs are also being proposed for the treatment of covid-19. also, a few medicines are recommended for the treatment of covid-19, which are given in table 1 [70-72]. 1.9 prevention and control strategies of covid19 the covid-19 is spreading worldwide so rapidly, while the infection rate of the virus is very high with a low mortality rate. the infection rate is higher than mers and sars-covs. older people and peoples table 2. medicines recommended for covid-19 medicines function hydroxychloroquine increase in the acidic levels of endosomes, prevents the endocytosis and stops the virus to enter in the host cell remdesivir with the help of rna template, it prevents the rna reproduction lopinavir and ritonavir prevents rna translation progression ribavarin prevents mrna capping and synthesis of viral rna darunavir stops the viral reproduction favipiravir from the rna template, it prevents the rna reproduction and also, prevents rna translation progression oseltamivir prevents rna translation progression arbidol prevents the outer membrane synthesis of the viral outer surface monoclonal antibodies prevents the viral infection umifenovir prevents virions synthesis through endosomal membrane barua et al. 7 with lower immunity are at higher risk of death. to stop this rapid spreading of the virus isolation is needed. till now there is no specific medicine and vaccine to combat the disease. oxygen therapy and symptomatic care strategies are currently used to treat. vaccination of prophylactic type is compulsory for the upcoming slowdown of covid linked widespread and epidemic. in the community, the main measure for interrupting the transmission of the disease is the isolation of the infected individuals. for example in china, the decision was taken by chinese medical authorities to quarantine suspected individuals for 14 days and isolated infected people [39]. till now there is doubt of the virus’s origin some says it came from animals like a bat [73], others say it came from snakes [74], so it is necessary to immediately stop eating these animals and stop coming in close contact with these animal’s fluid and or tissues. people's education is also needed for spreading the disease and to get quick treatment. individuals having symptoms like fever, shortness of breath, and or cough should immediately seek medical experts. for stopping the spread of the virus in large communities following things should be maintained like home isolation, mitigating social gatherings, temporary school closure, close nursing of symptomatic cases, wearing personal protective gears like a face mask, face shields, and carrying hand sanitizers, provision of life supports (e.g. oxygen supplier and mechanical ventilator), etc. in wuhan city, chinese authorities had taken lockdown measures seriously and strictly which stopped/slowed down the virus spreading [39, 75]. ways of travel showed be minimized except for compulsory medical care. setup of temperature scanning or checking is compulsory at the airport as well as at the border to recognize the suspected individuals. further research with this covid-19 virus is serious to find the outbreak source and to give proof of the upcoming epidemics [71]. few vaccines are used nowadays. the vaccines that are administered by who are shown in table 2 [76]. 2. indian strategy for covid-19 prevention and control the containment policies of india include detecting the positive cases and keeping them in isolation, social distancing, home quarantine, following additional medical guidance. currently in india, 29,822,764 confirmed cases and 389,452 death cases have been reported, which is shown in figure 6 [77]. present protocols of treatment contain analyzes of point-of-care molecular tests and also real-time pcr diagnosis. the person who is at higher risk of covid-19 such as forefront staffs and asymptomatic confirmed covid cases are given hydroxychloroquine or hcq drug (an antimalarial drug). the hydroxychloroquine medicine dozes are given as 400 mg two times a day for one day and later 400 mg once every week which will continue for seven weeks. though, pregnant women and kids below fifteen years are not suitable for this drug [78]. the critical patients are given hydroxychloroquine with azithromycin drug. the dozes of hydroxychloroquine and azithromycin drug are 400 mg two times a day for one day and later 200 mg two times a day which will continue for four days, and 500 mg one time a day which will continue for five days, respectively [79]. india introduced a mobile app named ‘arogya setu’ for tracing the activities of the people and also initiated plasma therapy to fight against the novel coronavirus. the government of india has initiated a complete lockdown and maintaining the social distance for the t a b le 2 . n a m e o f th e v a c c in e s re c o m m e n d e d b y w h o f o r c o v id -1 9 n a m e o f th e v a c c in e e m e r g e n c y u se l is ti n g d a te t h e p fi z e r/ b io n te c h v a c c in e 3 1 /1 2 /2 0 2 0 o x fo rd – a st ra z e n e c a c o v id -1 9 v a c c in e 1 5 /0 2 /2 0 2 1 ja n ss e n a d 2 6 .c o v 2 .s 1 2 /0 3 /2 0 2 1 m o d e rn a c o v id -1 9 v a c c in e 3 0 /0 4 /2 0 2 1 s in o p h a rm c o v id -1 9 v a c c in e 0 7 /0 5 /2 0 2 1 s in o v a c -c o ro n a v a c 0 1 /0 6 /2 0 2 1 barua et al. 8 prevention of covid-19 [78]. the lockdown schedule follows phase-i: march 25 – april 14 [85][80]; phaseii: april 14 – may 3; phase-iii: may 4 – may 17; and phase-iv: may 18 – may 31, 2020. in this pandemic situation, a supplementary test has been recommended as a rapid antibody test which is needed to keep in observation as the test reports are given after 7 to 10 days. as the laboratories are receiving a lot of samples for examining, pool testing is introduced in this placed so that the process does not take a longer time (five samples are examined at once). for in-vitro a famous drug for malaria hydroxychloroquine showed hindering effect for covid-19 for this reason this drug became the trial choice [81]. although, the use of this drug or in combination with another macrolide drug already showed adverse cardiovascular outcomes for the qt period [81, 82]. though, there is no proof that this drug is effective for this virus either alone or azithromycin combination, except there is a risk of ventricular arrhythmias has been shown by many researchers [83]. in india improving plasma therapy on various covid-19 infected patients is done on those who were having a saturation of oxygen in the bloodless than 90 % and rate of respiration greater than 30 breaths per minutes or having lungs infiltrations [84]. the selection of donors who were recovered after 28 days of having low viral load for plasma therapy are very specific i.e. they should be below 60 years old and should not have any pre-existing health conditions like hypertension, diabetes, and/or problems related to the heart [85, 86]. csir (council of scientific and industrial research of india already started working on more advanced vaccine development like the recombinant dna vaccine and rna vaccine for the c ovid-19 virus. the trials of acqh are already being started by csir as it showed acceptable results in the case of the treatment of dengue. this acqh is an extraction of a plant which are found in areas like madhya pradesh, jharkhand, and gujrat indian states [87]. furthermore, favipiravir drug clinical trial is also being done in india [88]. with the advancement of smartphone technology, india launched an app for both android and ios which is a useful intelligent software for awareness spreading of the coronavirus for the citizens of india [89]. this app provides various latest information like the total number of infected, the total number of the recovered, infected area, read zones, several peoples using this app near me within a radius of 50 km, 200 km as well as latest news and health services, etc. the software of the app uses smartphone bluetooth and gps to work properly, it tells the user whether he/she has come in contact with an infected individual or area by the database search of the country’s confirmed case area. the infected individual’s information is kept confidential because he/she does not face any social discrimination or rejection fear from the society peoples. due to lockdown and different types of prevention, the new active cases of covid-19 were able to control. although, in the mid-week of september 2020, the new active covid-19 positive cases increased it is observed that within mid-week february 2021, the new active cases decreased. suddenly a huge number of new active covid-19 cases was observed in the last week of march 2021 [77]. simultaneously with covid-19, a fungal infectious disease named “black fungus” was detected in the patients who recovered from covid-19 [90]. 3. second wave of covid-19 in india: causes and effects presently, india is going through a tough situation due to the second wave of covid-19. as the days are passing by, the number of active cases is rising, and therefore india is going through a major medical crisis. there is a high demand for oxygen supplies but due to the medical crisis, india is unable to fulfill its oxygen demand. few reasons for this deadly attack of covid19 for the second time in india can’t be neglected. as india is a third-world developing country with a huge population [91], there is a lack of proper implementation of rules and regulations. in 2020, people took covid-19 seriously and they used to wear figure 6. the present situation of covid-19 in india [worldometers, 18 june, 2021] barua et al. 9 masks properly but as time passed there was a lack in the seriousness of proper way of wearing masks and sanitization. these are the basic habits that will help the public to fight against covid-19 [92]. in maximum cases, it is noticed people are wearing single-layer cloth masks which increases the possibility of getting infected [93]. people should use double masking, a cloth mask above a three-layer mask, or wearing an n95 mask to avoid getting infected [94]. due to the financial condition of indian common people, individuals are bound to go to their respective workplace, therefore they have to avail the public transport which leads to increase in the number of active cases. frontline workers like police, health workers, and other social workers, are working their sweat out are mostly getting affected due to the direct contact with more public. there is no growth of antibodies in the previously covid-19 affected patients, due to which they are getting infected for the second time. in rural areas people share a common space to live in, therefore, when an infected person comes in contact with the non-infected person, for instant, using a common bathroom, the non-infected person gets infected. indian government imposes few rules on social activities and occasions, but commoners were unable to maintain it in a proper way (improper use of mask, lack in the use of sanitizer and surface disinfected, and lack in proper social distancing). due to the huge population, the indian government is facing a huge challenge to vaccinate every individual, which leads to an insufficient vaccine, which is increasing the active number of cases in india. although the indian government (both central and state) took many initiatives but also several others ngos started covid-care support. iiest-shibpur, howrah has started a covidcare unit in their campus [95], they also initiated to vaccinate their employees, students, and their families. 4. conclusions the novel coronavirus is spreading worldwide very rapidly. the infection from this novel coronavirus is much higher concerning than mers and sars. from the r0 value is found out that the infection rate of sars-cov-2 is very high. elderly people and those with weakened immune systems are at higher risk of dying and multi-organ failure. to cut the quick spread of the virus, social activities should be stopped and home lockdown and isolation are needed. children below 15 years of age get no or less infected from this virus. oxygen therapy and symptomatic care are the current treatment strategies. vaccination of prophylactic type is needed for upcoming inhibition of covid-19 related pandemic and epidemic. supplementary files supplementary file 1. acknowledgments the authors 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https://pubmed.ncbi.nlm.nih.gov/33047556/ https://pubmed.ncbi.nlm.nih.gov/33047556/ https://pubmed.ncbi.nlm.nih.gov/33047556/ https://pubmed.ncbi.nlm.nih.gov/32988954/ https://pubmed.ncbi.nlm.nih.gov/32988954/ https://pubmed.ncbi.nlm.nih.gov/32988954/ https://pubmed.ncbi.nlm.nih.gov/32988954/ https://pubmed.ncbi.nlm.nih.gov/33167047/ https://pubmed.ncbi.nlm.nih.gov/33167047/ 2 type of article: letter to the editor title: can chemical burns lead to autoimmune gastritis in the long term? authors: mohammad reza zabihi 1, mohammad akhoondian 2, samad karkhah 3, 4* affiliations: 1. department of immunology, school of medicine, tehran university of medical sciences, tehran, iran. 2. department of physiology, school of medicine, cellular and the molecular research center, guilan university of medical science, rasht, iran. 3. burn and regenerative medicine research center, guilan university of medical sciences, rasht, iran. 4. department of medical-surgical nursing, school of nursing and midwifery, guilan university of medical sciences, rasht, iran. *corresponding author: samad karkhah (msc) address: nursing and midwifery school of shahid dr beheshti, hamidyan shahrak, shahid dr beheshti ave., rasht, guilan, iran fax: +98-13-33550097; postal code: 41469-39841 tel: +98-9032598167; +98-13-33552088; email: sami.karkhah@yahoo.com emails and degree: samad karkhah (msc), email: sami.karkhah@yahoo.com (orcid: 0000-0001-9193-9176). short title: can chemical burns lead to autoimmune gastritis in the long term? number of tables: 0 number of figures: 1 journal of current biomedical reports jcbior.com volume 2, number 3, 2021 eissn: 2717-1906 1 review meconium microbial toxins and microbiota: a novel and non-invasive proposed diagnostic sample to anticipate the severity of neonates covid-19 behrouz khakpour taleghanie1, saba abedi2,3, adele jafari1,4, mojtaba hedayati ch3,5,* 1department of physiology, school of medicine, guilan university of medical sciences, rasht, iran 2student research committee, school of medicine, guilan university of medical sciences, rasht, iran 3microbial toxins physiology group, universal scientific education and research network, rasht, iran 4cellular and molecular research center, school of medicine, guilan university of medical sciences, rasht, iran 5department of microbiology, virology and microbial toxins, school of medicine, guilan university of medical sciences, rasht, iran abstract the novel coronavirus, termed severe acute respiratory syndrome coronavirus 2 (sars cov-2) is a major public health challenge all over the world and is the causative agent of coronavirus disease 2019 (covid-19). since december2019 the outbreak of covid-19 has become a major epidemic issue all over world. in this pandemic, preterm and term neonates with infected mothers are becoming more vulnerable each day. although we mostly witness asymptomatic neonates, getting symptomatic may not be a rarity in the future. after entrance of covid-19 into the body it could affect the balance of microbiota combination which can result in microbiota dysbiosis and eventually leads to immune imbalance. intrauterine microbiome dysbiosis in covid-19 positive mothers and transmission of lipopolysaccharide (lps) may restructure the environment of the developing fetus with possible short/long-term impact on the individual’s health and disease. therefore, assessing the changes in microbiome of neonates from infected mothers via exploring meconium could be valuable. it is also logical to measure lps level and balanced its levels by using prebiotics and probiotics as a supplemental therapeutic procedure to prevent medical challenges in future. the aim of this essay is to review the potential conception that detection of covid-19 and the meconium microbiota and lps quantity could be used as a source of prognostic information about the covid-19 severity in the fetus of pregnant women with covid-19. keywords: meconium, lps, covid-19, sars-cov-2, microbiota 1. covid-19 outbreak the novel coronavirus, severe acute respiratory syndrome coronavirus 2 (sars cov-2) termed is a great public health challenge, and is the causative agent of coronavirus disease 2019 (covid-19). since december 2019 the outbreak of covid-19 has become a major epidemic issue all over world [1, 2]. in this pandemic, when the world is in constant battle to stop the spread of infection and prevent mortality, *corresponding author: mojtaba hedayati ch, ph.d department of microbiology, school of medicine, guilan university of medical sciences, rasht, iran tel/fax: +98 13 33690006 email: mhedayatich@gums.ac.ir; mhedayatich@gmail.com https://orcid.org/0000-0002-8277-9841 received: may, 20, 2021 accepted: july, 05, 2021 preterm and term neonates with infected mothers are becoming more vulnerable each day. although we mostly witness asymptomatic neonates [3], getting symptomatic may not be a rarity in the future. 2. covid-19, microbial translocation and microbial toxins during pregnancy no proof of vertical transition of covid-19 has been found yet [4], affirming placenta as an efficient © the author(s) 2021 https://jcbior.com/ khakpour taleghanie et al. 2 obstacle even in case of severe infection. but some research declared that the virus is a potential risk factor for enforcing intense placental disruption independently of vertical transition [5]. shanes et al. analyzed the placentas of 16 women affected intensively with covid-19, the placentas indicated fetomaternal vascular poor perfusion and intervillous thrombus (ivt). this study proposes a malformed maternal diffusion reflecting a systemic inflammatory or hypercoagulable condition affecting placenta’s function which could be associated with adverse perinatal outcomes [6]. after entrance of covid-19 into the body it could affect the balance of microbiota combination which can result in microbiota dysbiosis and eventually leads to immune imbalance [7]. it is possible to observe simultaneous penetration of microbial toxic metabolites like bacterial lipopolysaccharide (lps), into intrauterine space. this potent cytokine storm inducer then might lead to immune imbalances in neonates, which are predictable. it is demonstrated that covid-19 spike protein attaches to bacterial lps and increases inflammatory activations [8]. n proteins of sars-cov-2 and middle east respiratory syndrome coronavirus (mers-cov) exasperate lps causing pneumonia through triggering masp-2 activity [9]. the presence and transmission of lps to the fetus and pregnancy fluids and their potency to induce lung inflammatory diseases has also been demonstrated in previous studies [10, 11]. based on these findings, we consider that it may be possible to measure the amount of lps in the body as a prognostic marker of covid-19 in pregnant mother or newborn baby. in order to measure toxic metabolites like bacterial lps and detection of covid-19, a safe and reliable test is highly required besides neonatal nasopharyngeal swabs (which could be highly invasive), assessing amniotic fluid, cord blood which are mostly negative. the hypothesis of this essay is to review the value of meconium microbiota and lps quantification as an origin of prognostic data toward the severity of covid-19 in the neonate of women with positive covid-19 (figure 1). the inutero bacterial situation could be manifested by microbial mixture of meconium. the presence of bacteria in the embryo’s bowel is recognized as a basis for microbial provocation and can supply an essential signal for a balanced immune function [12]. microbiome combination is a biologic material, which could be highly recommended as a clinical diagnostic routine test for diagnosis and follow up in infants with mothers infected with covid-19. figure 1. routes of microbial and lps translocation. illustration present probable known pathways for translocation of mother microbiota and microbial products to fetus. different population of bacteria, virus or microbial toxins could translocate and colonize in fetus meconium and make it a rich sample for analysis khakpour taleghanie et al. 3 3. microbial translocation: mechanism, immunity, physiology of emerging diseases and probable probiotic supportive role more than 30 years ago, for the first time the existence of some kinds of microbes in the amnion in the absence of any disruption was announced [13]. there are several hypotheses for the transplacental transition of bacteria and their products into utero. the most probable theory is bacterial transition from the mother’s bowel to blood circulation and then to the organs including uterine [14]. during pregnancy, maternal’s small intestinal epithelial cells (dendritic cells and micro fold cells) can actively pass through the epithelium of intestine, pick up the bacteria or their genome and deliver them to the amniotic liquid [1517]. this presumption is established by empiric studies in pregnant mice fed enterococcus faecium by mouth, this bacterium was detected in the blood of umbilical cord, amniotic fluid, meconium and placenta [18]. mother's microbiome could affect infant's immune system by means of its bacterial products. microbiota produce multiple substances that can interfere with different biological actions, immune modulating and energy producing functions [19]. the immune system recognizes microbial metabolites and pathogenassociated molecular patterns (pamps), the sensation of these molecules can cause direct influence on immune process [20-22]. hence, compensating microbiota imbalance could be beneficial for immunity of fetus for both short and long term, one easy and safe method for this aim is using prebiotic and probiotic products. it is indicated that using probiotics is an effective and promising approach to stop intestinal problems in neonates [23, 24]. maternal immune factors have a significant role in bacterial invasive actions into the amniotic fluid; this invasion can boost an inflammation cascade eventually leading in preterm labor [25]. hypothetically, swallowing amniotic fluid by fetus can make it gut susceptible to microbes and their metabolites translocation and permeability, which may be a significant contributor to early immune progress in neonates. it is demonstrated that, memory cd4+ and cd8+ t cells could be recognized at the end of third month in embryo’s intestines [26]. memory cd4+ t cells in embryo’s gut have been proved to collaborate with antigen presenting cells (apcs) and leads to generation of interferon gamma (ifn-γ), interleukin-2 (il-2) or tumor necrosis factor-α (tnfα), assisting intestine’s expansion [27, 28]. ultimately, it is presumed that early fetus access to microbial metabolites and lps may have a considerable influence on infant’s immune system in future and make them more susceptible to different diseases like diabetes mellitus, allergy [29], autism [30], obesity [31], and neurodegenerative diseases like alzheimer’s diseases [32]. on the other hand, many studies have shown that exposure to lps can cause symptoms of neurological diseases such as alzheimer's, which can be a late symptom in covid-19 patients. this may be a bridge between bacterial lps that can be released as a result of antibiotic treatments and the death of gram-negative bacteria in the body and contribute to the formation and deterioration of cytokine storms [33, 34]. it is possible that mother’s viral infections result in the gut microbiota change during pregnancy with enduring outcomes for the neonates. it is demonstrated that fetal brain growth can be affected by improper gut flora of mother which can cause longlasting neurological consequences. several studies on mice indicated that maternal viral infection can cause reduction in intestinal barrier integrity in the children, as a consequence considerable changes in intestinal microbial combination and microbial metabolism were observed leading to autism-like behavior and neuropathology. for instance, it was demonstrated that giving bacteroides fragilis to the mice in their pregnancy enhanced the gut microbiota combination and gut penetrance in the neonates and decreased the occurrence of autism [14, 17, 18]. multiple studies mentioned that the anal swabs from the individuals with covid-19 were positive (35, [35, 36], and the covid-19 could have been separated from the fecal specimen of covid-19 positive patients by various medical teams [37]. in a case report study in china, the anal swabs of a three-month-old girl was still positive nearly a month after the onset of the illness even when she had no symptom and 14 days after her oropharyngeal swabs were negative her anal swabs were still positive [38]. 4. comprehensive conclusion intrauterine microbiome dysbiosis in covid-19 positive mothers may restructure the condition of the embryo by viable short-term and long-term influence on the human’s health state. therefore, assessing the changes in microbiome of neonates from infected khakpour taleghanie et al. 4 mothers via exploring meconium could be valuable. it is also logical to measure lps level and balance it by using prebiotics, probiotics or symbiotic as a supplemental therapeutic procedure to prevent medical challenges in future. so far, there has been no study conducted to test physiological relation of maternal microbiota toxic metabolites like bacterial lps in covid-19 positive mothers and neonate’s meconium. we suggest to analyze meconium’s lps levels and gram-negative microbiota as a noninvasive, reliable and potential package test beside other virus detection methods of covid-19 in infants with covid-19 positive mothers. it could equip us with better strategies to keep newborns safe. because levels of mentioned microbiome and their products like lps, might act as a clock bomb during probable cytokine storm formation. author contributions mhch: conceptualization, funding acquisition, supervision, validation. sa: drafted the manuscript. aj: drafted the 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394:112814. 34. murata s, rezeppa t, thoma b, marengo l, krancevich k, chiyka e, et al. the psychiatric sequelae of the covid-19 pandemic in adolescents, adults, and health care workers. depress anxiety. 2021; 38(2):233-46. 35. ling y, xu sb, lin yx, tian d, zhu zq, dai fh, et al. persistence and clearance of viral rna in 2019 novel coronavirus disease rehabilitation patients. chin med j (engl). 2020; 133(9):1039-43. 36. holshue ml, debolt c, lindquist s, lofy kh, wiesman j, bruce h, et al. first case of 2019 novel coronavirus in the united states. n engl j med. 2020; 382(10):929-36. 37. han q, lin q, ni z, you l. uncertainties about the transmission routes of 2019 novel coronavirus. influenza other respir viruses. 2020; 14(4):470-1. 38. fan q, pan y, wu q, liu s, song x, xie z, et al. anal swab findings in an infant with covid-19. pediatr investig. 2020; 4(1):48-50. https://pubmed.ncbi.nlm.nih.gov/24226770/ https://pubmed.ncbi.nlm.nih.gov/24226770/ https://dx.doi.org/10.5812/ijp.100139 https://dx.doi.org/10.5812/ijp.100139 https://dx.doi.org/10.5812/ijp.100139 https://pubmed.ncbi.nlm.nih.gov/24416709/ https://pubmed.ncbi.nlm.nih.gov/24416709/ https://pubmed.ncbi.nlm.nih.gov/19997613/ https://pubmed.ncbi.nlm.nih.gov/19997613/ https://pubmed.ncbi.nlm.nih.gov/19997613/ https://pubmed.ncbi.nlm.nih.gov/19997613/ https://pubmed.ncbi.nlm.nih.gov/3536221/ https://pubmed.ncbi.nlm.nih.gov/3536221/ https://pubmed.ncbi.nlm.nih.gov/3536221/ https://pubmed.ncbi.nlm.nih.gov/30664737/ https://pubmed.ncbi.nlm.nih.gov/30664737/ https://pubmed.ncbi.nlm.nih.gov/30664737/ https://pubmed.ncbi.nlm.nih.gov/30770246/ https://pubmed.ncbi.nlm.nih.gov/30770246/ https://pubmed.ncbi.nlm.nih.gov/30770246/ https://pubmed.ncbi.nlm.nih.gov/30770246/ https://pubmed.ncbi.nlm.nih.gov/30770246/ https://pubmed.ncbi.nlm.nih.gov/26317856/ https://pubmed.ncbi.nlm.nih.gov/26317856/ https://pubmed.ncbi.nlm.nih.gov/26317856/ https://pubmed.ncbi.nlm.nih.gov/26317856/ https://pubmed.ncbi.nlm.nih.gov/28028357/ https://pubmed.ncbi.nlm.nih.gov/28028357/ https://pubmed.ncbi.nlm.nih.gov/28028357/ https://pubmed.ncbi.nlm.nih.gov/18281199/ https://pubmed.ncbi.nlm.nih.gov/18281199/ https://pubmed.ncbi.nlm.nih.gov/18281199/ https://pubmed.ncbi.nlm.nih.gov/26462180/ https://pubmed.ncbi.nlm.nih.gov/26462180/ https://pubmed.ncbi.nlm.nih.gov/26462180/ https://pubmed.ncbi.nlm.nih.gov/26462180/ https://doi.org/10.1016/j.bbr.2020.112814 https://doi.org/10.1016/j.bbr.2020.112814 https://doi.org/10.1016/j.bbr.2020.112814 https://doi.org/10.1016/j.bbr.2020.112814 https://doi.org/10.1016/j.bbr.2020.112814 https://pubmed.ncbi.nlm.nih.gov/33368805/ https://pubmed.ncbi.nlm.nih.gov/33368805/ https://pubmed.ncbi.nlm.nih.gov/33368805/ https://pubmed.ncbi.nlm.nih.gov/33368805/ https://pubmed.ncbi.nlm.nih.gov/32118639/ https://pubmed.ncbi.nlm.nih.gov/32118639/ https://pubmed.ncbi.nlm.nih.gov/32118639/ https://pubmed.ncbi.nlm.nih.gov/32118639/ https://pubmed.ncbi.nlm.nih.gov/32004427/ https://pubmed.ncbi.nlm.nih.gov/32004427/ https://pubmed.ncbi.nlm.nih.gov/32004427/ https://pubmed.ncbi.nlm.nih.gov/32129925/ https://pubmed.ncbi.nlm.nih.gov/32129925/ https://pubmed.ncbi.nlm.nih.gov/32129925/ https://pubmed.ncbi.nlm.nih.gov/32328338/ https://pubmed.ncbi.nlm.nih.gov/32328338/ https://pubmed.ncbi.nlm.nih.gov/32328338/ 2 type of article: letter to the editor title: bioinformatics as an efficient tool and approach for burn wound healing authors: mohammad reza zabihi 1, mohammad akhoondian 2, pooyan ghorbani vajargah 3, 4, samad karkhah 3, 4* affiliations: 1. department of immunology, school of medicine, tehran university of medical sciences, tehran, iran. 2. department of physiology, school of medicine, cellular and the molecular research center, guilan university of medical science, rasht, iran. 3. department of medical-surgical nursing, school of nursing and midwifery, guilan university of medical sciences, rasht, iran. 4. burn and regenerative medicine research center, guilan university of medical sciences, rasht, iran. *corresponding author: samad karkhah (msc) address: nursing and midwifery school of shahid dr beheshti, hamidyan shahrak, shahid dr beheshti ave., rasht, guilan, iran fax: +98-13-33550097; postal code: 41469-39841 tel: +98-9032598167; +98-13-33552088; email: sami.karkhah@yahoo.com emails and degree: samad karkhah (msc), email: sami.karkhah@yahoo.com (orcid: 0000-0001-9193-9176). short title: bioinformatics as an efficient tool and approach for burn wound healing number of tables: 0 number of figures: 0 journal of current biomedical reports jcbior.com volume 2, number 1, 2021 eissn: 2717-1906 1 editorial letter covid-19 and respiratory tract viral co-infections: choosing the screening method amin mohsenzadeh1, hoda sabati2,* 1faculty of science, islamic azad university of ardabil, iran 2biotechnology and biological science research center, faculty of science, shahid chamran university of ahvaz, iran keywords: covid-19, sars-cov-2, diagnostic, co-infection, secondary infection the new pandemic pathogen, severe acute respiratory syndrome coronavirus 2 (sars-cov-2) or coronavirus disease 2019 (covid-19), causes a wide range of symptoms that differ from mild cold‐like symptoms to pneumonia and death. this virus has been an international public health emergency since december, 2019 due to its high infectious rate. beyond the pathogenesis of covid-19, viral co-infections play a vital role in the occurrence and development of this pandemic by increasing the difficulties of diagnosis, treatment and even the disease symptoms and mortality. regarding clinical presentation, transmission and coincidence this disease is very similar to middle east respiratory syndrome (mers), severe acute respiratory syndrome (sars) and seasonal influenza which makes it difficult to accurately detect [1-3]. the covid-19 detection and diagnostic methods are mainly divided into two groups, a laboratory-based (such as serological tests, and real-time reversetranscriptase polymerase chain reaction (rt-pcr)) and lung computed tomography (ct) scan or chest radiography. accuracy of these methods related to their sensitivity and specificity. previous studies showed that only some of them had both sensitivity and specificity over 50% in detecting sars-cov-2 infection. therefore, the use of appropriate diagnostic * corresponding author: hoda sabati, msc biotechnology and biological science research center, faculty of science, shahid chamran university of ahvaz, iran tel/fax:+98 922 6092051 email: h.sabati@yahoo.com https://orcid.org/0000-0002-4473-7245 received: january, 03, 2021 accepted: january, 05, 2021 methods with sufficient accuracy is of particular importance in screening covid-19 [4-6]. several reports indicate that viral interactions have some effect on the course of infectious diseases. therefore, high co-infection rate might be due to the overlap between the emergence of covid-19 and other respiratory viruses. this overlap emphasizes the importance of the covid-19 screening methods, regardless of other positive results for other viruses in the initial trials [7]. although co-infection with dual respiratory viruses is uncommon in adults, but there were cases have been reported. previous study showed that influenza a virus was one of the common viral pathogens causing co-infection among patients with covid-19 which may have caused initial falsenegative results of rt-pcr for sars-cov-2 [8]. in a case report, co-infection of metapneumovirus and sars-cov-2 has been investigated [9]. huang et al. reported a unique case of influenza b virus co-infection with sars-cov-2 in taiwan [10]. other viruses that can cause co-infection include: coronavirus, rhinovirus/enterovirus, respiratory syncytial virus (rsv), parainfluenza [11-13]. therefore, it is necessary to limit the risk of secondary infections by other viruses that can cause false-negative results for sars-cov-2 on screening methods and increase the risk of https://jcbior.com/ mohsenzadeh et al. 2 mortality rate in covid-19 patients. recognition of possible viruses causing co-infection among covid19 patients helps clinicians to choose appropriate screening method, proper management and therapeutic [7, 14]. author contributions all authors contributed equally to this manuscript, and approved the final version of manuscripts. conflict of interests the authors declare that they have no conflicts of interest. ethical declarations not applicable. financial support none to be declared. references 1. sharma a, tiwari s, deb mk, marty jl. severe acute respiratory syndrome coronavirus-2 (sars-cov-2): a global pandemic and treatment strategies. int j antimicrob agents. 2020; 56(2):106054. 2. gerges harb j, noureldine ha, chedid g, eldine mn, abdallah da, chedid nf, et al. sars, mers and covid-19: clinical manifestations and organ-system complications: a mini review. pathog dis. 2020; 78(4). 3. chen x, liao b, cheng l, peng x, xu x, li y, et al. the microbial coinfection in covid-19. appl microbiol biotechnol. 2020; 104(18):7777-85. 4. harahwa ta, lai yau th, lim-cooke ms, al-haddi s, zeinah m, harky a. the optimal diagnostic methods for covid-19. diagnosis (berl). 2020; 7(4):349-56. 5. floriano i, silvinato a, bernardo wm, reis jc, soledade g. accuracy of the polymerase chain reaction (pcr) test in the diagnosis of acute respiratory syndrome due to coronavirus: a systematic review and meta-analysis. rev assoc med bras. 2020; 66(7):880-8. 6. lisboa bastos m, tavaziva g, abidi sk, campbell jr, haraoui l-p, johnston jc, et al. diagnostic accuracy of serological tests for covid-19: systematic review and meta-analysis. bmj. 2020; 370:m2516. 7. khodamoradi z, moghadami m, lotfi m. co-infection of coronavirus disease 2019 and influenza a: a report from iran. arch iran med. 2020; 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https://pubmed.ncbi.nlm.nih.gov/26746130/ https://pubmed.ncbi.nlm.nih.gov/32957928/ https://pubmed.ncbi.nlm.nih.gov/32957928/ https://pubmed.ncbi.nlm.nih.gov/32957928/ https://pubmed.ncbi.nlm.nih.gov/32957928/ journal of current biomedical reports jcbior.com volume 3, number 2, 2022 eissn: 2717-1906 1 review evaluation of the effect of vaccination on transmissibility and pathogenicity of omicron variant and its comparison with other sars-cov-2 variants neda sinaei1, hamed hekmatnezhad2,*, nafise dani3, mona keivan4, ashkan roozitalab5 1department of biotechnology, iranian research organization for science and technology, tehran, iran 2department of basic sciences, sari agricultural sciences and natural resources university, sari, iran 3department of biology, science and research branch, islamic azad university, tehran, iran 4student research committee, school of medical, kermanshah university of medical sciences, kermanshah, iran 5department of biomedical and diagnostic sciences, college of veterinary medicine, knoxville, tennessee, usa abstract the new variant of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has once again sounded the alarm on healthcare systems worldwide and has caused concern in some countries. this variant has been identified in south africa and initially called b.1.1.529 and later renamed omicron by the who. the transmissibility and immune evasion in the omicron variant (b.1.1.529) is higher than the previous variants. compared to the previous dominant variant, which was called the delta variant, the omicron variant has a very high transmission power but luckily, omicron's symptoms are less serious. according to the who, the omicron variant has the potential to re-infect people who already have other variants of sars-cov-2. omicron contains at least 32 mutations in the spike protein also other proteins that are required for viral replication and it is twice the size of delta variant. half of the mutations in this variant occur in the area of the virus through which they bind to the cells of the human body and cause infection. the omicron variant likely developed in one person during chronic infection with an immune system deficiency (possibly untreated hiv/aids). it is possible that injecting a booster dose of existing vaccines and subsequently increasing antibody levels will provide adequate protection and an appropriate barrier against omicron. the purpose of this article is to evaluate the omicron variant and compare it with other sars-cov-2 variants and the effect of a booster dose in preventing disease progression. keywords: sars-cov-2, omicron, b.1.1.529, covid-19, vaccines 1. introduction the fire under the ashes of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) flared up again, this time from south africa. the new sars-cov-2 variant was detected in specimens collected in botswana on november 11, 2021, and in south africa on november 14, 2021 [1]. the who *corresponding author: hamed hekmatnezhad, msc department of basic sciences, sari agricultural sciences and natural resources university, sari, iran tel/fax: +98 911 9915699 email: hamedhekmatnejad@yahoo.com https://orcid.org/0000-0002-5716-9240 received: january, 04, 2021 accepted: april, 07, 2022 reported the identified variant as variant b.1.1.529 on november 24, 2021. the new variant was initially called b.1.1.529 and was renamed omicron by who later [2]. in this review article, we have tried to examine the new variant of sars-cov-2 called omicron to take the necessary measures to reduce its prevalence. © the author(s) 2022 https://jcbior.com/ sinaei et al. 2 2. origins of the omicron variant the omicron variant likely developed in one person during chronic infection with an immune system deficiency (possibly untreated hiv/aids) [3]. the beta variant was identified in south africa last year may also have originated from an hiv-infected person. the first reports of omicron were of young people who usually had mild symptoms of covid-19 due to their young age. south africa has 7.7 million people living with hiv in 2019, the highest rate in the world [4]. in another hypothesis, one of the synthetic pathogens of covid-19 for laboratory mice could be the cause of omicron. the new variant of the sarscov-2 may have been transmitted to humans from domestic animals or wild mice [5]. 3. comparison of omicron variant with other variant of concern the omicron variant (lineage b.1.1.529) joins the four previous sars-cov-2 variants as a variant of concern (voc). these four variants include the alpha variant from the united kingdom (lineage b.1.1.7), the beta variant from south africa (lineage b.1.351), the gamma variant from brazil (lineage p.1), and the delta variant from india (lineage b.1.617.2) [6]. in the last two years, various variants such as alpha (b.1.1.7), beta (b.1.351), gamma (p.1), delta (b.1.617.2), and other types such as epsilon (b.1.427) / b.1.1429), zeta (p.2), eta (b.1.525), theta (p3), iota (b.1.526) and kappa (b.1.617.1), were caused concern in all countries. recently, the delta variant has caused widespread deaths worldwide, which by vaccination have reduced. research has shown that the omicron variant easily escapes the trap of antibodies, thus likely to have a significant spread in people, especially vaccinated people [7, 8]. 4. comparison of omicron variant with delta variant 4.1 transmissibility not much information is available about the transmissibility of omicron, but its replacing with delta as the dominant species will increase concerns [1]. importantly, due to the small number of patients in south africa, it is difficult to determine the rate of omicron transmission. studies of omicron spike protein mutations suggest that the omicron variant may have a faster transmission rate than the original sars-cov-2 virus, but it is difficult to compare with the delta type. the omicron variant is predicted to be more contagious than the delta variant but has milder symptoms than the delta variant [9]. 4.2 the severity of the disease presently, the severity of the disease is unclear in omicron infection. given the small number of cases attributed to the omicron variant to date, it's far hard to evaluate the severity of the disease. data from infected people in south africa show that there aren’t abnormal symptoms associated with the omicron variant, and in some people, it is asymptomatic or may have mild symptoms [10]. 5. mutations as mentioned, the cdc and who have separately classified the new sars-cov-2 variants into two groups: variants of concern (vocs) and variants of interest (vois). table 1 show the mutations of the spike protein of the vocs and vois proteins [11, 12]. the vois are variants that contain specific biomarkers linked with modifications. vois can lower the antibiotic neutralization caused by natural infection or vaccination, and can also reduce the effectiveness of a vaccine [13]. as mentioned, the rate of mutations in omicron is significantly higher. in this variant, there are at least 32 mutations in the spike protein and other proteins that are required for virus replication, such as nsp12 and nsp14.2. in contrast, in the highly infectious variant of delta, there were 16 mutations, of which 9 mutations occurred in the virus protein spike [14]. there are 30 amino acid substitutions in the spike protein of omicron, including three small deletions and one small insertion. totally, 15 of the 30 amino acid substitutions are in the receptor-binding domain (rbd) which facilitates the transmission of the virus into cells (table 2). there are also other changes and deletions in other regions of the genome. at least three omicron mutations help the virus escape detection by immune system antibodies [12, 15]. 6. expansion of omicron variant in the world omicron cases in many countries such as south africa, botswana, netherlands, portugal, united kingdom (england and scotland), australia, hong kong, canada, denmark, austria, italy, belgium, czech republic, france, sweden, spain, us, and iran sinaei et al. 3 have been approved. based on mathematical models, omicron is expected to cause over half of all coronavirus infections in the european union in the coming months [16]. 7. effectiveness and safety of vaccines the total dose of vaccine available worldwide to date is approximately 10 billion and 936 million, of which 4 billion and 467 million have been fully vaccinated. research on the effectiveness and safety of vaccines in recipients after a few months of vaccination shows that a third dose of the vaccine, especially in some populations such as the elderly and people with weak immune systems, is necessary to maintain community safety. regarding the effect of vaccination on omicron, currently there is no data to assess the serum ability of vaccinated individuals or those who have previously had sars-cov-2 disease. in immunization resulting from vaccination, the spike protein is the primary target. in the omicron variant, the rate of mutations in the spike protein is higher than in other variants. based on the number and location of these mutations and their comparison with previous variants, serum neutralizing activity have been expected to decrease significantly in vaccinated or previously infected individuals. injections of the third dose have been expected to reduce the incidence. vaccination will also reduce hospitalization and mortality [17, 18]. according to preliminary data, sinopharm, sputnik, and johnson & johnson vaccines are less safe than the omicron variant, but mrna-based vaccines, such as pfizer, moderna, and astrazeneca are safer against the omicron variant. mrna-based technology in vaccine development plays a more effective role in sars-cov2 inhibition [19, 20]. according to research at the kirby institute, neither the two doses of astrazeneca nor pfizer were able to elicit a strong immune response to neutralize omicron in the samples tested. for this reason, it is necessary to inject a booster dose more than before table 1. the mutations of the spike protein of the vocs and vois proteins variants of concern (vocs) types of variant mutations of the spike protein alpha (b.1.1.7) 69/70/144del, n501y, a570d, p681h, t716i, s982a, d1118h beta (b.1.351) l18f, d80a, d215g, r246i, k417n, e484k, n501y, d614g, a701v gamma (p.1) l18f, t20n, p26s, d138y, r190s, h655y, t1027i, d614g, k417t, e484k, n501y delta (b.1.617.2) t19r, g142d, 156/157del, r158g, l452r, t478k, d614g, p681r, d950n omicron (b.1.1.529) 69–70/142–144/211del a67v, t95i, y145d, l212i, ins214epe, t547k, d614g, h655y, n679k, p681h, n764k, d796y, n856k, q954h, n969k, l981f variants of interest (vois) epsilon (b.1.427) l452r, d614g epsilon (b.1.429) s13i, w152c, l452r, d614g zeta (p.2) l18f, t20n, p26s, f157l, e484k, d614g, s929i, v1176f eta (b.1.525) a67v, 69/70/144del, e484k, d614g, q677h, f888ll5f iota (b.1.526) t95i, d253g, s477n, e484k, d614g, a701v theta (p.3) 141/142/143del, e484k, n501y, p681ht95i, g142d kappa (b.1.617.1) e154k, l452r, e484q, d614g, p681r, and q1071h table 2. amino acid substitutions in the spike protein of omicron and rbd substitutions amino acid substitutions in the spike protein of omicron rbd substitutions del69-70, a67v, del142-144, y145d, del211, l212i, ins214epe, t547k, d614g, h655y, n679k, p681h, n764k, d796y, n856k, q954h, n969k, t95i, l981f g339d, s371l, s373p, s375f, k417n, n440k, g446s, s477n, t478k, e484a, q493r, g496s, q498r, n501y, y505h sinaei et al. 4 [8]. in the united states, three covid-19 vaccines, pfizer, moderna, and johnson & johnson’s, have been approved by the who to prevent covid-19 disease. pfizer or moderna are preferred. people may get johnson & johnson’s janssen covid-19 vaccine in some countries depending on the circumstances (table 3) [21]. the pfizer and moderna vaccines companies have did announced that they can produce the omicron vaccine in 100 days. the uk health security agency (ukhsa) has suggested that vaccine immunity within 25 weeks of receiving the second dose is less than 10% for the omicron type and 40% for the delta variant. according to research at the university of oxford, a triple vaccination program against omicron is required. mutations in omicron are similar to other voc, so vaccination may inhibit the omicron variant. as mentioned, there are more than 30 mutations in the spike protein of the omicron variant, so researchers are focusing on omicron sequencing data. based on this information, immunization from vaccination can to some extent prevent sars-cov-2 from entering cells. cellular immunity will develop after vaccination and then t lymphocytes attack the virus-infected cells and by producing perforin and protease enzymes (caspase), induce apoptosis. in general, t lymphocytes are involved in the prevention of severe disease, and with the completion of research, vaccine efficacy data may indicate that the new omicron variant will not significantly cause death in vaccinated populations. currently, the most important and best solution is to increase vaccination and booster dose injection. but billions of people around the world are not vaccinated. only 7% of the african population has received both doses of the vaccine [22, 23]. 8. conclusion after overcoming the high mortality rate by the delta variant, the sudden emergence of the omicron variant with a high number of mutations has caused concern. in this study, omicron was shown to be about ten times more infectious than the other variants and twice as infectious as the delta variant. also, the ability to vaccine-escape in omicron is more than other variants and twice more than the delta variant. we showed that the booster dose injection would positively affect the severity of the disease, and we also tried to investigate spike protein mutations in different variants and omicron. currently, there is little information about the new variant of omicron, so masks, gloves, three-layer masks, quarantine of infected people, and vaccination (preferably mrna vaccines) should still be used to prevent the spread of the disease. the risk of death from people with the omicron variant isn't yet fully understood, but in the elderly, people with a history of diseases such as high blood pressure, diabetes, and those who haven't been vaccinated are at higher risk for death. authors’ contributions ns, hh conducted the study design. nd, mk, ar data collection. hh, nd, mk, ar drafting the article. ns, hh critical revisions. all authors read and approved the final version of article. conflict of interests there is no conflict of interest. ethical declarations not applicable. table 3. people who are eligible for a booster dose vaccines types who receives the booster dose? when should receive the booster dose? which vaccine can be given as a booster? pfizer people 18 years and older normally at least 6 months after the first dose of the covid-19 vaccine pfizer or moderna moderna people 18 years and older normally at least 6 months after the first dose of the covid-19 vaccine pfizer or moderna johnson & johnson’s people 18 years and older normally at least 2 months after receiving your j&j/janssen covid-19 vaccination pfizer or moderna sinaei et al. 5 financial support self-funded. references 1. fan y, li x, zhang l, wan s, zhang l, zhou f. sars-cov-2 omicron variant: recent progress and future perspectives. signal transduct target ther. 2022; 7(1):141. 2. world health organization classification of omicron (b.1.1.529): sars‐cov‐2 variant of concern. accessed november 26, 2021. availible from: https://www.who.int/news/item/26-112021-classification-of-omicron-(b.1.1.529)-sars-cov-2-variant-ofconcern 3. tarcsai kr, corolciuc o, tordai a, ongrádi j. sars-cov-2 infection in hiv-infected patients: potential role in the high mutational load of the omicron variant emerging in south africa. geroscience. 2022:1-9. 4. roomaney ra, van wyk b, cois a, 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https://doi.org/10.1016/j.vacun.2022.06.003 https://doi.org/10.1016/j.vacun.2022.06.003 https://doi.org/10.1016/j.vacun.2022.06.003 https://pubmed.ncbi.nlm.nih.gov/34929158/ https://pubmed.ncbi.nlm.nih.gov/34929158/ https://doi.org/10.52547/jcbior.2.1.3 https://doi.org/10.52547/jcbior.2.1.3 https://doi.org/10.52547/jcbior.2.1.3 https://doi.org/10.52547/jcbior.2.1.3 journal of current biomedical reports jcbior.com volume 1, number 1, 2020 1 original research bacterial etiology and antibiotic susceptibility pattern of female patients with urinary tract infection referred to imam khomeini hospital, ahvaz, iran, 2019 sousan akrami1.2, reza abouali2, mohammad mostafa olapour2, rozhin heidary lal abady2, hamid yazdaninejad2, arshid yousefi-avarvand3,* 1 department of microbiology, faculty of medicine, ahvaz jundishapur university of medical sciences, ahvaz, iran 2 student research committee, ahvaz jundishapur university of medical sciences, ahvaz, iran 3 department of medical laboratory sciences, school of para medicine, ahvaz jundishapur university of medical sciences, ahvaz, iran abstract urinary tract infection (uti) is one of the most frequent infections among women, and if untreated could lead to severe complications. the treatment of uti is difficult due to the appearance of pathogens with increasing resistance to antimicrobial agents. this study thus aimed to determine the bacterial etiological pathogens of uti and the antibiotic sensitivity pattern of the pathogens isolated. this descriptive cross-sectional study was performed from march to september 2019 on a total of 339 women referred to imam khomeini teaching hospital in ahvaz, iran. mid-stream urine samples were collected from the patients and were cultured. the presence of significant bacteriuria (> 105 cfu/ml) was determined using the plate count method. the antimicrobial susceptibility test was done by the standard disk diffusion method. the most frequently isolated pathogens were escherichia coli (54.8%), klebsiella (18.2%), pseudomonas aeruginosa (9.9%), proteus (8%), and acinetobacter (5.1%). e. coli, as the most common pathogen of utis, showed the most resistance to cephalosporins and the least resistance to imipenem. according to the findings, e. coli was the most common cause of uti in our region. considering the rate of uti, and the importance of preventing its severe complications, a survey of regional resistance patterns and timely treatment can control the development of its resistant bacteria. keywords: urinary tract infection; antibiotic resistance; uropathogens 1. introduction bacterial infections pose serious threats to public health and affect millions of people annually [1]. urinary tract infection (uti), as one of the most common bacterial infections, is known as the second most common cause of infection in the human body [2, 3]. thus, the lack of proper diagnosis and treatment of utis can lead to severe complications, such as urinary tract disorders, wounds in renal parenchyma, * corresponding author: dr. arshid yousefi-avarvand, ph.d department of medical laboratory sciences, school of para medicine, ahvaz jundishapur university of medical sciences, ahvaz, iran tel/fax: +98 61 33738317 email: arshid.yousefi5@gmail.com http://orcid.org/0000-0002-3987-9820 received: july, 26, 2020 accepted: august, 09, 2020 hypertension, and uremia. in pregnant women, uti can also lead to preterm labor and even abortion [4, 5]. the urinary tract is naturally devoid of any microorganisms, and infection occurs when any bacteria, virus, fungus, or parasite infects the urinary tract [2]. the rate of utis in developing countries is estimated to be at least 250 million cases per year. this infection is usually caused by bacteria, especially gram-negative bacteria, such as escherichia coli https://jcbior.com/ akrami et al. 2 which is seen in all age groups in both sexes, though it is more likely to occur in young women [6, 7]. over the half of women develop uti at least once in their lifetime, primarily in the lower urinary tract, which includes the urethra and bladder. therefore, identifying the type of bacteria that cause uti and prescribing the appropriate antibiotics to prevent its main complications such as urinary tract disorders, premature birth, and abortion seem indispensable [8]. antimicrobial sensitivity testing lab reports, intended to determine the bacterial etiological pathogens of uti, are usually released within 48 hours after receiving the samples in the laboratory. for this reason, in most cases, experimental treatment is recommended by the physicians, as antibiotic resistance pattern studies of pathogens and epidemiologic studies can be helpful in the treatment of utis [9]. in addition, as the resistance pattern in any infectious area in the body changes over time, new studies are thus needed to determine the etiological pathogens of the infection. given the prevalence of utis and the importance of preventing its severe complications, this study was thus intended to determine the most common strains causing utis and the antibiotic resistance pattern of such strains in the city of ahvaz. 2. materials and methods 2.1. urine samples the present study was performed on 341 patients admitted to imam khomeini teaching hospital in ahvaz, iran, during a six-month period from april to september 2019. to that end, the samples collected from midstream urine of the patients were transferred to the laboratory. then, they were cultured in blood agar and macconkey agar medium (merck, germany) and were incubated at 37 °c for 24 hours. cultures containing more than one bacterial strain were considered as contamination and excluded. all the isolates were identified using the conventional bacteriologic methods (i.e., microscopical and appropriate biochemical tests). 2.2. antibiotic susceptibility antimicrobial sensitivity tests were carried out by the disc diffusion technique using muller hinton agar (merck, germany) in accordance with the clinical and laboratory standards institute (clsi) recommendations [10]. imipenem (10 µg), ampicillin (10 µg), amikacin (30 µg), cefazolin (30 µg), ceftriaxone (30 µg), cephalexin (30 µg), cefixime (5 µg), gentamicin (10 µg), vancomycin (30 µg) and penicillin (10 units) all from padtanteb, iran. 3. results totally, 352 bacterial strains were isolated from the 339 urine samples of women screened for bacterial utis in this study. data analysis showed that the highest prevalence was for e. coli (54.8%), while enterococcus faecalis (1.1%) was the least abundant (table 1). table 2 shows the resistance pattern of isolates to different antibiotics. the results of antibiotic susceptibility test of the bacterial isolates indicated that a high proportion of the test organisms were sensitive to amikacin and imipenem. amikacin was, in turn, found to be very effective against all the isolates. besides, most of the isolates were sensitive to imipenem, and high-level resistance was seen in ceftriaxone, cephalexin, and cefazolin. table 1. distribution of organisms isolated from female patients with utis organism number (%) escherichia coli 193 (54.8) klebsiella pneumoniae 64 (18.2) pseudomonas aeruginosa 35 (9.9) proteus spp. 28 (8) acinetobacter spp. 18 (5.1) staphylococcus aureus 10 (2.8) enterococcus faecalis 4 (1.1) akrami et al. 3 4. discussion the arbitrary use of antibiotics in many cases causes uti not to be isolated, which in turn may lead to the failure of treatment [11]. to determine the antibiotic resistance pattern of isolates from a urine culture, there are several reports in different parts of the world, including iran. for instance, raeeszadeh et al. studied 114 patients with urinary duct infections and shows that the most commonly isolated pathogen was e. coli (74%), while the least isolated one was related to pseudomonas spp. (1%). also, the most and the least susceptibility in e. coli were for nitrofurantoin and sulfamethoxazole, respectively [12]. in another research, jabrodini et al. studied 1300 urine samples to determine the etiological pathogens causing uti and their antibiotic resistance profile. their results showed that 247 samples (19%) were positive for bacterial growth, 148 urine samples (11.3%) were positive for e. coli and klebsiella pneumoniae. besides, the most and the least antibiotic resistance in e. coli was found to be to nalidixic acid (59.3%) and nitrofurantoin (22.2%), respectively. for k. pneumoniae strains, the most and the least antibiotic resistance was observed in nitrofurantoin (67.5%) and ciprofloxacin (25%) [13]. similarly, studying 462 urine samples collected from patients with uti in bangladesh, reported that 100 of the samples were found to be culture positive. indeed, e. coli (70%) was predominant, followed by klebsiella spp. (11%) and enterococcus spp. (6%). the results also revealed that about 96% of uropathogens were sensitive against imipenem, and 75% against amikacin. moreover, the most antibiotic resistance in e. coli was to nalidixic acid (84.3%) [14]. in other research, shokoohi et al. studied 1400 urine samples from outpatients in hamadan, 235 samples were positive, of which 105 cases (44.7%) were related to men, and 130 cases (55.4%) were women. e. coli isolated from 141 (60%) cases and was the most common organism. in this study, the resistance of e. coli to ceftriaxone and cefixime was 55% and 50%, respectively. the study showed that the most effective antibiotic for e. coli is amikacin (70% sensitivity) [15]. as main limitations of retrospective studies, we only access to the results of locally available antibiotics. also, the results obtained from a regional hospital and may not be generalized to entire area. overall, e. coli isolates showed high resistance to conventional antibiotics for the treatment of utis. in t a b le 2 . a n ti b io ti c s u sc e p ti b il it y p a tt e rn s o f o rg a n is m s o r g a n is m s c e p h a le x in c e fa z o li n c e fi x im e c e ft r ia x o n e a m p ic il li n p e n ic il li n v a n c o m y c in im ip e n e m g e n ta m ic in a m ik a c in s % r % s % r % s % r % s % r % s % r % s % r % s % r % s % r % s % r % s % r % e sc h e ri c h ia c o li 3 4 6 4 3 5 6 5 4 6 5 4 7 2 2 8 9 2 8 6 5 3 5 8 6 1 4 k le b si e ll a s p p . 3 3 6 7 2 8 7 2 1 8 8 2 3 8 6 2 8 6 1 4 4 6 5 4 8 2 1 8 p se u d o m o n a s a e ru g in o sa 9 5 5 6 6 3 4 7 1 2 9 p ro te u s sp p . 4 6 5 4 1 7 8 3 3 6 6 4 1 0 0 0 6 7 3 3 7 6 2 4 a c in e to b a c te r sp p . 3 3 6 7 1 0 0 0 7 0 3 0 8 2 1 8 s ta p h y lo c o c c u s a u re u s 2 0 8 0 6 0 4 0 e n te ro c o c c u s fa e c a li s 1 0 0 0 2 5 7 5 1 0 0 0 5 0 5 0 akrami et al. 4 our study, the best antibiotics to treat e. coli utis were found to be imipenem and amikacin. the findings also showed that the level of antibiotic resistance in different regions in human body depends on different factors, including antibiotic use patterns. for this reason, periodic monitoring of antibiotic resistance is recommended to control the infection at its source. author contributions conception or design of the work ay, sa; data collection: ra, rh; data analysis and interpretation mmo, hy, ra; drafting the article: sa; critical revision of the article: ay. all authors read and approved the final version of manuscript. conflict of interests authors declare there is no conflict of interest. ethical declarations this article does not contain any studies with human participants and only samples leftovers in laboratory were used. the study design was approved by the regional ethics committee of ahvaz jundishapur university of medical sciences (ir.ajums.rec.1398.837). financial support this work was supported by a grant number 98s81 from vice chancellor for research, ahvaz jundishapur university of medical sciences. references 1. bloom de, cadarette d. infectious disease threats in the twenty-first century: strengthening the global response. front immunol. 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https://pubmed.ncbi.nlm.nih.gov/28248946/ https://pubmed.ncbi.nlm.nih.gov/17378940/ https://pubmed.ncbi.nlm.nih.gov/17378940/ https://pubmed.ncbi.nlm.nih.gov/17378940/ https://pubmed.ncbi.nlm.nih.gov/17378940/ http://rjms.iums.ac.ir/browse.php?a_id=3404&sid=1&slc_lang=en http://rjms.iums.ac.ir/browse.php?a_id=3404&sid=1&slc_lang=en http://rjms.iums.ac.ir/browse.php?a_id=3404&sid=1&slc_lang=en http://rjms.iums.ac.ir/browse.php?a_id=3404&sid=1&slc_lang=en https://journal.rums.ac.ir/browse.php?a_id=4049&sid=1&slc_lang=en https://journal.rums.ac.ir/browse.php?a_id=4049&sid=1&slc_lang=en https://journal.rums.ac.ir/browse.php?a_id=4049&sid=1&slc_lang=en https://journal.rums.ac.ir/browse.php?a_id=4049&sid=1&slc_lang=en https://journal.rums.ac.ir/browse.php?a_id=4049&sid=1&slc_lang=en https://journal.rums.ac.ir/browse.php?a_id=4049&sid=1&slc_lang=en https://www.banglajol.info/index.php/jbas/article/view/15681 https://www.banglajol.info/index.php/jbas/article/view/15681 https://www.banglajol.info/index.php/jbas/article/view/15681 https://www.banglajol.info/index.php/jbas/article/view/15681 http://psj.umsha.ac.ir/article-1-474-en.html http://psj.umsha.ac.ir/article-1-474-en.html http://psj.umsha.ac.ir/article-1-474-en.html http://psj.umsha.ac.ir/article-1-474-en.html http://psj.umsha.ac.ir/article-1-474-en.html journal of current biomedical reports jcbior.com volume 3, number 2, 2022 eissn: 2717-1906 1 original research the characterization of oxaliplatin-induced peripheral neuropathy using electromyography in gastrointestinal cancer patients babak bakhshayesh eghbali1, sara ramezani1, hamid saeidi saedi2, nazanin rahman amlash3, ehsan kazemnezhad leili4, hamidreza hatamian1, cyrus emir alavi1,* 1neuroscience research center, school of medicine, guilan university of medical sciences, rasht, iran 2gi cancer screening and prevention research center, school of medicine, guilan university of medical sciences, rasht, iran 3student research committee, neuroscience research center, school of medicine, guilan university of medical sciences, rasht, iran 4road trauma research center, school of nursing and midwifery, guilan university of medical sciences, rasht, iran abstract oxaliplatin-induced peripheral neuropathy (oipn) is a common dose-dependent chemotherapy complication in gastrointestinal cancer (gic). this side effect may restrict therapeutic dose elevation of oxaliplatin. here, oipn frequency and determinants of neuropathy appearance in oxaliplatin-treated gic patients. a total of 102 gic patients who underwent chemotherapy with fluorouracil, folinic acid and oxaliplatin (folfox4) regimen participated in this longitudinal study. electromyography (emg) was accomplished for ulnar, radial, sural, peroneal nerves and superficial peroneal nerve (spn) before, 3, and 6 months after treatment. national cancer institute-common toxicity criteria v.3 and clinical version of the total neuropathy score were used for the neuropathy diagnosis at six months after treatment onset. of all entered patients, twelve people discontinued this study, and five patients passed away. about 85 patients remained three and six months after chemotherapy onset. approximately 95% of patients three months after chemotherapy demonstrated oipn manifestations. finally, data for 81 patients having neuropathy were analyzed. mean age of patient 64.0±10.9 years. there were about 3.7%, 30.9%, 63% grade iii, ii, i of neuropathy, respectively. interestingly, a significant decrease in action potential (ap) amplitude of spn, sural and radial nerves but not ulnar and peroneal was observed after treatment onset. however, only the ulnar nerve indicated a substantial deceleration of nerve conduction. age, sex, weight, past medical diseases, smoking and acute neuropathy were not significantly associated with oipn. the occurrence of oipn is detectable by electrophysiological changes of spn, radial, and sural nerves at three and six months after starting chemotherapy with the folfox4 regimen. keywords: oxaliplatin, neuropathy, gastrointestinal cancer, chemotherapy, electromyography 1. introduction platinum compounds such as cisplatin, carboplatin, and oxaliplatin are among the alkaline agents that limit deoxyribonucleic acid (dna) *corresponding author: cyrus emir alavi, md neuroscience research center, school of medicine, guilan university of medical sciences, rasht, iran tel/fax: +98 13 33325783/+98 13 33339842 email: cyrusemiralavi@yahoo.com https://orcid.org/0000-0003-1664-0822 received: november, 22, 2021 accepted: february, 12, 2022 synthesis. cisplatin is used to treat especially metastatic gastrointestinal cancers (gics) [1-3]. oxaliplatin is used as a platinum analog and as a major component of a standard chemotherapy approach © the author(s) 2022 https://jcbior.com/ https://jcbior.com/ bakhshayesh eghbali et al. 2 with the composition of fluorouracil, folinic acid and oxaliplatin called folfox4 regimen in a variety of malignancies such as metastatic gic [4]. oxaliplatininduced peripheral neuropathy (oipn) is one of the most common side effects of the folfox4 regimen that occurs both during and after treatment and interferes with the chemotherapy process [5]. chemotherapy-induced peripheral neuropathies (cipns) including sensory, motor, and autonomic neuropathies are defined as the damage, inflammation, and degeneration of the peripheral nerve caused by chemotherapy [6]. based on the evidence, patients with cipn experienced some symptoms such as numbness, tingling, proprioception disorders, pain, and weakness of limbs. particularly, autonomic manifestations may be represented as some physiological abnormalities consisting of thermoregulation disturbance, blood pressure changes, and intestinal motility disorder and loss of involuntary reflexes [7]. the occurrence of cipn is dependent on the length of the sensory nerve and can be observed in the axons of myelinated and unmyelinated neural fibers. evidently, effective factors on the cipn development include age, dosage, and cumulative dose, and treatment duration, concurrent usage with other neurotoxic chemotherapeutic agents, alcohol consumption and diabetes [7]. it seems that improvement of oipn symptoms may be begotten very slowly and partially in the majority of folfox4-treated patients. likewise, restricting the dose increase of oxaliplatin is recommended immediately after oipn symptoms appear to prevent the stabilization of oipn manifestations. accordingly, early diagnosis of oipn possesses an especial clinical significance [4, 7]. recently, some studies have shown a direct association of acute neuropathy development with the occurrence and severity of cumulative neurotoxicity [8, 9]. reducing the dose of oxaliplatin is not an effective approach to decrease the severity of neuropathy because this approach is started when neuropathy has been stabilized. besides, the detection of acute toxicity may be beneficial for the prediction of neuropathy development. however, there is very little evidence to support a direct link between serum levels of this oxaliplatin and neurotoxicity. some studies have shown that there is no linear relationship between the blood level of the drug and the possibility of toxicity before cumulative dose occurrence, but after reaching the cumulative dose, the toxicity becomes dose-dependent [10]. mechanistically, permanent neural transduction in the length of the axon brings about the continuous discharge of voltage-dependent sodium channels, leading to peripheral neuropathy due to platinum compounds [11]. therefore, early diagnosis of neuropathy before the onset of clinical symptoms via electrodiagnostic test is very helpful [12]. given the influence of genetic and environmental factors on the oipn occurrence and severity, it seems that complications of folfox4 may be different in several ethnicities and geographical areas. so far, it has been provided few reports on the oipn in iranian gic patients. hence, this study aimed to investigate the occurrence and severity of peripheral neuropathy in gic patients treated with the folfox4 regimen after three and six months of treatment onset. here, the findings of the electrodiagnostic evaluation for the sensory and motor nerves before and after treatment were compared, as well. 2. materials and methods 2.1 study design and sample size a longitudinal study was designed. we used a consecutive sampling method where all patients figure 1. flow chart of the longitudinal study for gic patients referred to emg unit bakhshayesh eghbali et al. 3 presenting at a single institution were screened for study participation. the sample size was calculated based on the study of argyriou et al. that reported the oipn in gic 64% of patients [13], taking into account the 95% confidence level and measurement error of 0.1. about 10% was added to the final sample size to compensate for possible dropout. therefore, the final sample size was 102 who were entered in the study. however, twelve patients refused and five of those were dead. about 95% of 85 patients who remained in this study at three months after treatment onset reported the oipn. at the end of the study period, data for 81 oipn patients were analyzed (figure 1). 2.2 inclusion criteria of participants all patients with gic were treated with the folfox4 chemotherapy regimen for the four treatment courses. those continued this study until three months of chemotherapy onset. participants were evaluated using emg before chemotherapy began. 2.3 exclusion criteria of study the patients who passed away after three months of chemotherapy onset and the patients without available emg data at three months after chemotherapy onset were excluded. 2.4 study procedure this study was approved by the guilan university of medical sciences ethics committee (ir.gums.rec.1394.560) and was conducted in compliance with the guidelines of the declaration of helsinki. the written informed consent was obtained from the patients prior to their participation in the study. the study was conducted in razi educational, therapeutic and research in 2017 center. before starting chemotherapy, patients were referred to the electromyography (emg) unit to measure nerve conduction velocity (ncv) on the ulnar, radial, sural, and superficial peroneal nerve in an antidromic manner. sensory nerve action potential (ap) amplitude and velocity were calculated. motor conduction study of the common peroneal nerve, compound muscle ap amplitude and velocity assessment was also performed at the baseline (t0), at three months (t1) and six months (t2) of chemotherapy onset. patients were evaluated for neuropathy by one neurologist using and clinical criteria after six months of starting the chemotherapy course. neuropathy is clinically measured by two scales, national cancer institute-common toxicity criteria v.3 (nci-ctcv3) and the clinical version of the total neuropathy score (tnsc). during chemotherapy, patients were asked about the symptoms of acute neuropathy. grade one, two and three of neuropathy were considered as mild, moderate and severe neuropathy. 2.5 statistical analysis after collecting the data, statistical analysis was performed using spss v21 (ibm, new york, usa). fisher's exact and pearson's chi-squared tests were applied for nominal and categorical variables. independent t-test was used to compare continuous variables. the ap amplitude changes and ncv was compared over time via repeated-measures anova. post hoc analyses (bonferroni pairwise comparisons) were subsequently performed for the pairwise comparison. the level of significance was set at 0.05 for all statistical procedures. 3. results in this study, after a dropout of about 16.7%, 81 folfox4-treated gic patients with neuropathy were studied in terms of electrodiagnostic properties of sensory and motor nerves before and after 3 and 6 months of treatment. 42% of the samples were female and 58% were male. the mean and standard deviation (sd) of the age and weight were 64±10.9 years and 69.5±8.8 kg, respectively. among the total patients, underlying diseases including diabetes (n=25), hypertension (n=16), hyperlipidemia (n=20), ischemic heart disease (n=13), smoking (n=15) and symptoms of acute neuropathy during treatment (n=17) has been reported. supplementary figure 1 delineated the severity of neuropathic symptoms at six months of chemotherapy onset. it was reported that about 3.7% (n=3) grade three 30.86% (n=25), grade two, 62.96% (n=51) grade one and 2.47% (n=2) grade zero at six months after treatment onset, successively. based on the repetitive measure anova test, a significant difference was shown in the ap amplitude mean of spn, radial and sural nerves over time (p <0.001). there was no significant difference in the ap amplitude mean of the ulnar nerve (p=0.132) and bakhshayesh eghbali et al. 4 peroneal (p=0.624) nerve over time. see more information in table 1. supplementary figure 2 depicts the mean of ap amplitude changes percentage of the nerves over times. it was revealed that the mean of ap amplitude changes percentage of spn (23.99 ± 26.46), sural (25.72±29.04) and radial (16.53±27.02) nerves from t0 to t1 were dramatically different as compared to those in spn (2.62±5.29), sural (3.48±2.91) and radial (2.35±1.43) from t1 to t2 (p <0.001). similarly, a substantial difference was displayed in the mean of ap amplitude changes percentage of the same nerves from t0 to t2 relative to t1 to t2 (p <0.001). however, there was no a remarkable difference in the mean of ap amplitude changes percentage of spn, sural and radial nerves from t0 to t1 in comparison with those in spn (27.03±8.69), sural (27.89±9.66) and radial (17.98±5.4) nerves from t0 to t2 (p >0.05). as shown in table 2 there was no significant difference in ncv mean of peroneal (p=0.693), sural (p=0.263), radial (p=0.693), nerves, and spn (p=0.849) over time. however, there was a substantial reduction (p=0.023) of the ncv mean for ulnar nerve after three (61.83±21.12) and six (60.67±20.03) months of chemotherapy onset relative to baseline (66.09±25.86). the results of pairwise comparisons illustrated a significant attenuation of ncv mean at three (p=0.042) and six months after treatment onset versus baseline (p=0.006). according to table 3, it was not found a significant association of severity of neuropathy with diabetes, hyperlipidemia, hypertension, acute neuropathy, and a history of ischemic heart disease and smoking (p >0.05), age (p=0.667), weight (p=0.441) and gender (p=0.555). 4. discussion in this study, we investigated the effect of chemotherapy with folfox4 on the electrodiagnostic features of sensory and motor nerves in gic patients. according to the study of velasco et al. [14], the mean age of the subjects was 64 years. in contrast to this prior study, the majority of patients in our study were male. the findings of our study indicated that the frequency of each of the underlying diseases was less than one-third of the total patients. about 21% of the patients of this study had acute neuropathy symptoms during treatment. according to nci-ctcv3 and tnsc, 3.7% of patients had grade table 1. the ap amplitude mean of indicated nerves overtime in gic patients with neuropathy induced by folfox4 regimen variable time mean ± sd p value* peroneal nerve baseline 4.09±2.29 0.624 after 3 months 4.13±2.34 after 6 months 4.17±2.33 spn baseline 15.55±9.48 <0.001 after 3 months 11.2±7.61 after 6 months 10.7±7.57 sural nerve baseline 18.74±12.09 <0.001 after 3 months 13.45±10.13 after 6 months 13.01±9.91 ulnar nerve baseline 23.27±18.67 0.132 after 3 months 22.8±18.42 after 6 months 22.08±17.85 radial nerve baseline 24.58±13.85 <0.001 after 3 months 20.38±13.67 after 6 months 20.15±14.18 *repeated-measeure anova table 2. the ncv mean of indicated nerves overtime in gic patients with neuropathy induced by folfox4 regimen variable time mean ± sd p value peroneal nerve baseline 49.03±7.37 p=0.603 p1,2=0.999 p1,3=0.999 p2,3=0.999 after 3 months 48.95±7.30 after 6 months 49.25±7.25 spn baseline 71.67±33.47 p=0.849 p1,2=0.999 p1,3=0.999 p2,3=0.999 after 3 months 72.55±26.99 after 6 months 72.49±28.36 sural nerve baseline 73.28±30.07 p=0.163 p1,2=0.365 p1,3=0.421 p2,3=0.999 after 3 months 71.49±26.80 after 6 months 70.94±28.90 ulnar nerve baseline 66.09±25.86 p=0.023 p1,2=0.142 p1,3=0.042 p2,3=0.006 after 3 months 61.83±21.12 after 6 months 60.68±20.03 radial nerve baseline 55.44±9.59 p=0.693 p1,2=0.999 p1,3=0.999 p2,3=0.999 after 3 months 55.49±8.33 after 6 months 55.15±8.73 bakhshayesh eghbali et al. 5 three of neuropathy. these results corroborated previous reports in this regard [13, 14]. the percentage of the patients with grades two and three of neuropathy in our study was approximately 34%. however, another study exhibited grade two of neuropathy in almost 20% of gic patients treated with the folfox4 regimen [15]. overall, the neuropathy rate in our study was 95% while others have reported the neuropathy incidence by 68% and 82% of total patients receiving folfox4 chemotherapy [16]. an explanation for this result may be that various ethnicities may be differentially prone to experience the neuropathy presumably due to the differences in their diet, genetic and climate [17]. another finding of our study was that hypertension and smoking increased by 20% and 18% the frequency of moderate and severe neuropathies in folfox4-treated gic patients, respectively. diabetes, hyperlipidemia, and ischemic heart disease resulted in an enhancement of moderate and severe neuropathies frequency by less than 10%. unexpectedly, we witnessed that the percentage of mild oipn in the patients with a diabetes history was higher than that in non-diabetic patients. nevertheless, there is no association of the severity of neuropathies with none of past medical histories in this study. this result of our study was inconsistence with some prior studies which have suggested an association between the incidence of clinical neuropathy and past medical diseases such as diabetes [18]. it is believed that high blood glucose can harm the peripheral nerves presumably through some pathophysiological mechanisms such as vascular dysfunction, hyperosmolarity and edema of the nerve trunk [19]. albeit hyperglycemic instability and subsequently loss of axonal integrity due to preexisting diabetes may increase the risk of cipn, it is thought that the cipn severity might be influenced the rate of neural regeneration in cancer patients with diabetes history [20, 21]. although some investigations showed a relationship between neuropathy and age [14], but there was no association between neuropathy severity and age, weight and gender of patients in the present study. the rate of acute neuropathy in this study was lower than in other studies [22, 23], which is possible to be related to ethnic discrepancies and geographic differences. as an important finding of our study, a significant decrease was disclosed in ap amplitude of spn, sural and radial nerves. however, ncv decrease was clarified only in the ulnar nerve. herein, park and his colleagues displayed the reduction of ap amplitude in the sural and radial nerves [12]. decreased ap amplitude in these nerves can be suggested as determinants of high-grade neuropathy. according to the finding of our study, the previous studies have not table 3. the association of neuropathy severity in folfox4-treated gic patients with underlying diseases and acute neuropathy ns status mild neuropathy moderate to severe neuropathy p-value age (mean±sd) 63.6±11.2 64.7±10.6 0.667 sex n (%) male 32(68.1) 15(31.9) 0.550 female 21(61.8) 13(38.2) weight (mean±sd) 68.9±8.1 70.5±9.9 0.441 diabetes n (%) yes 18(72.0) 7(28.0) 0.406 no 35(62.5) 21(37.5) hypertension n (%) yes 8(50.0) 8(50.0) 0.147 no 45(69.2) 20(30.8) hyperlipidemia n (%) yes 11(55.0) 9(45.0) 0.258 no 42(68.9) 19(31.1) ischemic heart disease n (%) yes 7(53.8) 6(46.2) 0.338 no 46(67.6) 22(32.4) smoking n (%) yes 12(80.0) 3(20.0) 0.189 no 41(62.1) 25(37.9) acute neuropathy n (%) yes 12(70.6) 5(29.4) 0.615 no 41(64.1) 23(35.9) bakhshayesh eghbali et al. 6 yet confirmed the clinical importance of ncv in the diagnosis of folfox4-induced neuropathy. oxaliplatin neurotoxicity in the folfox regimen is known as a dose-limiting indicator. dose reduction happens after neuropathy development and axon damage. therefore, reducing the dose of the chemotherapeutic agent is useless. moreover, it seems that the application of clinical examination and grading systems based on the nci-ctcv3 and tnsc may underestimate neuropathy development in gic patients receiving folfox4 regimen [24, 25]. accordingly, electrodiagnostic features might possess a clinical utility for the early detection of neuropathy in patients treated with oxaliplatin before the occurrence of toxicity induced by oxaliplatin. notably, performing emg at least after 3 and 6 months of treatment onset may be suggested to detect seemingly healthy patients in terms of neurology. electrophysiological property of peripheral nerves appears to be a sensitive measure for the diagnosis of neuropathy caused by chemotherapy. in addition, the electrodiagnostic findings may be a prognostic marker for oxaliplatin-induced neurotoxicity, refined treatment strategies, and facilitate neuroprotective strategies. this study had some limitations. our study was conducted with a little sample size. it is suggested to design a large-scale study in the future. furthermore, we cannot investigate the serum levels of inflammation and oxidative stress markers in the patients. besides, we did not measure chemotherapyinduced toxicity at various time points of study. it is seriously recommended to design a next study that incorporates the above considerations. the occurrence of neuropathy due to the folfox4 therapeutic approach can be detected by emg at least three months after chemotherapy onset. the trend of ap amplitude changes for the sural and radial nerves, and spn at various time points of treatment might be significant indicators of neuropathy development after chemotherapy before chemotherapy-induced toxicity happens. the data obtained from emg may have a clinical utility to identify the patients treated with the folfox4 regimen and are susceptible to neuropathy. supplementary files supplementary file 1. authors’ contributions conception or design of the work: bbe, sr, hss, nra, cea; data collection: bbe, sr, hss, nra, hh; data analysis and interpretation: ekl, sr, nra; drafting and critical revision of the manuscript: bbe, sr, hss, nra, and cea. all authors read and approved the final version of the manuscript. conflict of interests there is no conflict of interest. ethical declarations all applicable international, national, and/or institutional guidelines for the study of human participants were performed by the authors. the study was approved by the ethics committee of guilan university of medical sciences that conforms to the provisions of the declaration of helsinki. acknowledgments the authors would like to thank the all staff of the neuroscience research center and the clinical research development unit of poursina hospital at guilan university of medical sciences, rasht, who helped us in performing the data collection and study processes. financial support this project was not funded references 1. dasari s, tchounwou pb. cisplatin in cancer therapy: molecular mechanisms of action. eur j pharmacol. 2014;740:364-78. 2. johnstone tc, suntharalingam k, lippard sj. the next generation of platinum drugs: targeted pt (ii) agents, nanoparticle delivery, and pt (iv) prodrugs. chem rev. 2016;116(5):3436-86. 3. köberle b, schoch s. 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revista dor. 2015;16(3):215-20. 9. gordon-williams r, farquhar-smith p. recent advances in understanding chemotherapy-induced peripheral neuropathy. f1000research. 2020;9. 10. pasetto lm, d’andrea mr, rossi e, monfardini s. oxaliplatinrelated neurotoxicity: how and why? crit rev oncol hematol. 2006;59(2):159-68. 11. lazić a, popović j, paunesku t, woloschak ge, stevanović m. insights into platinum-induced peripheral neuropathy–current perspective. neural regen res.. 2020;15(9):1623. 12. park sb, lin cs, krishnan av, goldstein d, friedlander ml, kiernan mc. long-term neuropathy after oxaliplatin treatment: challenging the dictum of reversibility. oncologist. 2011;16(5):708. 13. argyriou aa, polychronopoulos p, iconomou g, chroni e, kalofonos hp. a review on oxaliplatin-induced peripheral nerve damage. cancer treat rev. 2008;34(4):368-77. 14. velasco r, bruna j, briani c, argyriou aa, cavaletti g, alberti p, et al. early predictors of oxaliplatin-induced cumulative neuropathy in colorectal cancer patients. j neurol neurosurg psychiatry. 2014;85(4):392-8. 15. shimizu t, satoh t, tamura k, ozaki t, okamoto i, fukuoka m, et al. oxaliplatin/fluorouracil/leucovorin (folfox4 and modified folfox6) in patients with refractory or advanced colorectal cancer: post-approval japanese population experience. int j clin oncol. 2007;12(3):218-23. 16. kemeny n, garay ca, gurtler j, hochster h, kennedy p, benson a, et al. randomized multicenter phase ii trial of bolus plus infusional fluorouracil/leucovorin compared with fluorouracil/leucovorin plus oxaliplatin as third-line treatment of patients with advanced colorectal cancer. j clin oncol. 2004;22(23):4753-61. 17. tahrani aa, altaf qa, piya mk, barnett ah. peripheral and autonomic neuropathy in south asians and white caucasians with type 2 diabetes mellitus: possible explanations for epidemiological differences. j diabetes res. 2017;2017:1273789. 18. sempere-bigorra m, julián-rochina i, cauli o. chemotherapy-induced neuropathy and diabetes: a scoping review. curr oncol. 2021;28(4):3124-38. 19. dewanjee s, das s, das ak, bhattacharjee n, dihingia a, dua tk, et al. molecular mechanism of diabetic neuropathy and its pharmacotherapeutic targets. eur j pharmacol. 2018;833:472523. 20. hershman dl, till c, wright jd, awad d, ramsey sd, barlow we, et al. comorbidities and risk of chemotherapy-induced peripheral neuropathy among participants 65 years or older in southwest oncology group clinical trials. j clin oncol. 2016;34(25):3014-22. 21. khoshnoodi ma, ebenezer gj, polydefkis m. epidermal innervation as a tool to study human axonal regeneration and disease progression. exp neurol. 2017;287:358-64. 22. tofthagen c, donovan ka, morgan ma, shibata d, yeh y. oxaliplatin-induced peripheral neuropathy’s effects on healthrelated quality of life of colorectal cancer survivors. support care cancer. 2013;21(12):3307-13. 23. mizrahi d, park sb, li t, timmins hc, trinh t, au k, et al. hemoglobin, body mass index, and age as risk factors for paclitaxel-and oxaliplatin-induced peripheral neuropathy. jama netw open. 2021;4(2):e2036695-e. 24. gilchrist ls, tanner l. the pediatric-modified total neuropathy score: a reliable and valid measure of chemotherapyinduced peripheral neuropathy in children with non-cns cancers. support care cancer. 2013;21(3):847-56. 25. sałat k. chemotherapy-induced peripheral neuropathy—part 2: focus on the prevention of oxaliplatin-induced neurotoxicity. pharmacol rep. 2020;72(3):508-27. https://www.scielo.br/j/rdor/a/b4s8vxhvfnygfjgpkkvrhkf/?lang=en https://www.scielo.br/j/rdor/a/b4s8vxhvfnygfjgpkkvrhkf/?lang=en https://www.scielo.br/j/rdor/a/b4s8vxhvfnygfjgpkkvrhkf/?lang=en https://pubmed.ncbi.nlm.nih.gov/32201575/ https://pubmed.ncbi.nlm.nih.gov/32201575/ https://pubmed.ncbi.nlm.nih.gov/32201575/ https://pubmed.ncbi.nlm.nih.gov/16806962/ https://pubmed.ncbi.nlm.nih.gov/16806962/ https://pubmed.ncbi.nlm.nih.gov/16806962/ https://pubmed.ncbi.nlm.nih.gov/32209761/ 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https://pubmed.ncbi.nlm.nih.gov/33587134/ https://pubmed.ncbi.nlm.nih.gov/33587134/ https://pubmed.ncbi.nlm.nih.gov/33587134/ https://pubmed.ncbi.nlm.nih.gov/33587134/ https://pubmed.ncbi.nlm.nih.gov/22993026/ https://pubmed.ncbi.nlm.nih.gov/22993026/ https://pubmed.ncbi.nlm.nih.gov/22993026/ https://pubmed.ncbi.nlm.nih.gov/22993026/ https://pubmed.ncbi.nlm.nih.gov/32347537/ https://pubmed.ncbi.nlm.nih.gov/32347537/ https://pubmed.ncbi.nlm.nih.gov/32347537/ journal of current biomedical reports jcbior.com volume 1, number 1, 2020 1 original research evaluation the intestinal level of lcn2/ngal in patients with clostridium difficile infection in the south of iran seyedeh mahsan hoseini-alfatemi1, hadi sedigh ebrahim-saraie2,* 1 pediatric infections research center, research institute for children's health, shahid beheshti university of medical sciences, tehran, iran 2 razi clinical research development unit, razi hospital, guilan university of medical sciences, rasht, iran abstract clostridium difficile is a gram-positive obligate anaerobic bacterium that recognized an important pathogen of humans. the present study aimed to evaluate the intestinal level of neutrophil gelatinaseassociated lipocalin (ngal) in patients with c. difficile infection (cdi) as a disease activity marker in the south of iran. this cross-sectional study conducted from october 2017 to june 2018 on patients referred to nemazee and amir hospital in shiraz, south of iran. patients less than two years old were excluded from the study. the study population was consist of 46 cases (symptomatic patients that confirmed as a cdi), and 21 control individuals (asymptomatic patients colonized by c. difficile). c. difficile isolates were identified by conventional microbiological producers and amplification of housekeeping gene by pcr method. the level of ngal was determined by enzyme-linked immunosorbent assay (elisa) according to the instructions of the kit manufacturer. the results showed that the level of ngal in symptomatic patients' (case group) was higher than asymptomatic carriers (control group), 5.9 ng/ml vs. 4.1 ng/ml; however the observed difference was not statistically significant. also, in both groups, the mean level of ngal was significantly higher in patients with gastrointestinal diseases than others. in summary, despite all the limitations, the results of the present study indicate that the intestinal level of ngal is a biological indicator of intestinal inflammation, regardless of cdi. however, further study needs to elucidate the role of ngal in inflammation caused by bacterial infections. keywords: clostridium difficile; cdi; lipocalin-2; ngal; gastrointestinal diseases 1. introduction clostridium difficile is a gram-positive obligate anaerobic bacterium that recognized an important pathogen of humans. the organism can cause serious diseases such as pseudomembranous colitis (pmc), toxic megacolon, and perforation of the colon [1, 2]. c. difficile is responsible for the majority of cases of nosocomial diarrhea [3]. due to spore production, *corresponding author: dr. hadi sedigh ebrahim-saraie, ph.d razi clinical research development unit, razi hospital, guilan university of medical sciences, rasht, iran tel/fax: +98 13 33542460 email: seddigh.hadi@gmail.com, seddigh.hadi@gums.ac.ir https://orcid.org/0000-0001-8339-5199 received: august, 03, 2020 accepted: august, 15, 2020 they are often highly resistant to sterilizations and disinfectants which become them a successful healthcare-associated pathogen [4]. neutrophil gelatinase-associated lipocalin (ngal) also known as lipocalin-2 (lcn2) belongs to a family of small secretory proteins that are expressed by different types of cells, the richest source of which is neutrophils [5, 6]. the results of experiments show a https://jcbior.com/ hoseini-alfatemi et al. 2 systematic increase in the expression level of lcn2 in various models of colitis, including human inflammatory bowel diseases (ibd) [7]. besides, human lipocalin has been reported to be increased in patients with ulcerative colitis [7]. recently, it has been shown that lcn2 is a real-time indicator for kidney disease and meets all the criteria required for a biomarker [8]. expression of lcn2 in adipose tissue has been increased in various experimental models of obesity and obese individuals, indicating that lcn2 may be involved in inflammatory disorders [7]. chassaing et al. introduced the lcn2 in the stool as an indicator to identify inflammation caused by inflammatory bowel disease, which could be linked to various intestinal infections and used as a biomarker index [7, 9]. the present study aimed to evaluate the intestinal level of ngal in patients with c. difficile infection (cdi) as a disease activity marker in the south of iran. 2. materials and methods 2.1. study design this cross-sectional study conducted from october 2017 to june 2018 on patients referred to nemazee and amir hospital in shiraz, south of iran. patients less than two years old were excluded from the study. the study population was consist of 46 cases (symptomatic patients that confirmed as a cdi), and 21 control individuals (asymptomatic patients colonized by c. difficile). c. difficile were identified by conventional microbiological producers including characteristics on cycloserine-cefoxitin fructose agar (ccfa) (mast diagnostic, uk) and amplification of triose phosphate isomerase (tpi) housekeeping gene by pcr method [10, 11]. this study was following the declaration of helsinki and ethical permission was sought from the institutional ethics committee of shiraz university of medical sciences (approval no. ir.sums.rec.1396.s872). 2.2. measurement of ngal level the level of ngal was determined by enzymelinked immunosorbent assay (elisa) according to the instructions of the lipocalin-2/ngal human elisa kit manufacturer with a detection limit of 0.02 ng/ml (biovendor, czech republic). the samples were diluted to a ratio of 1:10 according to the kit protocol and previous studies in this field. after performing the elisa test preparation steps, the optical density of each well was read with an elisa reader at a wavelength of 450 nm and the results were reported in ng/ml. the final result was calculated by multiplying the dilution coefficient at the concentration reported by the device. 2.3. statistical analysis the analysis was performed by using spsstm software, version 21.0 (ibm co., armonk, ny, usa). the results are presented as descriptive statistics in terms of relative frequency. values were expressed as the mean ± standard deviation (continuous variables) or percentages of the group (categorical variables). the paired t-tests were used to compare means, and a p value <0.05 was considered as statistically significant. 3. results totally, 46 symptomatic cases consist of 52.2% males and 47.8% females with a mean age of 31.4±24.6 year, and 21 asymptomatic controls consist of 52.4% males and 47.6% females with a mean age of 43.1±23.1 year were included. the distribution of underlying diseases are shown in table 1. the results showed that the level of lcn2 in symptomatic patients' (case group) was higher than asymptomatic carriers (control group), 5.9 ng/ml vs. 4.1 ng/ml; however the observed difference was not statistically significant (figure 1). to determine the relationship between ngal levels and gastrointestinal diseases, the mean level of ngal in symptomatic patients with ulcerative colitis, gastroenteritis, colorectal cancer, and crohn's was compared with the patients with non-gastrointestinal diseases. as shown in figure 2, the mean level of ngal was significantly higher in patients with gastrointestinal diseases than others (12.6 ng/ml vs. 3.0 ng/ml; p <0.001). also, in asymptomatic carriers (figure 3), the mean level of ngal was significantly higher in patients with gastrointestinal diseases than in others (8.1 ng/ml vs. 2.0 ng/ml; p <0.009). 4. discussion in recent years, the prevalence of cdi has increased significantly and has become a growing health concern, especially in hospitals [12]. hospitalacquired cdi can be a consequence of the development of the disease due to colonization with c. difficile or exposure to a new strain transmitted from patients or the environment [13]. hoseini-alfatemi et al. 3 table 1. the distribution of underlying diseases among studied patients underlying disease case (symptomatic) control (asymptomatic) frequency percent frequency percent ulcerative colitis (uc) 8 17.4 1 4.8 acute lymphoblastic leukemia (all) 5 10.9 2 9.5 liver transplantation 5 10.9 3 14.3 gastroenteritis 5 10.9 4 19.0 acute myeloid leukemia (aml) 1 2.2 kidney failure 4 8.7 2 9.5 diabetes mellitus (dm) 2 4.3 metabolic disorder 2 4.3 pneumonia 2 4.3 autoimmune hepatitis 1 2.2 chronic lymphocytic leukemia (cll) 1 2.2 1 4.8 colon cancer 1 2.2 crohn's 1 2.2 1 4.8 glaucoma 1 2.2 liver neoplasms 1 2.2 multiple myeloma 1 2.2 myelodysplastic syndromes (mds) 1 2.2 non-hodgkin lymphoma (nhl) 1 2.2 optic glioma 1 2.2 osteosarcoma 1 2.2 wilm's tumor 1 2.2 chronic myelogenous leukemia (cml) 2 9.5 aspiration pneumonia 1 4.8 gi vasculitis 1 4.8 guillain-barre 1 4.8 kidney transplantation 1 4.8 mushroom poisoning 1 4.8 total 46 100 21 100 figure 1. comparison the intestinal level of ngal in case (symptomatic) and control (asymptomatic carriers) groups figure 2: comparison the intestinal level of ngal in patients with gastrointestinal diseases and other patients among the case group (symptomatic) hoseini-alfatemi et al. 4 in a study by chassaing et al., suggested that the lcn2 in feces can be an indicator for the identification of inflammation caused by ibd in mouse models. also, they suggest that lcn2 could be linked to other intestinal infections, but to introduce as a biomarker needs to future examination [7]. in the present study, there was no statistically significant difference between the levels of lcn2 among case and control groups. however, the intestinal level of lcn2 in both groups was significantly higher in people with gastrointestinal diseases. previously, xiu guo et al. had shown that in infected animal models with pathogenic strains of escherichia coli, the level of lcn2 in the jejunum increased rapidly [14]. these results indicate that lcn2 as a proinflammatory factor can be expressed independently of the presence of bacterial infection, but the presence of any intestinal inflammation can significantly promote its production. it has been suggested that alteration in the level of lcn2 can balance the level of the intestinal microbiome and the host response to infection [7, 15]. it has also been shown that increasing lcn2 levels helps to limit infection by inhibiting siderophore receptors and reducing pathogen access to iron [14]. the main limitations including the fact that the present study is a cross-sectional study that was performed only on patients with antibiotic-related diarrhea and not all diarrhea patients were tested for the presence of c. difficile. in summary, despite all the limitations, the results of the present study indicate that the intestinal level of ngal is a biological indicator of intestinal inflammation, regardless of cdi. however, further study need to elucidate the role of ngal in inflammation caused by bacterial infections. author contributions conception or design of the work: hs; data collection: hs; data analysis and interpretation: mh, hs; drafting the article: mh, hs; critical revision of the article: mh, hs. all authors read and approved the final version of manuscript. conflict of interests the authors declare that they have no conflicts of interest. ethical declarations the study design approved by the local ethics committee of shiraz university of medical sciences (approval no. ir.sums.rec.1396.s872). financial support this study was supported by shiraz university of medical sciences with grant no. 96-14947. references 1. edwards an, 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https://pubmed.ncbi.nlm.nih.gov/26573915/ https://pubmed.ncbi.nlm.nih.gov/26573915/ https://pubmed.ncbi.nlm.nih.gov/26573915/ https://pubmed.ncbi.nlm.nih.gov/26573915/ https://pubmed.ncbi.nlm.nih.gov/29029438/ https://pubmed.ncbi.nlm.nih.gov/29029438/ https://pubmed.ncbi.nlm.nih.gov/29029438/ https://pubmed.ncbi.nlm.nih.gov/29029438/ https://www.ncbi.nlm.nih.gov/pubmed/27458605 https://www.ncbi.nlm.nih.gov/pubmed/27458605 https://www.ncbi.nlm.nih.gov/pubmed/27458605 https://www.ncbi.nlm.nih.gov/pubmed/27458605 journal of current biomedical reports jcbior.com volume 3, number 4, 2022 eissn: 2717-1906 1 original research evaluation of aerobic bacterial etiology and antibiotic susceptibility pattern of diabetic foot infections in rasht, the north or iran mohammad mahdi hanaei1, mahsa motiei2, ronia tavasolian3, sara khoshdoz2, hossein hemmati2, mohammad sadegh esmaeili delshad2, edris mehmandoust4, hadi sedigh ebrahim-saraie4,* 1department of internal medicine, shaheid beheshti university of medical sciences, tehran, iran 2razi clinical research development unit, razi hospital, guilan university of medical sciences, rasht, iran 3department of clinical sciences and nutrition, university of chester, chester, united kingdom 4department of microbiology, school of medicine, guilan university of medical sciences, rasht, iran abstract diabetic foot ulcers (dfu) are one of the most frequently occurred complications of patients with poor-controlled diabetes mellitus. diabetic foot infection (dfi) is increasing as a common problem and more than half of dfus will be eventually infected. here, we aimed to evaluate aerobic bacterial etiology and antibiotic susceptibility pattern of dfis. this descriptive cross-sectional study was conducted at razi educational and remedial hospital in the north of iran. from march to august, 2020 patients who were diagnosed as diabetic foot ulcer and attended to razi hospital included in the study. in this study samples were collected from infected areas of diabetic foot ulcers. standard microbiological methods were used to identify the isolates. the disc diffusion method was used to determine antimicrobial susceptibility on mueller-hinton agar following the clinical and laboratory standards institute (clsi) recommendations. thirty-one patients enrolled in this study. they included 17 females (54.8%) and 14 males (45.2%). the mean age of patients was 62.8 years, ranging from 40 to 93 years old. totally, six types of aerobic bacteria were isolated from patients. the most prevalent type was escherichia coli (41.9%), followed by klebsiella spp. (16.1%). the most effective antibiotic against gram-negative bacteria were aminoglycosides. while, staphylococcus aureus isolates mostly susceptible to tested antibiotics. also, none of the isolated s. aureus were methicillin-resistance. the results of antimicrobial sensitivity showed that aminoglycosides might be suitable agents for empirical therapy in iran. regular monitoring of culture and sensitivity reports is required to select drugs for empiric treatment. keywords: diabetic foot ulcer, wound, antibiotic resistance, iran 1. introduction diabetic foot ulcers are one of the most frequently occurred complications of patients with poorcontrolled diabetes mellitus and it leads to hospitalization of diabetic patients, which significantly increases the costs of this disease [1]. diabetics are 25% more likely to develop diabetic foot ulcers, and almost *corresponding author: hadi sedigh ebrahim-saraie, ph.d department of microbiology, school of medicine, guilan university of medical sciences, rasht, iran tel/fax: +98 13 33542460 email: seddigh.hadi@gmail.com http://orcid.org/0000-0001-8339-5199 received: january, 02, 2022 accepted: may, 15, 2022 every 30 seconds, one diabetes-affected limb occurs worldwide. global prevalence of diabetic foot is 6.3%; however, this rate greatly varied geographically [2]. association of several risk factors are documented with higher occurrence of diabetic foot, including male gender, type 2 diabetic, longer diabetic duration, © the author(s) 2022 https://jcbior.com/ hanaei et al. 2 elderly, low body mass index, hypertension, and smoking history [3]. although diabetic foot ulcers are multifactorial, the most common causes are poor blood sugar control, calluses, foot deformity, improper foot care, improper footwear, peripheral neuropathy and poor blood circulation, dry skin and so on [4]. in iran, diabetes is progressive and studies show a high prevalence of peripheral neuropathy in diabetic patients ranging from 28% to 75%. despite the high rate of premature mortality in diabetic foot patients, they fear of severe amputation than death [5]. diabetic foot infection is increasing as a common problem and more than half of dfus will be eventually infected. several studies have shown, in the development of mild infections, mono microbial causes and in severe cases, poly microbial causes have played a role [6]. namely, aerobic gram-positive cocci, gram-negative bacilli (including pseudomonas spp., escherichia coli, enterobacter spp. and citrobacter spp.) are known as a common cause in mono microbial infection. also, several anaerobes including bacteriodes spp., peptostreptococcus spp., fusobacterium spp. and clostridium spp. are reported as causes of poly microbial factors. during the last decades the inappropriate use of commercial antibiotics in humans and agriculture, along with the evolution and expansion of mobile genetic resistance elements resulting in the increase of multidrug resistance (mdr) [7]. early diagnosis of bacterial etiology of dfis and the targeted use of appropriate antibiotics promote wound healing and reduce the risk of amputation and mortality in diabetic patients. so we aimed to evaluate aerobic bacterial etiology and antibiotic susceptibility pattern of dfis in rasht, the north or iran. 2. materials and methods 2.1 study subjects this descriptive cross-sectional study was conducted at razi educational and remedial hospital in the north of iran. from march to august, 2020 patients who were diagnosed as diabetic foot ulcer and attended to razi hospital included in the study. both male and female patients with known diabetes at any age, regardless of their foot ulcer stage were included. the exclusion criteria were patients with immunodeficiency or who receive immunosuppressive drugs (like corticosteroids), taking broad-spectrum antibiotics for the last two days before sampling, missed information, contaminated samples and patients who do not want to be included in this study. approved consent forms were obtained from patients. the study design was approved by the ethics committee of the guilan university of medical sciences (reg no. ir.gums.rec.1398.410) and followed the declaration of helsinki. 2.2 microbiological methods in this study samples were collected from infected areas of diabetic foot ulcers. before sampling debrided wounds with a sterile scalpel and then rinsed with normal saline. after a few minutes when normal saline was dried on wound, we used a sterile swab to collect the sample from depth of the necrotic wounds or from the lateral margin of them and then swabs were placed into sterile tubes. samples was cut into a sterile container contained 1 ml tryptic soy broth and transferred to the laboratory as soon as possible. following that, 10 µl of tube content was cultured into a blood agar, chocolate agar, and macconkey agar. the blood agar and macconkey agar plates were incubated aerobically at 37ºc for 24-48 hours. at 37ºc, chocolate agar plates were incubated in an atmosphere supplemented with carbon dioxide (a candle jar). standard microbiological methods were used to identify the isolates, including morphological analysis, gram staining, catalase, oxidase, coagulase tests, sugar fermentation, and other available biochemical tests. 2.3 susceptibility testing the disc diffusion method was used to determine antimicrobial susceptibility on mueller-hinton agar (merck, germany) following the clinical and laboratory standards institute (clsi) recommendations. clsi recommendations were followed to select antimicrobial discs (padtan teb, iran), control strains, and interpretation of results for each pathogen. plates were incubated at 35-37° c for 16-18 hours. we expounded the results of antibiotic susceptibility based on standard instructions of clsi guidelines. 2.4 statistical analysis statistical analysis was performed by the spss™ software (version 21). the results are presented in the form of descriptive statistics based on relative hanaei et al. 3 frequency. frequencies and percentages were used to summarize categorical variables, while median and interquartile range (iqr) values were used to describe continuous variables. 3. results thirty-one patients enrolled in this study. they included 17 females (54.8%) and 14 males (45.2%). the mean age of patients was 62.8 years, ranging from 40 to 93 years old. totally, six types of aerobic bacteria were isolated from patients. table 1 shows the frequency and percent of each organism. the most prevalent type was escherichia coli (41.9%), followed by klebsiella spp. (16.1%). antibiotic susceptibility pattern for each bacteria is presented in table 2. the most effective antibiotic against gram-negative bacteria were aminoglycosides. while, staphylococcus aureus isolates mostly susceptible to tested antibiotics. also, none of the isolated s. aureus were methicillin-resistance. 4. discussion foot infection in diabetic patients is a common, complex, and costly problem [8]. 20% of cases of hospitalization of diabetic patients are related to diabetic foot ulcers, and about 50% of these ulcers are likely to become infected [9]. diabetic foot infections are usually polymicrobial, and aerobic and anaerobic bacteria are isolated from these infections [10]. various studies have shown that staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, proteus, klebsiella, enterobacter, and enterococcus are the most common bacteria isolated from diabetic foot ulcers. however, the pattern of antibiotic resistance of pathogens can vary depending on the geography, prevalence of microorganisms, and antibiotic consumption [11]. this study evaluated microbiological characteristics, and antimicrobial susceptibility of infected diabetic foot ulcers in diabetic patients referred to a hospital in north of iran. a total of 31 patients who met the inclusion criteria were included in the study for five months (march to august 2020). our study showed that various aerobic bacteria could be isolated from dfi samples. all our patients were older than 40 years. most of the patients with foot infections in the study of lipsky, zhang, and rastogi were older than 50 years, and in the study of perim, they were more aged than 60 year [8, 12-14]. in general, foot lesions commonly occur among elderly patients with diabetes and those with sensory neuropathy [8]. previous studies have shown that the prevalence of foot infection is higher among male patients than in female patients [15-17]. differences in lifestyle, occupation, and more physical activity of men than women may cause their feet to bear more pressure [18]. however, following perim’s study, we also did not find a difference between the prevalence of infection in male and female patients, which may be due to the limited number of patients. aerobic gramnegative bacteria (mainly enterobacteriaceae and occasionally pseudomonas aeruginosa or other gramnegative species) are usually isolated along with grampositive cocci in patients with chronic infections [8]. in our study, the prevalence of aerobic gram-negative bacteria was 67.7%, and aerobic gram-positive bacteria was 9.7%. the most common microorganisms isolated were escherichia coli and klebsiella. in two studies conducted on patients with diabetic foot ulcers in india and kuwait, aerobic gramnegative organisms (51.4% and 51.2%) and aerobic gram-positive organisms (33.3% and 32.3%) were the most common pathogens isolated respectively [19]. in other studies, most bacteria isolated from diabetic foot ulcers were gram-negative [17, 20-22]. while 80.3% of the aerobic microorganisms isolated from dfi were gram-positive in amini's study [23]. similar to our results, the most common bacteria isolated in several studies was escherichia coli [9, 17, 19, 23, 24]. while several studies in iran have reported gram-positive bacteria, especially staphylococcus aureus, as the most common organism isolated from dfi [10, 18, 23, 25]. differences in the profile of microbes infecting diabetic foot ulcers can be due to differences in environmental factors such as hygiene habits, geographic region, and study time [9, 19]. also, the table 1. the aerobic bacterial etiology of dfi bacteria frequency percent e. coli 13 41.9 klebsiella 5 16.1 pseudomonas spp. 2 6.5 staphylococcus aureus 2 6.5 micrococcus spp. 1 3.2 enterobacter spp. 1 3.2 no growth 4 12.9 contamination 3 9.7 total 31 100.0 hanaei et al. 4 difference in the microbial profile of diabetic foot ulcers and their sensitivity pattern can be due to the difference in sample volume, microbial detection method, source of infection, prescribing inappropriate doses of antibiotics, arbitrary use of antibiotics, and prescribing antibiotics by non-specialists [10, 19, 23]. based on our findings, the enterobacteriaceae family was resistant to most of the tested antibiotics (3189%), which is partially consistent with the results of the study conducted by perim et al. [8]. aminoglycosides had an acceptable sensitivity against gram-negative bacteria, which is consistent with the results of other studies [8, 23, 24]. therefore, this antibiotic can be suitable for use in experimental treatment. amikacin was the most effective antibiotic against pseudomonas isolates. also, similar to our results, in some recent studies, high resistance of gram-negative bacilli to ciprofloxacin has been reported [9, 18]. therefore, ciprofloxacin as an experimental antibiotic may not be appropriate in this situation. it should be considered that most e. coli strains isolated are probably non-pathogenic and do not require treatment. therefore, clinicians should focus on pathogenic organisms (especially table 2. antibiotic susceptibility pattern antibiotic pattern enterobacterales p. aeruginosa staphylococci no. % no. % no. % cfz s 3 15.8 i 4 21.1 r 12 63.2 caz s 3 15.8 1 50 i 1 5.3 0 0 r 15 78.9 1 0 imi s 4 21.1 0 0 i 3 15.8 0 0 r 12 63.2 2 100 amk s 10 52.6 2 100 i 3 15.8 0 0 r 6 31.6 0 0 gen s 10 52.6 1 50 2 100 i 1 5.3 0 0 0 0 r 8 42.1 1 50 0 0 cip s 2 10.5 0 0 2 100 i 0 0 0 0 0 0 r 17 89.5 2 100 0 0 sxt s 5 26.3 2 100 i 1 5.3 0 0 r 13 68.4 0 0 taz s 7 36.8 1 50 i 4 21.1 0 0 r 8 42.1 1 50 pen s 2 100 i 0 r 0 ery s 0 i 0 r 2 100 tet s 2 100 i 0 r 0 fox s 2 100 i 0 r 0 hanaei et al. 5 staphylococcus aureus and pseudomonas aeruginosa) and use high-dose and narrow-spectrum antibiotics recommended in international guidelines [18]. to prevent wound infection, proper management of diabetes, foot care, and compliance with hygiene principles are essential [24]. antimicrobial treatment without determining the cause of infection eradicates sensitive microorganisms and can cause the selection and spread of resistant organisms [18]. therefore, it is necessary to evaluate different micro-organisms infecting the wound on a routine basis and to know the antibiotic sensitivity patterns of infectious wound isolates in patients with a diabetic foot infection. this knowledge is crucial to achieving optimal clinical outcomes, planning treatment of these patients with appropriate antibiotics, limiting the emergence of amr strains, and reducing healthcare costs [8, 26]. the report of the current study has limitations because this study was conducted in only one hospital in iran and the sample size was small. culture was not performed for anaerobic bacteria. also, the wounds were not examined for multi-microbial or monomicrobial infections, and there was no history of previous antibiotic use by the patients. therefore, larger-scale validation studies with more data should be conducted before generalizing the findings. the difference in the microbial pattern of diabetic foot infection in various studies shows that experimental treatment in each country should be selected according to the most common pathogen specific to the region and its antimicrobial sensitivity. also, considering the isolation of all types of microorganisms from these wounds, it is essential to start experimental antibacterial treatment to cover gram-positive and gram-negative bacteria. the results of antimicrobial sensitivity showed that aminoglycosides might be suitable agents for empirical therapy in iran. regular monitoring of culture and sensitivity reports is required to select drugs for empiric treatment. similar studies can help to understand the diversity of microorganisms responsible for dfi and the antibiotic resistance pattern of each region. authors' contributions concept and study design: hh, me, em, hs. methods, data collection, and experimental work: sk, em, hh, me. results analysis and conclusions: mh, mm, rt, hs. manuscript preparation and editing: mh, mm, rt, hs. all authors read and approved the final version of the manuscript. conflict of interests no potential conflict of interest was reported by the authors. ethical declarations the study design was approved by the ethical committee at the guilan university of medical sciences [ir.gums.rec.1398.410]. financial support guilan university of medical sciences. references 1. akkus g, sert m. diabetic foot ulcers: a devastating complication of diabetes mellitus continues non-stop in spite of new medical treatment modalities. world j diabetes. 2022; 13(12):1106-21. 2. zhang p, lu j, jing y, tang s, zhu d, bi y. global epidemiology of diabetic foot ulceration: a systematic review and meta-analysis (†). ann med. 2017; 49(2):106-16. 3. al-rubeaan k, al derwish m, ouizi s, youssef am, subhani sn, ibrahim hm, et al. diabetic foot complications and their risk factors from a large retrospective cohort study. plos one. 2015; 10(5):e0124446. 4. wang x, yuan cx, xu b, yu z. diabetic foot ulcers: classification, risk factors and management. world j diabetes. 2022; 13(12):1049-65. 5. sobhani s, asayesh h, sharifi f, djalalinia s, baradaran hr, arzaghi sm, et al. prevalence of diabetic peripheral neuropathy in iran: a systematic review and meta-analysis. j diabetes metab 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outcomes at a tertiary care hospital. international surgery journal. 2020; 7(5):1607-10. 25. pitocco d, spanu t, di leo m, vitiello r, rizzi a, tartaglione l, et al. diabetic foot infections: a comprehensive overview. eur rev med pharmacol sci. 2019; 23(2 suppl):26-37. 26. sekhar s, unnikrishnan m, rodrigues gs, vyas n, mukhopadhyay c. antimicrobial susceptibility pattern of aerobes in diabetic foot ulcers in a south-indian tertiary care hospital. the foot. 2018; 37:95-100. journal of current biomedical reports jcbior.com volume 3, number 2, 2022 eissn: 2717-1906 1 original research epidemiology of organophosphate poisoning in the north of iran hamid mohammadi kojidi1, mohammad habibullah pulok2, banafshe felezi-nasiri3, maryam yaseri1, enayatollah homaie rad3,* 1razi clinical research development unit, razi hospital, guilan university of medical sciences, rasht, iran 2nova scotia health authority, dalhousie university, halifax, ns, canada 3social determinants of health research center, guilan university of medical sciences, rasht, iran abstract the use of pesticides as one of the main agricultural poles has been increased in iran in recent years. organophosphate poisoning has harmful the consequences for human health. this study present clinical and laboratory evidences on the patients exposed to agricultural insecticides poisoning and the cause of these poisons. we collected clinical data from the patients referred to razi hospital, rasht, iran who were poisoned with organophosphorus toxins. for this purpose, a checklist was prepared, and data were collected for 414 patients between 2011 and 2016. the results showed that the most cases of poisoning were men (73%) and about 27.2% of the patients was in the age group of 45-60 years (highest frequency in age groups). the most frequent symptoms were vomiting (65%), nausea (61%), abdominal pain (39%), and perspiration (27%). there was also a decrease in consciousness (16%) and sialorrhea (16%). totally, 186 (46.2%) patients were exposed to organophosphorus toxins by respiratory and 215 (53.4%) orally. out of the 414 samples, 102 (33%) had abnormal creatine phosphokinase (cpk) enzymes and 114 (34.5%) abnormal lactate dehydrogenase (ldh). mean hospital length of stay (los) was 3.3 days. we found significant relationship of los with heart failure, hypertension, and addiction. to better manage the process of treatment of agricultural poisoned patients and to reduce the waste of limited resources available, careful consideration should be given to the type of pesticide used by the patient to prevent overdose and unintentional use of antidote. keywords: organophosphate pesticides, mortality, poisoning, length of stay, guilan 1. introduction the application of insecticides has dramatically grown for agricultural development in low and middle income countries in the recent decades due to population growth. one of the most important side effects of increased use of pesticides is poisoning which could be very detrimental for human health and ecological balance. poisoning account for about 0.15% of disability adjusted life years (0.17% in low income countries) and it is one of the leading causes of death [1]. in iran, more than 3.7 million life-years of total population were lost due to poisoning [1]. poisoning is *corresponding author: enayatollah homaie rad, ph.d poursina hospital, guilan university of medical sciences, rasht, iran tel/fax: +98 13 33332498 email: homaierad@gmail.com http://orcid.org/0000-0002-9064-0380 received: december, 12, 2021 accepted: may, 03, 2022 also one of the major risk factors of hospitals admission in many countries. for example, about five million people are poisoned every year and receive health care services in hospitals in the united states [2]. controlling pathogens and risk factors of diseases through the use of pesticides is very beneficial, but these toxins themselves might cause illness and death. these problems are caused by various direct and indirect exposures to pesticides. the prevalence of poisoning with chemical pesticides in developing countries is 13 times higher than in industrialized countries. it is estimated that about 85% of global © the author(s) 2022 https://jcbior.com/ mohammadi kojidi et al. 2 pesticide productions are used in developing countries [3]. frequent and unprotected contact with various types of insecticides cause risk of liver and kidney failure as well as cardiovascular disease and respiratory problems [4]. there is evidence that women who live in the environment with poisonous water have significant delay in intrauterine growth compare to others [5]. studies also reported that parquet toxins, the oregano-phosphorus toxins, maneb and mancozib, lead to parkinson's disease [6]. people living near areas exposed to insecticides are more likely to suffer parkinson's disease [7]. studies found that people who are exposed by herbicides are 4 times and those who are exposed by insecticides are 3 times more liked to suffer parkinson [4, 8]. organophosphorus compounds are one of the major pesticides used in agriculture and are responsible for more than 80% of poisoning with agricultural pesticides in iran [9]. these compounds have become popular due to their effects as insecticides as well as low impact on environment and very low harmful radicals. they have been replaced by other insecticides, including organochlorines. these poisons are absorbed to body through the respiratory tract, digestive system, the mucosa, and skin. there are many types of organophosphate toxins in iran, which are classified into high toxicity, moderate toxicity and low toxicity [10, 11]. in recent years, many solutions have been implemented to prevent and combat health-related problems of poisoning caused by exposure to organophosphates in iran. in general, the best way to curb such poisonings is to educate general public, especially the farmers. guilan province is one of the main centers of agricultural product and pesticide use, which has a higher rate of plant protection. due to high population density in this province, the risk of exposure of people to such poisons is greater than other provinces. therefore, the aim of this study was to provide with clinical and laboratory evidences about the patients exposed to agricultural pesticide poisoning, and the causes of these poisonings. we documented the situation in guilan province and discussed ways to reduce the incidence of poisoning. the results of this study could be used for preventive and therapeutic implementations to reduce morbidity and mortality. 2. materials and methods this was a cross sectional study included 414 patients referred to razi hospital of rasht (capital of guilan province) between 2011 and 2016, diagnosed with an organophosphate poisoning problem. razi hospital is the major and unique hospital for treating patients affected by poisoning. majority of the poisoned people from all over of the province are referred for poisoning care in this hospital. in this study, we used data from health information system (his) and medical records of the patients. first, international classification of disease (icd) codes of organophosphate poisoning was identified from the icd book. this followed the extraction of next medical codes of target patients. we developed a checklist to collect data. the checklist consisted of three parts. the first part included sociodemographic information the patients as age, sex, marital status, location, etc. the second part included information about disease and clinical records such as the history of underlying illnesses, addiction, drug use, and a history of suicide. the third part was on, clinical information such as increased body secretion, sweating, sore throat, tearing, bronchus, urinary incontinence, stool, nausea, diarrhea, mycosis and bradycardia as muscarinic symptoms, weakness, muscle cramps, fasciculation, respiratory paralysis, and tachycardia as signs of nicotine, as well as changes in the creatine phosphokinase (cpk) and lactate dehydrogenase (ldh) enzymes. five researchers reviewed and revised the contents and design to validate the checklist. data were collected by referring to the medical records department of razi hospital. completed records were checked for the accuracy at first. after deleting incomplete and irrelevant samples (11 ones), data were entered to excel software. the ethical clearance was approved by the deputy research of guilan university of medical sciences. we used descriptive statistics and regression models to analyze the data of study. all estimations were performed using stata/se software version 13.1 (statacorp lp, tx, usa). 3. results table 1 presents descriptive statistics of the patients included in this study. it shows that about 73% were male and 102 (25%) people were from urban regions. totally, 96 (23%) people were not married. mohammadi kojidi et al. 3 the age distribution shows that 42 people (10%) were between 10-20 years old, 90 (22%) patients were between 20-30 years old, 99 ones (24.26%) were between 30-45 years old, 111 ones (27.21%) were was 42.89 (%16.97). figure 1 depicts the frequency of each clinical symptom among the poisoned patients. vomiting (65%), nausea (61%), abdominal pain (39%), perspiration (27%), decreased consciousness (16%), and sialorrhea (16%) had the highest frequency of symptoms. in addition, 21 (5.19%) poisoned people died and 384 (94.81%) were treated. furthermore, the frequency of exposure to poisoning is shown in the table 2. 186 people (46.27%) were exposed to organophosphate pesticides from breathing, and 215 (53.48) were exposed to these toxins through eating. only one person (0.25%) had exposure to the toxin through the eye. table 2 shows the status of the cpk and ldh enzymes in patients. also, 207 (67%) of the samples had a normal cpk enzyme and for others cpk enzyme had increased. in addition, 216 patients had normal ldh (65.45%) enzyme and others had increase in ldh (34.45%) enzyme. table 3 shows the prescription of atropine and pralidoxime in patients exposed to organophosphate pesticides. as outlined in the table 3, for 240 (60.15%) patients atropine was prescribed and for 159 (39.85%) ones pralidoxime were prescribed for treatment; both of these drugs were prescribed for 111 (28%) patients. table 4 shows that the average length of stay (los) of patients exposed to organophosphate pesticides was 3.30 days. the mean duration of los for atropine administration was 1.98 days and it was pralidoxime 2.07 days for pralidoxime. the difference in mean los between this two groups was statistically significant (p <0.001). table 1. descriptive statistics of the participating patients variables frequency percentage female 111 27.21 male 297 72.79 living: urban region 102 25 living: rural region 306 75 not married 96 23.53 married 312 76.47 age 10-20 years 42 10.29 20-30 years 90 22.06 30-45 years 99 24.26 45-60 years 111 27.21 60> years 66 16.18 table 2. the status of the cpk and ldh enzymes in patients enzyme status frequency percentage cpk normal 207 66.99 increased 102 33.01 ldh normal 216 65.45 increased 114 34.55 mohammadi kojidi et al. 4 results from the poisson regression models on the relationship between individual, exposure and clinical factors of patients and hospital length of stay are presented in table 5. there were no significant relationships of age, gender, exposure type with hospital length of stay. the incidence rate ratio (irr) of health failure was 1.901 (p =0.000), indicated that poisoned people with heath failure stayed in hospitals about two times more than others. in addition, the patients suffering from addiction (irr= 1.436), utilizing atropine (irr= 1.404), having intubation (irr= 2.189), and having hyper tension (irr= 2.471), urinary incontinence (irr= 1.797), sialorrhea (irr= 1.335) and sweat (irr= 1.303) had significantly higher length of stay in the hospital. 4. discussion the results of this study showed that nausea and vomiting were the most common clinical symptoms of the patients who were exposed to organophosphate figure 1. frequency of clinical symptoms of patients exposed to organophosphate pesticides (total 414 patients). table 3. prescription of atropine and pralidoxime in patients exposed to organophosphate pesticides drug prescription frequency percentage atropine prescribed 240 60.15 not prescribed 159 39.85 pralidoxime prescribed 159 39.85 not prescribed 240 60.15 both prescribed 211 28.03 table 4. the average length of stay of patients exposed to organophosphate pesticides variable length of stay sd significant level atropine only 1.980 1.464 p <0.001 pralidoxim only 2.067 1.893 total 3.303 2.816 mohammadi kojidi et al. 5 pesticides. abdominal pain and sweating were the next reported symptoms. nausea, vomiting, and headache were found to be the most frequent symptoms of the patients in yazd, iran in 2012 [12]. talaei et al. found that abnormal pulmonary sounds, meiosis, increased salivation, and vomiting were the most frequent in tehran [8]. in addition, shayeghi et al. concluded that headache, dizziness, and nausea had the highest frequency in patients' clinical symptoms [11]. in other studies, the most common complication of patients was nausea and vomiting and increased salivation and salivary secretion and the least complication was incontinence in the urine. coughing, sputum excretion and wheezing were the highest prevalence in exposed pesticide pesticides [13-15]. studies also found relationships between dizziness table 5. relationship between individual, exposure and clinical factors of patients and hospital length of stay variable irr se p 95 % ci age 1.002 0.003 0.519 0.996 1.008 gender (ref) 0.843 0.087 0.100 0.688 1.033 exposure gastrological 0.963 0.132 0.782 0.736 1.259 underling disease others 1.216 0.156 0.129 0.945 1.564 diabetes 1.206 0.205 0.271 0.864 1.683 heath failure 1.901 0.244 0.000 1.478 2.445 addiction 1.436 0.177 0.003 1.128 1.829 suicide 1.237 0.138 0.056 0.994 1.538 pralidoxim 1.178 0.124 0.120 0.958 1.448 atropine 1.404 0.133 0.000 1.165 1.691 cpk 1.000 0.000 0.378 0.999 1.000 ldh 1.000 0.000 0.180 1.000 1.001 intubation 2.189 0.475 0.000 1.430 3.351 vertigo 0.984 0.151 0.915 0.729 1.328 loss of consciousness 1.093 0.143 0.497 0.846 1.413 epilepsy 0.586 0.246 0.202 0.257 1.332 weakness 1.041 0.152 0.782 0.782 1.388 hypertension 2.471 0.614 0.000 1.518 4.023 tachycardia 0.518 0.203 0.093 0.240 1.117 bradycardia 0.621 0.179 0.098 0.353 1.092 diarrhea 0.824 0.098 0.101 0.653 1.039 urinary incontinence 1.797 0.384 0.006 1.182 2.730 miosis 0.965 0.158 0.828 0.699 1.331 vomiting 0.839 0.130 0.257 0.620 1.136 abdominal pain 0.746 0.078 0.005 0.609 0.915 nausea 1.317 0.195 0.063 0.985 1.760 bronchorrhea 0.619 0.146 0.042 0.390 0.982 sialorrhea 1.335 0.184 0.036 1.019 1.749 tearing 0.831 0.127 0.224 0.616 1.120 sweat 1.303 0.146 0.018 1.047 1.623 constant 1.619 0.274 0.004 1.163 2.256 note: irr=incidence rate ratio, se=standard error, and ci= confidence interval mohammadi kojidi et al. 6 and vomiting due to organophosphate toxicities and some cardiovascular symptoms such as low blood pressure, bradycardia, and arrhythmia, as these symptoms lead to a worse prognosis [16]. therefore, severe complications and mortality could be increased in poisoned people if they are poorly tracked specially in patients suffering cardiovascular diseases. findings of ours study revealed that 5% of patients exposed to organophosphate toxins died. a study conducted in khorramabad city of iran found that about 7% died out of 153 exposed to organophosphate and oran glycerides, of which 3% were exposed to organophosphate pesticides [2]. in a study in zanjan, it was found that 2 (3%) out of 57 children were exposed to organophosphate died [9]. mean age of the exposed group was 42.89 years. also, 45 and 60 years old group had the highest frequency of organophosphate poisoning. in a study conducted in iran, the age group of 15 to 30 years was the most frequent people who exposed to organophosphate toxins. however, in a study conducted in fasa, the average age of exposed individuals was 45.6 years old [2, 15, 17]. the findings of this study showed that 46% of the patients were exposed to inhalation and 53% were digested with toxin and only one had eye exposure. about 92% of exposures were deliberated and only 8% were unwanted in mahmoudi study [2]. in another study, about 60% of the poisonings were respiratory, 23% ocular, and the rest were edible related to occupational poisoning [13]. in mashhad, it was also found that 14% of the exposures were accidental, 5% were occupational and the rest were suicidal [10]. in saudi arabia, 69% of the cases were digested and 15% through inhalation and other exposed through the skin [18]. we found that about 33% of patients of the study had increased cpk enzyme and 35% had increased ldh enzyme. cpk is found primarily in muscle. in the serum, it increases when damage occurs to muscle cells. this enzyme usually increases six hours after the lesion. if the tissue lesion is not very serious, the enzyme level reaches its peak 18 hours later and returns to its normal level 2 to 3 days later. the activity of ldh in the cell is 500 in terms of its serum level, and since ldh is a cytoplasmic enzyme, it can be a sensitive marker for tissue damage [19, 20]. the findings revealed that the los in the hospital was 3.30 days. the mean los of atropine treated patients was 1.98 and pralidoxime administration was 2.06 days. on average los of the patients exposed to organophosphate pesticides was 6 days and 11 days in india and japan, respectively [21, 22]. priyendu et al., found that the los was 9 days in saudi arabia. the results from the regression model indicated the importance of chronic diseases management of organophosphate poisoning. poisoned patients suffering hypertension, hearth failure, and addiction were more severe than others. this suggest that these people should have more notice than others [23]. organophosphate pesticides have many usages. for example, it is used in agriculture and pest management in farms. famers are exposed to this type of pesticides as well as pesticide producer factory workers [9]. therefore, it is very important to use protective equipment and safety tools in these occupational group. unfortunately, most of guilanian farmers work in their own farms privately without any provision and control. farming ministry could inform farmers about the dangers of exposure to organophosphate pesticides and can educate farmers about the ways of being protected from the dangers of organophosphate poisoning. it has been argued that poisoning of agricultural workers with pesticides, including organophosphorus pesticides, is more prevalent in less developed countries [24, 25]. in addition, this could lead to direct exposure to poisoning in famers and indirect effects on agricultural products consumer [26]. therefore, training farmers in the proper use of the pesticides can be very effective. these types of trainings need between sectoral collaborations and ministry of health must alert other related organizations about the side effects of using these toxins. meanwhile, checking the medical records of individuals during supply of the pesticides can be effective. this study had some limitations. firstly, there were missing in patients' information and biographies, and clinical history and prescribing drugs were illegible in some cases. in addition, we could not access data from other hospitals or cities in the province of guilan, and the study was limited to the razi hospital in rasht. however, all patients in other hospitals were also transferred to razi hospital in rasht. guilan province is a province covered by agricultural fields and there is a large amount of access to agricultural pesticides and organophosphates in the province. the share of agriculture work is high in this mohammadi kojidi et al. 7 province and there is increasing rate of toxins. therefore, training of farmers to properly use these toxins as well as utilizing prohibiting equipment could be effective to lessen the harmful health effects. authors’ contributions hm, my, eh conducted the study design. bf data collection. bf, mh data sorting and analysis. hm, mh, eh drafting the article. hm, my, eh critical revisions. all authors read and approved the final version of article. conflict of interests there is no conflict of interest. ethical declarations the ethical clearance was approved by the deputy research of guilan university of medical sciences (registered no. 1987). financial support self-funded. references 1. mew ej, padmanathan p, konradsen f, eddleston m, chang ss, phillips mr, et al. the global burden of fatal self-poisoning with pesticides 2006-15: systematic review. j affect disord. 2017; 219:93-104. 2. mahmoudi ga, asaee r. epidemiologic 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sleeve gastrectomy on serum lncrna levels in obese patients onur tokgun1,*, kubilay inci2, büşra çelikkaya2, pervin elvan tokgun1, aysegul cort donmez3, onur birsen4 1department of medical genetics, faculty of medicine, pamukkale university, denizli, turkey 2department of cancer molecular biology, institute of medical sciences, pamukkale university, denizli, turkey 3medical biochemistry, faculty of medicine, pamukkale university, denizli, turkey 4general surgery, faculty of medicine, pamukkale university, denizli, turkey abstract obesity is a disease associated with excessive fat accumulation in the body, which body mass index (bmi) is ≥30 kg/m. bariatric surgery technique is one of the most common treatment options for obesity with the advantage of faster weight loss in a short time. lncrnas play a role in adipogenesis and metabolic diseases, including obesity, type 2 diabetes, cardiovascular disease (cvd), osteoarthritis, and hypertension, so they are significant targets for therapeutic options. in this study, we aimed to determine lncrnas and specific parameters that show different expressions in the plasma of patients with obesity. we included fifteen patients with bmi ≥30 kg/m2 before and <30 kg/m2 after laparoscopic sleeve gastrectomy (lsg) in the study. total rnas, including lncrnas and other noncoding rnas, were isolated from plasma samples of patients, and eight lncrnas (h19, neat1, hotair, anril, malat1, atb, sngh5, uca1) were quantified by real-time pcr. gene ontology, kegg, and relation of obesity analysis were utilized. unpaired student's t-test pearson correlation analysis was used for statistical analysis. we observed a statistically significant increase in the expression levels of all lncrnas in the patients with the post-operative bmi change. we have added a new dimension to the biomarker studies related to obesity and the clinical follow-up of patients undergoing lsg surgery. further studies are required for enlighting the molecular mechanisms. keywords: lsg surgery, lncrna, obesity, bmi 1. introductıon in consequence of the requirement of modern lifestyle habits, obesity incidence has increased worldwide in recent years. world health organization identified that obesity is a disease associated with excessive fat accumulation in the body, linked to substantial risks for metabolic disorders and the pandemic process [1]. in diagnosis, body mass index is used worldwide [2], and a patient with body mass index (bmi) ≥30 kg/m is accepted as an obese person *corresponding author: onur tokgun, ph.d pamukkale university, school of medicine, department of medical genetics, pamukkale-denizli, turkey tel/fax: +902582961687/+905556410300 email: onur_tokgun@hotmail.com https://orcid.org/0000-0003-0537-9032 received: december, 31, 2021 accepted: january, 26, 2022 [1]. however, the current treatment option in the long term is ineffective depending on metabolic reactions robust homeostatic mechanisms that facilitate further weight gain [3]. furthermore, bmi is an advisable parameter for diagnosing obesity, while having some critical limitations in diagnosing obesity at the individual level [4]. the bmi affects individual factors, including age, sex, and age-related factors, and it does not reflect body fat distribution [5]. hence, there is a need for effective and specific biomarkers in early © the author(s) 2022 https://jcbior.com/ https://tureng.com/tr/turkce-ingilizce/during%20the%20recent%20years tokgun et al. 2 diagnosis. subcutaneous and visceral adipose tissues are metabolically more active. they secrete cytokines and hormones that exert metabolic disturbances such as insulin resistance and chronic low-grade inflammation at a higher rate. therefore, obesity is a disease processed by complex pathways [6]. one of the most common treatment options for obesity is bariatric surgery techniques, particularly laparoscopic procedures, as an effective treatment. laparoscopic sleeve gastrectomy (lsg) is one of the most common bariatric operations that effectively treat obesity and severe obesity-associated diseases. lsg is a surgery method performed by removing approximately 80% of the lateral aspect of the stomach vertically. studies reported that lsg has many advantages involving faster weight loss in a short time no foreign material as a gastric band. however, lsg can cause complicated biological problems due to the change of many biochemical parameters in the body and rapid weight alteration [3]. non-coding gene is a significant part of the genome that serve primarily as regulatory functions. long non-coding rnas (lncrnas) are usually longer than 200 nucleotides that modulate gene expression [7] and demonstrate organand lineage-specific lncrnas in the human body [8]. furthermore, studies revealed that lncrnas play roles in adipogenesis and metabolic diseases, including obesity, type 2 diabetes, cardiovascular disease (cvd), osteoarthritis, and hypertension [9]. recently, increased results indicated that lncrnas have participated in crucial cellular mechanisms in the pathogenesis of human metabolic diseases. specific regulations of metabolically sensitive lncrnas in mice have been demonstrated [10]. on the other hand, the association between the expression pattern of meg3, h19, and obesity parameters, essential genes in adipose tissues from obese women have been reported [5]. therefore, lncrnas are significant targets for therapeutic options. however, no studies reveal the relationship between circulating lncrnas and obesity parameters after alteration bmi index through lsg. therefore, in our study, we established the effects and correlation of circulating lncrnas associated with metabolic process on standard obesity parameters. furthermore, we aimed to determine lncrnas and specific parameters that show different expressions in the plasma of patients with obesity. 2. materıals and methods 2.1 sample collection the ethics committee approved all protocols used in this study of pamukkale university hospital with the number e-60116787-020-110884. informed consent was taken from each patient applied for the study. blood samples were collected between january 2020 and april 2021. we did not include individuals whose bmi values did not fall into the expected level in the study. briefly, 15 patients with bmi ≥30 kg/m2 before lsg and < 30 kg/m2 after lsg were accepted for the study. the blood samples were collected in 5 ml k2edta tubes three days before and six months after surgery. the plasma samples were prepared as follows: blood samples were centrifuged at 1500 rpm for 10 minutes at +4oc. serum was divided into 15 ml falcon tubes and centrifuged at 1500 rpm for 15 min at +4oc. the collected plasma samples were stored at 80oc. 2.2 molecular analysis total rnas, including lncrnas and other noncoding rnas, were isolated by trizol ls reagent (invitrogen, usa) and rneasy mini kit (qiagen, germany) to manufacturers' instructions. spectrophotometric measurements were performed at 260, 280, and 230 nm using the nanodrop instrument (thermo, usa). 260/280 and 260/230 ratios were evaluated for purity and quality and observed as 1.8-2.1 and 1.8, respectively. the cdna synthesis reaction was performed using miscript ii rt kit (qiagen, germany). pcr reactions were set in the sensequest qpcr instrument (sensoquest gmbh, germany) using the following protocol: 37°c for 60 minutes and 95°c for 5 minutes. cdna samples were stored at -20°c for the next stages of the experiments. real-time quantitative pcr (qrt-pcr), reactions were prepared using 2x magic sybr mix (procomcure biotech, austria) for the quantitative analysis of lncrnas, including h19, neat1, hotair, anril, malat1, atb, sngh5, uca1 [11], and performed using cfx96 biorad instrument. u6 small nuclear rna was the internal control. the reaction was performed in 3 replicates for each sample. the fold change in the expression of the lncrnas was calculated using the formula 2^-δδct. tokgun et al. 3 2.3 investigate the relationships between obesity and lncrnas collected data were analyzed using the graphpad prism 8.0.1 program and the excel-based pcr array data analysis software provided by the qiagen company. we utilized the go analysis, the pathway analyses, and disease-lncrna association analysis according to gene set enrichment analysis (gsea) (https://www.gsea-msigdb.org/gsea/index.jsp) and david database (https://www.gseamsigdb.org/gsea/index.jsp). in addition, the kegg (kyoto encyclopedia of genes and genomes) database (http://www.genome.ad.jp/kegg) with fisher's exact test was also used to identify pathways with p <0.05. finally, the evaluated enrichment score of the paths is determined as previously described [12]. 2.4 statistical analysis the graphs, calculations, and statistical analyses were performed using graphpad prism software version 8.0.1 (graphpad software, san diego, ca, usa). unpaired student's t-test was used for comparisons differentially expressions of lncrnas in the obese patients after lsg operation. a receiver operating characteristic (roc) curve was employed for each lncrna. their respective 95% confidence intervals (ci) were calculated to evaluate the sensitivity and specificity for obese patients. pearson correlation coefficient analysis was utilized to determine the relationship between expression levels of lncrnas and bmi index alteration. statistical results with *p <0.05, **p <0.01, ***p <0.001, or ****p <0.0001, were considered to be statistically significant. 3. results fifteen patients (n=12 women, n=3 men) who had undergone lsg were included in our study. the mean age of the patients was 34.13±13.23. the mean preoperative weight of the operated patients was 121.66±21.82, while the mean post-operative weight was 85.06±19.36 (p <0.0001). in addition, the mean bmi changes before and after the operation were 49.03±20.34 19.36±9.1, respectively. besides, statistically significant differences were observed in the glucose, tsh, t3, crp, and triglyceride levels after the surgical process (table 1). h19, neat1, hotair, anril, malat1, atb, sngh5, and uca1 lncrna expression levels were investigated by qrt-pcr analysis from serum samples taken from patients two times as before and six months after the operation. qrt-pcr analysis results revealed a statistically significant increase in all lncrna expressions in the samples six months after the operation (figure 1). atb and hotair expression were regarded as the lncrnas with the most statistically significant increase among all lncrnas with increased expression. the data we obtained from the gene ontology analysis showed that lncrnas play a role in obesity and related mechanisms (supplementary figure 1). when t3, glucose, tsh, crp, and triglyceride levels were analyzed before and after the operation, it was observed that there was a blind decrease after the surgical operation. auc values were above 0.8 for all analysis groups (supplementary figure 2). when the lncrna expression changes observed in the operated patients were analyzed according to the bmi changes, it was observed that there was an increase in the expression levels of all lncrnas. furthermore, these changes were statistically significant (figure 2). 4. discussion lncrnas are generally identified as transcripts longer than 200 nucleotides compared to types of short rna such as mirnas, sirnas, and pirnas [9]. they display different expression patterns according to tissues and conditions in the human body. moreover, their variable expression responds to numerous stimuli and timely expression at specific developmental requirements [10]. lncrnas are being widely studied to play a role in adipogenesis and metabolic diseases and obesity and obesity-associated as type 2 diabetes, and cvd [13]. furthermore, lncrnas have been reported to affect the chronic positive energy balance, including overeating and low energy expenditure [1, 13], but new studies are required in this area. therefore, in the present study, we investigated the relationship between the potential alteration of circulating lncrna and the physical and biochemical parameters of obesity after lsg. lately, there have been many experiments and research related to the role of lncrnas in various diseases and mechanisms. in contrast, the action of the vast majority of lncrnas remains unclear [10]. with the advancements of high-throughput techniques, transcriptome data of lncrnas possesses an enormous amount of information related to tokgun et al. 4 patients with metabolic diseases such as obesity [7]. the results obtained from studies consist of a combination of rna-seq, and computational methods that are currently working to identify novel candidate lncrnas as potential biomarkers and possible therapeutic targets [14]. therefore, sun et al. demonstrated that the expression pattern of three lncrnas is different in the blood components and adipose tissue of obese patients against the control group, including non-obese persons [14]. however, there is a deficiency in the correlation between expression alteration of lncrnas and obesity-related parameters. on the other hand, yao et al. described lncrnas as potential biomarkers in metabolic syndrome [6]. in the same direction as previous studies, in our research, we determined a correlation between alteration in the expression pattern of especial lncrnas and metabolic parameters of obesity in the patients the transition from obese to non-obese according to the bmi index. moreover, our results indicated that lncrna h19, neat1, hotair, malat1, atb, uca1, and sngh5 correlate with alteration of bmi index. thus factors can influence them in energy metabolism and obesityassociated physical activity; there is a need for elucidating to functional roles of these lncrnas in both in vitro and in vivo studies. current studies showed the role of lncrnas in obesity-associated biological mechanisms, including adipogenesis (lncrna h19, neat1) [15], lipid metabolism (neat1) [16], white adipogenesis (hotair), and adipose regulation (malat1) [17]. furthermore, in the present study, kegg pathway enrichment results and pearson correlation analysis indicated that lncrna anril, atb, uca1, and sngh5 and these lncrnas mentioned in the literature have the potential to be biomarkers in the follow-up of obese patients after lsg. to study the disease relation and biological functions of these lncrnas, we utilized the go analysis, the pathway analyses, and disease-lncrna association analysis by working gsea and david database. these analysis results demonstrated that these lncrnas play an essential role in the obesity and disease linked to obesity, including diabetes mellitus, insulin resistance, and hypertension. furthermore, we showed connections between lncrnas including h19, table 1. the clinical characteristic feature of patient group variable group total n = 15 significant level age (years) 34.13±13.23 gender 12, female 3, male height (cm) 161.93±15.61 surgery condition before after p value weight (kg) 121.66±21.82 85.06±19.36 p <0.0001 bmi 49.03±20.34 19.36±9.1 p <0.0001 glucose (mmol/l) 122.06±82.05 81.2±8.38 p <0.0001 tsh (mu/l) 3.46±2.65 2.09±0.91 p <0.0001 t3 (nmol/l) 3.41±0.51 2.55±0.39 p <0.0001 crp 0.9±0.65 0.65±0.84 p <0.0001 triglyceride (mmol/l) 167.92±105.84 96.84±24.34 p <0.0001 *the p values refer to comparisons between “before surgery” and “after surgery” groups in the top part of the table. **data are represented as mean ± sd. ***bmi; body mass index, tsh; thyroid stimulating hormone, t3; triiodothyronine. tokgun et al. 5 neat1, hotair, malat1, atb, uca1, sngh5, and genes in the obesity-associated pathways. consistent with our findings, a study on the obese women group reported that lncrna meg3 and h19 were associated with a metabolic profile in the adipose tissue of obese women. obesity is an epidemic factor for several chronic diseases involving type 2 diabetes, coronary heart disease, and different types of cancer [4]. epidemiological studies demonstrated that classical anthropometric measures including height, weight, head circumference, and bmi are used to identify obesity. however, the underlying biological mechanisms are less understood as obesity is a complex disease including biochemical and genetic factors. the current knowledge of the significant roles figure 1. expression level of circulating h19, neat1, hotair, anril, malat1, atb, sngh5, uca1 in before and after surgery. expression of lncrnas were detected by qpcr and normalized by u6 expression. (*p <0.05, **p <0.01, ***p <0.001, or ****p <0.0001). figure 4. correlation of malat1 (a), h19 (b), neat1 (c), hotair (d), anril (e), atb (f), uca1 (g) and sngh5 (h) expressions with δbmi (δbmi:prebmipostbmi). tokgun et al. 6 of obesity-related biomarkers in disease development is inadequate [18, 19]. an increasing number of epidemiological studies have investigated the relationship between biomarkers associated with obesity and disease risk. therefore, there is a needing to discover consistent and reliable biomarkers associated with obesity and changing biochemical parameters [19-21]. even though many investigations have been continued in metabolomics, epigenetic, transcriptomic, and proteomic studies are also required. together with the present study, we have added a new dimension to the biomarkers related to obesity and the clinical follow-up of patients undergoing lsg surgery. however, we have realized that our research has several limitations, including a larger sample cohort that further verifies in the broader working group elucidation of molecular mechanisms that relate potential lncrnas and associated pathways. together with our study, we present new research topics in the literature to illuminate these blind spots. supplementary files supplementary file 1. authors’ contributions study conception: ot, acd, ob; study design: ot, ki, bc, pet; supervision: ot, ob, acd; materials: ob; data collection and/or processing: ot, ki, bc, pet; statistical analysis and/or data interpretation: ot, ki; literature review: ot, ki, bc; manuscript preparation: ot, ki, pet, and critical review: ot. all authors read and approved the final version of manuscript. conflict of interests the authors disclosed no conflict of interest during the preparation or publication of this manuscript. ethical declarations the ethics committee approved all protocols used in this study of pamukkale university hospital with the number e-60116787-020-110884. informed consent was taken from each patient applied for the study. financial support this study was partially supported by the scientific research coordination unit of pamukkale university under the project number "2019bsp002o". references 1. blüher m. obesity: global epidemiology and pathogenesis. nat rev endocrinol. 2019; 15(5):288-98. 2. whitlock g, lewington s, sherliker p, clarke r, emberson j, halsey j, et al. body-mass index and cause-specific mortality in 900 000 adults: collaborative analyses of 57 prospective studies. lancet. 2009; 373(9669):1083-96. 3. nguyen nt, varela je. bariatric surgery for obesity and metabolic disorders: state of the art. nat rev gastroenterol hepatol. 2017; 14(3):160-69. 4. nimptsch k, konigorski s, pischon t. diagnosis of obesity and use of obesity biomarkers in science and clinical medicine. metabolism. 2019; 92:61-70. 5. daneshmoghadam j, omidifar a, akbari dn, karimi z, emamgholipour s, shanaki m. the gene expression of long noncoding rnas (lncrnas): meg3 and h19 in adipose tissues 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https://pubmed.ncbi.nlm.nih.gov/34703027/ https://pubmed.ncbi.nlm.nih.gov/34703027/ https://pubmed.ncbi.nlm.nih.gov/34703027/ https://pubmed.ncbi.nlm.nih.gov/28919064/ https://pubmed.ncbi.nlm.nih.gov/28919064/ https://pubmed.ncbi.nlm.nih.gov/28919064/ journal of current biomedical reports jcbior.com volume 1, number 2, 2020 eissn: 2717-1906 1 review current applications of the microbiome engineering and its future: a brief review zahra malayejerdi1, omid pouresmaeil1,* 1department of medical laboratory sciences, varastegan institute for medical sciences, mashhad, iran abstract in the human body there are many microorganisms with a variable genetic content. these microorganisms play an important role in the metabolism, homeostasis, immune system and generally human health. over the millions of years, different microorganisms adapted to each other, and different environmental communities formed on earth. microbial communities, known as microbiome, could exist in living or non-living environments, such as human body and plants, as well as in soil, oceans, and air. the main purpose of microbiome engineering is mostly human microbiome and is now used in the treatment of diseases such as clostridium difficile infection, inflammatory bowel disease, obesity, etc. the research data in this thesis were collected from the main medical article sources including web of science, google scholar, pubmed and scopus. articles on microbiome which published during 20102019 were reviewed. the widespread impacts of the microbiome on the ecosystems and the increased attention to microbiome recognition are factors contributing to the creation of microbiome engineering science, and recent advances in genome sequencing and metagenomic science have made microbiome analysis apart from cultivation process. microbiome engineering has advantages and disadvantages. so, according to the positive aspects and efforts to increase applications, this science could lead to advances in microbial engineering, and have positive effects on human health. although microbiome engineering is a new field, there has been lots of progressions in recent years that can be an important strategy for improving human health by microbial manipulation leading to the changing of microbial population. keywords: microbiome, human gut microbiome, microbiome engineering, microbiota 1. introduction there are many microorganisms in the human body with a wide variety of genetic content. these microbes play an important role in metabolism, homeostasis, immune system, and human health. however, most microbes cannot be cultured in a laboratory environment except for a limited number of bacteria [1]. the mankind has almost the same genetic patterns; just a little difference in dna lead to phenotypic diversity among the human population, * corresponding author: omid pouresmaeil, msc department of medical laboratory sciences, varastegan institute for medical sciences, mashhad, iran tel/fax: +98 915 3752499 email: omidpb71@gmail.com http://orcid.org/0000-0002-0548-2294 received: august, 14, 2020 accepted: september, 18, 2020 but the human microbiome metagenomic, which is, the whole genome of microbes living in the body, is completely variable and unique. therefore, understanding the diversity of healthy microbiome is a major challenge in microbiome research and dates back to the 1960s and continues through the human microbiome project [2, 3]. the human microbiome project is a project launched by the national institute of health (nih) to identify and analysis of all types of microorganisms in the human and the term https://jcbior.com/ malayejerdi et al. 2 “microbiome” was used first time by joshua lederberg in 2001 [4, 5]. through evolution; over millions of years, different organisms have adapted to each other, and different environmental communities have formed on earth. microbial communities, known as microbiome, can exist in living or non-living environments such as the human body, animals, and plants, as well as in soil, oceans, and air. the microorganisms that make up microbiome are actively compatible with the hosts and interactions affect on characteristics of the ecosystems in which they live. it can also have a wide range of effects on physiology (host or environmental conditions). therefore, the widespread impact of the microbiome on the ecosystem and increased attention to microbiome recognition are factors affecting the development of microbial engineering. recent advancements in the sequencing of genome and metagenomic science have made it possible to analyze microbiome independent of culture techniques [6]. the term “microbiome” refers to all microorganisms [including archaea, bacteria, eukaryotes, and viruses], genomes and their environmental conditions, and the term “bio” it is an environmental community it has a special climate, plants and animals where they live. in other words, a microbiome is a combination of metagenomic, metabonomic, metatranscriptomic, and metaproteomic that is described along with comprehensive environmental or clinical information. metagenomic, metabonomic, metatranscriptomic and metaproteomic are sciences that have been formed around the axis of microbiome engineering. microorganisms in the environment are called microbiota. the term microbiota was first coined by lederberg and mccray; human microbiota is made up of many microorganisms, including microbes on the surface of the skin, genital tract, and gastrointestinal tract [6]. the microbiome is a complex term that is sometimes mistakenly used instead of microbiota but, they have different meanings. the term metagenomic is used to describe the sequence of microbial dna; nowadays, the 16srrna marker is used. more than 1,000 intestinal bacterial species have already been discovered; bacteroides have previously been the most common intestinal bacteria, classified into five genera: alistipes, prevotella, paraprevotella, parabacteroides, and odoribacter. metataxonomics is the science that describes the whole microbiota and their phylogenetic relationships through the analysis of their genome. the term microblome is an analytical method in which the metabolic profile of a microbiota is estimated and described. metatranscriptomics analyzes transcriptional mrnas, and metaproteomics addresses the characteristics of all microbiota proteins, first developed by rodrigue-vaiera [7, 8]. microbiome engineering it tries to shape the microbiota to change ecosystems, the characteristics of ecosystems are determined by the main microbiome. the unique feature of each microbiome is the result of the interaction of microorganisms and host communities, as well as the response to metabolites produced by microbial communities. therefore, disruption of microbiomes can cause extensive changes in the host and the environment, the main goal of microbiome engineering is the more human microbiome and today it is used in the treatment of diseases. the human microbiome project to a large extent advanced metagenomic microbial properties in the human body, it describes the intestines, mouth, skin, lungs, nose, and urinary system. the information obtained from the human microbiome project has made microbial engineering dramatically provide solutions to restore the microbial balance with therapeutic functions [2, 6]. 2. methods in the present study, related articles were collected from main sources such as web of science, google scholar, pubmed, and scopus. the selected articles were published between 2010 and 2019, due to the remarkable progress that microbiome engineering has made during this period. microbiome engineering has reached its peak in the coming years and has a very bright future ahead. 3. discussion 3.1 fecal microbiota transplantation jang lee et al. after receiving healthy feces from a healthy donor, prepared a suspension and injected it into the gastrointestinal tract of the recipient. they reported fecal microbiota transplantation (fmt) or fecal transplantation was performed to heeling clostridium difficile infection and restoring the gut microbiome; it is also reported to be effective in treating inflammatory bowel disease (ibd), diarrhea, food poisoning, or other gastrointestinal diseases such as rheumatoid arthritis, autism, cancer, weight loss and even person's mood and behavior [6, 9-11]. malayejerdi et al. 3 3.2 reduce muscle pain and probiotic scheiman et al. by study on the gut microbiome reported that veillonella atypica has an important role to convert the acetic acid to propionate [12]. lactic acid is caused by prolonged exercise and causes muscle pain, while propionate is used by cells to improve exercise ability. the study has shown that v. atypica can increase running time in marathon runners [12]. yatsonko et al. noted the effects of the microbiome in infants. over time, as a result of contact with the environment, more bacteria enter the baby's body, and the organisms entering to the body in the first days of life will most likely remain for the rest of life. depending on the type of delivery and nutrition condition, the microbiome may be different. the study noted that the type of delivery affects on baby's immune system. natural delivery leads to stronger immune system development in babies [2]. jernberg et al. assessed the role of antibiotics and probiotics in association with human microbiome. this study states that antibiotics are the most effective agents on intestinal homeostasis. probiotics are usually a combination of bacteria that produce lactic acid, such as lactobacilli, bifidobacteria, which are important for an appropriate health level [1]. 3.3 stress and depression nofeld et al. experimented on female mice to show the absence of normal microbiota could lead to increased anxiety behaviors. bilateral communication between the gastrointestinal tract and the brain is important for maintaining intestinal homeostasis. this connection is made by the central nervous system. disruption of these systems can lead to changes in response to stress and general behaviors. the results declared that normal microbiota reduces anxiety [13, 14]. serotonin is known as a neurotransmitter that affects the feeling of happiness and regulates mood. in fact, more than 90% of the serotonin produced is in the gut [15]. o'mahoni et al. declared a relation between the intestinal microbiome and the synthesis of serotonin and its effect on depression. they noted that the intestinal microbiome could be a factor in regulating intestinal serotonin production. many intestinal bacteria can produce small molecules such as serotonin, epinephrine, and norepinephrine. clostridium sporogenes can produce tryptophan, stimulating the production of serotonin. escherichia coli and other bacteria such as enterococci can produce serotonin and epinephrine. these hormones regulate many of the functions of the nervous system in the brain by producing other hormones affecting anxiety and stress [3, 15-17]. the study mentioned intestinal microbial disorders, impaired intestinal serotonin synthesis, leading to depression [3, 15-17]. microbiome engineering with a specific metabolic network makes it possible to produce specific products; so, the metabolic activities of each microbiome leads to a special type of biological product with unique properties. 3.4 future and limitations of microbiome engineering microbiome engineering can produce or detect intelligent microbes that have new and unique properties, which can be used to treat some diseases [6]. for example in experiments fmt have been used to treat many diseases, including c. difficile, to restore the gut microbiome [6]. additionally, some bacteria can be generated which selectively could potentially use to eradicate strains of dangerous microbiota in the body. for example e. coli has been engineered to interrupt signaling between microbes as a means of altering the microbiota [18]. despite extensive research on the human microbiome, understanding of the microbiota ecosystem is still limited and the dynamics of the microbiome and the interactions between microbes are not well understood [4, 6]. 4. conclusions although microbiome engineering passes several developments in recent years, it still needs lots of researches and studies. studies on human microbiomes which have been performed using metagenomic, highlight the undeniable functional role of microbiome in the body, and it provides new clinical therapeutic methods related to the microbiome. microbiome engineering is an important strategy for improving human [6]. author contributions all authors contributed equally to this manuscript and approved the final version of manuscripts. conflict of interests authors declare there is no conflict of interest. malayejerdi et al. 4 ethical declarations not applicable. financial support none. references 1. kim bs, jeon ys, chun j. current status and future promise of the human microbiome. 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https://pubmed.ncbi.nlm.nih.gov/27449598/ https://pubmed.ncbi.nlm.nih.gov/27449598/ https://pubmed.ncbi.nlm.nih.gov/27449598/ journal of current biomedical reports jcbior.com volume 1, number 2, 2020 eissn: 2717-1906 1 erratum erratum: immunogenicity, antigenicity and epitope mapping of salmonella invh protein: an in silico study behzad dehghani1,*, iraj rasooli2,3 1 shiraz hiv/aids research center, institute of health, shiraz university of medical sciences, shiraz, iran 2 department of biology, shahed university, tehran, iran 3molecular microbiology research center, shahed university, tehran, iran there was an error in the author list of the published article. two authors (t hashempour, z hasanshahi) requested to remove from the authors lists. after obtaining the agreement of the authors and the corresponding author, editor-in-chief accept the corrections as listed below. the correct author list is: behzad dehghani, iraj rasooli we apologize for any inconvenience this may have caused. * corresponding author: behzad dehghani, msc 2nd floor, voluntary counseling and testing center, lavan ave, delavaran-e basij blvd, khatoun sq, shiraz, fars, iran tel/fax: +98 71 37386272 email: deghanibehzad@gmail.com https://orcid.org/0000-0002-4895-9419 https://jcbior.com/ https://orcid.org/0000-0002-4895-9419 journal of current biomedical reports jcbior.com volume 3, number 4, 2022 eissn: 2717-1906 1 original research evaluation of the antibacterial effect of nickel oxide nanoparticles against bacteria involved in dental caries chanour tavakol1, sara nasiri2, elahe keshavarz3, hossein ghafori4, hirad rokni5, meysam hasannejad6, milad jafari sayadi7, fereshte naser alavi7, hasan pourmoshtagh8,* 1student research committee, school of medicine, tehran university of medical sciences, tehran, iran 2heshmat hospital, guilan university of medical sciences, rasht, iran 3department of sciences, farhangian university, rasht, iran 4department of microbiology, faculty of biological sciences, islamic azad university, tonekabon branch, tonekabon, iran 5school of dentistry, islamic azad university of medical sciences, tehran, iran 6department of microbiology, school of medicine, guilan university of medical sciences, rasht, iran 7razi clinical research development unit, razi hospital, guilan university of medical sciences, rasht, iran 8department of pediatrics, loghman-hakim hospital, shahid beheshti university of medical sciences, tehran, iran abstract tooth decay is one of the most common diseases in the oral cavity and is one of the most widespread diseases in the human population. this study aimed to determine the antibacterial effect of nickel oxide nanoparticles against bacteria involved in tooth decay. in this study, the disk diffusion method was used to determine the antibiotic susceptibility and the microdilution broth method was used to determine the minimum inhibitory concentration (mic). nanoparticles were also synthesized in two molecular size (a: 8.1 and b: 12 nm) by the sol-gel method. the mic of the first nanoparticle for streptococcus sanguinis and streptococcus mutans was 31.25 and 125 μg/ml, respectively. the mic of the second nanoparticle for s. sanguinis was 125 μg/ml. in the case of s. mutans up to a concentration of 500 μg/ml, no growth inhibition was observed. the results showed that nickel oxide nanoparticles have acceptable antibacterial properties against s. mutans and s. sanguinis, which can be used in dental materials to prevent dental caries. however, this requires the determination of cellular toxicity and its side effects in future studies. keywords: nanoparticles, nickel oxide, streptococcus sanguinis, streptococcus mutans 1. introduction tooth decay is one of the most common diseases in the oral cavity and is one of the most widespread diseases in the human population. about half of people 6 to 19 years old who are economically disadvantaged have decayed teeth [1]. tooth decay is also a problem for adults, with more than 90% of people over the age of 40 having it. a quarter of people over the age of 60 have lost all their teeth permanently *corresponding author: hasan pourmoshtagh, md department of pediatrics, loghman-hakim hospital, shahid beheshti university of medical sciences, tehran, iran tel/fax: +98 912 6402128 email: hpourmoshtagh@gmail.com http://orcid.org/0000-0001-8478-8226 received: april, 30, 2022 accepted: july, 30, 2022 due to the impact of caries on their self-esteem and involvement in nutritional problems [2]. bacteria are the main cause of tooth decay. the main mechanism of bacteria in causing tooth decay is the formation of plaque on tooth enamel. plaque is actually a kind of biofilm that is made up of one or more types of microorganisms that can grow at different levels. the formation of dental biofilm is not necessarily a sign of © the author(s) 2022 https://jcbior.com/ tavakol et al. 2 disease, but the formation of biofilm is an important mechanism of disease for bacteria [3]. streptococcus mutans is a gram-positive and aerobic cocci in the oral cavity flora in humans. this bacterium is the most important cause of tooth decay. s. mutans damages tooth enamel by fermenting sucrose and producing lactic acid. the bacterium also uses sucrose to make dental plaque. s. mutans is known to produce lactic acid as part of its metabolism [4, 5]. unlike most edible microorganisms, s. mutans grows in low acid conditions and becomes the dominant bacterium in cultures with a permanent decrease in ph. in addition, contrary to the characteristics of plaque, whose metabolism is significantly slowed down at such a ph, the metabolism of s. mutans is actually improved [4, 6]. streptococcus sanguinis is a gram-positive, aerobic cocci and normal oral flora and is found in dental plaque. s. sanguinis is widely present in the oral cavity [7-9]. studies show that s. sanguinis has two genes which produces glucan. glucan is also formed in the presence of glucose and various forms of sucrose. glucan is the major biofilm polymer in s. sanguinis and increases the adhesion of bacteria to hydroxyapatite, promoting biofilm development [10, 11]. when particles are prepared in nanoscale (less than 100 nm) they can react with organic and inorganic molecules more easily due to the increase in surface area [12]. the main advantages of hydroxyapatite (ha) nanoparticles are their similarity to tooth mineral structure, biological activity and biocompatibility [13, 14]. in one design, the effect of hydroxyapatite magnesium carbonate nanopowder was used to prevent and repair primary caries lesions and the results showed that mgha nanoparticles are adsorbed on the enamel surface and by changing the mechanical properties of the tooth, a new layer is formed in enamel surface and filling all cavities improves tooth remineralization [15]. nickel oxide is of interest due to the chemical and magnetic properties of an oxide. nickel oxide nanoparticles are widely used. these nanoparticles are also used as an effective catalyst for the oxidation of many organic compounds. nickel oxide nanoparticles as an antibacterial agent have also been effective on some bacteria. numerous methods for the preparation of nickel oxide nanoparticles are known. the size distribution and morphology of nanoparticles depend on the way they are synthesized. among the nanoparticle synthesis methods, we can mention the most common methods called sol-gel method [16]. removing plaque from teeth reduces the risk of problems such as tooth decay, gum disease and bad breath. since aerobic and anaerobic bacteria are an important part of dental plaque, new substances with antibacterial properties are essential to prevent their growth. metal nanoparticles show significant antibacterial activity. this property is due to the very small size and surface to volume ratio of these particles. in this regard and the lack of similar studies, we aimed to investigate the antibacterial effect of nickel oxide nanoparticles against bacteria involved in tooth decay. 2. materials and methods 2.1 bacterial strains the studied strains were standard atcc of s. mutans, and s. sanguinis which was lyophilized from the center for genetic and biological resources of iran. to prepare the strain for the experiment, the stored strain was first inoculated in 5 ml of tryptone soy broth (merck, germany) and incubated for 24 hours at 37 °c. then, a fresh culture of a bacterial suspension equal to half mcfarland standard was prepared in müller-hinton broth medium and used for further experiments. 2.2 synthesis of nickel oxide nanoparticles at first, 100 ml of 2 m ammonium hydroxide solution dropwise to 50 ml of 0.5 m solution of ni (no3) 2.6h2o stirred by magnetic magnet for 4 h at 100 °c. c was added. the solution was kept at room temperature for 24 hours. the prepared light green suspension was centrifuged and then the dry precipitate was calcined at 300 and 700 °c (17) [17]. the structure and morphology of the synthesized nanoparticles were identified by ftir and tem techniques. the size of synthetic nanoparticles was estimated to be about 5 to 15 nanometers with this technique. the synthesized nanoparticle powder was first weighed by a sensitive scale on sterile aluminum foil. then pour one gram of nanoparticles in a sterile tube 16 x 20 mm screw cap, and add 10 ml of sterile distilled water. after vortexing, we mixed it well in the sonicator and at room temperature for 10 minutes. then the desired serial dilutions were prepared. all tavakol et al. 3 steps were performed in full compliance with sterile conditions. 2.3 determination of antibiotic susceptibility in order to determine the antibacterial susceptibility, well diffusion method on mullerhinton agar (mha; merck, germany) described by nanda et al. were applied. in each well 25 μl of nanoparticles in desired concentrations (500, 250, 125, and 62.5 µg/ml) was added [18]. finally, the plates were transferred to an incubator at 35-37 °c and the results were evaluated after 16-18 hours. finally, the results were reported based on the measurement of the diameter of the growth inhibition zone by a millimeter ruler. determination of minimum inhibitory concentration (mic) was performed by standard broth microdilution based on the clinical and laboratory standards institute (clsi) recommendation [19]. first, 96-well microplates different concentrations of nanoparticles (500-15.625 µg/ml) were provided using müller-hinton broth (merck, germeny) medium containing 5% sheep blood agar. after providing the required concentrations, 10 μl of the diluted microbial suspension was added to each well (according to clsi method), the optimum concentration of bacteria for inoculation into the wells is 106 cfu/ ml. following 24 h incubation at 37 °c, the wells were inspected for microbial growth, and the mic results were recorded based on the lowest concentration that did not produce visual growth. 2.4 statistical analysis analysis was performed by using spsstm software, version 22.0 (ibm corp., usa). the results are presented as descriptive statistics in terms of relative frequency. 3. results morphological studies determined the size of calcifying nanoparticles at 300 °c (np1) and 400 °c (np2) were 8.1 and 12 nm, respectively. as the calcination temperature decreases, the size of the nanoparticles decreases, and as the size decreases, the surface-to-volume ratio increases. according to the table 1, the growth inhibition zone for the concentration of 500 to 62.5 µg/ml of nanoparticles for s. sanguinis varied from 12 mm to 17 mm. for s. mutans, the minimum zone diameter was 10 mm at the 250 µg/ml of nanoparticles and the maximum zone diameter was 14 mm at both 125 and 62.5 µg/ml of nanoparticles. based on table 2, the amount of the first mic nanoparticle for s. sanguinis and s. mutans was 31.25 and 125 μg/ml, respectively. 4. discussion according to our results, it was illustrated that s. sanguinis represented a higher antibacterial effects than s. mutans. numerous studies have been performed on the effects of nanocomposites on bacteria that cause tooth decay. similar to our study, a study by khodaee et al., the antibacterial and antibiofilm effects of silver nanoparticles against dental plaque microorganisms, s. mutans and agrigatebacter actinomycetemcomitans represented that the active ingredient in silver nanoparticles showed a greater antibacterial effect than nickel oxide nanoparticles [20]. in the study of emrani et al., the results showed that the mic for biosynthesized nanoparticles of licorice extract against s. mutans, actinomyces viscosus and lactobacillus rhamnosus bacteria were 1.56, 6.25, and 50 µg/ml, respectively (p ≤0.05), and the mic for nanoparticles biosynthesized with peppermint extract was 12.5, 12.5, and 200 µg/ml (p ≤0.05), respectively [21]. both expression and mint showed greater antibacterial effect than nickel oxide nanoparticles. in another study conducted by vahid dastjerdi et al., it was found that the largest diameter of the growth inhibition disk in the disk diffusion method for s. mutans, s. sanguinis, streptococcus salivarius, streptococcus sobrinus and enterococcus faecalis at a concentration of 100 micrograms per milliliter and equal to 14, 18, 15, 18, and 10.5 µg/ml, respectively. the mic values for s. mutans, s. sanguinis, s. salivarius, s. sobrinus and e. faecalis were 50, 25, 6.25, 25, 25, and 50 µg/ml, respectively [22]. similar to the previous study on nanoparticles biosynthesized with licorice and peppermint, in this study the results showed that the aqueous extract of pomegranate flower showed slightly more antibacterial properties than nickel oxide nanoparticles, and perhaps this hypothesis suggests that the use of biological compounds along with nanoparticles increases their effectiveness, which of course requires further studies to prove this. tavakol et al. 4 in another study conducted by sadeghi et al., the mic of silver and chlorhexidine nanoparticles in relation to s. sanguinis was 25 and 16 µg/ml, respectively, and for actinomyces viscosus was 4 and 64 µg/ml, respectively. comparing the results of this study with the present study, both types of nickel oxide nanoparticles showed more antibacterial effect than chlorhexidine and less antibacterial effect than silver nanoparticles [23]. an investigation by lu et al., in which the mic of 5 nanometer silver nanoparticles for anaerobic bacteria such as actinomycetemcomitans, fusobacterium nucleotum, streptococcus mitis, s. mutans, and s. sanguinis were 50, 50, 25, 25, 25 and 25 µg/ml, respectively [24]. comparing the results of this study with our study, showed that the first type of nickel oxide nanoparticles showed less antibacterial effect against s. mutans and more antibacterial effect against s. sanguinis than silver nanoparticles. in the study of aflatoonian et al., the minimum growth inhibitory concentration and the minimum bactericidal concentration of nanoparticles on pseudomonas aeruginosa and e. faecalis strains were determined using broth microdilution method. visible-ultraviolet spectroscopy showed an absorption peak in the range of 370 nm. transmission electron microscopy images showed the synthesis of more spherical zinc oxide nanoparticles with a size of less than 50 nm. the lowest inhibitory concentrations of zno nanoparticles against p. aeruginosa and e. faecalis were determined to be 6.25 and 12.5 µg/ml, respectively [25]. a study by davari et al. represented that copper and zinc oxide nanoparticles at concentrations of 0.1 and 0.5% at 15 and 30 days compared to the control group significantly reduced the number of bacteria. zinc oxide nanoparticles at 0.5% in the composite composition showed the highest and both oxidized nanoparticles and zinc at the concentration of 0.05% showed the lowest amount of antimicrobial properties [26]. despite the promising effects of nickel oxide nanoparticles, further study to investigate the toxicity on different human cell lines is suggested as the most important limitation of the recent study. the results showed that nickel oxide nanoparticles have acceptable antibacterial properties against s. mutans and s. sanguinis, which can be considered as preventative agents for tooth decay in materials and compounds used in dentistry. authors' contributions concept and study design: ek, mh, fn, hp; methods, data collection and experimental work: ek, mj, mh; results analysis and drafting: ct, sn, hg, hr, hp; critical revisions: ct, mh, hr, fn, hp. all authors read and approved the final version. conflict of interests none to be declared. ethical declarations the study deign was approved by the ethical committee at the guilan university of medical science [ir.gums.rec.1399.569]. table 1. diameter of zone inhibition by well-diffusion nanoparticle streptococcus sanguinis streptococcus mutans 500 µg/ml 250 µg/ml 125 µg/ml 62.5 µg/ml 500 µg/ml 250 µg/ml 125 µg/ml 62.5 µg/ml np (1) 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https://pubmed.ncbi.nlm.nih.gov/26171362/ https://pubmed.ncbi.nlm.nih.gov/26171362/ https://pubmed.ncbi.nlm.nih.gov/26171362/ https://jida.ir/article-1-1149-en.html https://jida.ir/article-1-1149-en.html https://jida.ir/article-1-1149-en.html https://jida.ir/article-1-1149-en.html https://pubmed.ncbi.nlm.nih.gov/23440430/ https://pubmed.ncbi.nlm.nih.gov/23440430/ https://pubmed.ncbi.nlm.nih.gov/23440430/ http://eprints.zbmu.ac.ir/3014/ http://eprints.zbmu.ac.ir/3014/ http://eprints.zbmu.ac.ir/3014/ http://eprints.zbmu.ac.ir/3014/ https://jmds.mums.ac.ir/article_14273.html?lang=en https://jmds.mums.ac.ir/article_14273.html?lang=en https://jmds.mums.ac.ir/article_14273.html?lang=en https://jmds.mums.ac.ir/article_14273.html?lang=en journal of current biomedical reports jcbior.com volume 3, number 2, 2022 eissn: 2717-1906 1 original research the effect of n2o gas on serum cobalamin and homocysteine in patients undergoing orthopedic surgery amir mohammad azizi kolachahi1, mahmood khosravy2, aidin badanara marzdashty3, mahmood abedinzade4,* 1student research committee, school of paramedicine, guilan university of medical sciences, rasht, iran 2department of hematology and medical oncology, school of medicine, arak university of medical sciences, arak, iran 3department of anesthesia and operating room, guilan university of medical sciences, rasht, iran 4department of physiology, school of paramedicine, guilan university of medical sciences, rasht, iran abstract nitrous oxide (n2o) is an inhaled anesthetic drug that uses during surgery and recently proposed as a treatment for mood disorder in a research setting. exposure to n2o during surgery can cause hematopoietic and neural side effects. the purpose of this study was to the designation of serum cobalamin, homocysteine and cbc factors changes after exposure to nitrous oxide during orthopedic surgery. this study has been done on 30 patients undergoing orthopedic operations from 2016 until 2018. all patients are scheduled for orthopedic surgery. all patients received nitrous oxide during their anesthesia period. blood samples were taken from patients 48 hours before surgery and 24 hours after surgery. all samples froze at -20°c immediately after collecting. then samples transferred to the university laboratory with regards to keeping a cold chain for analysis. paired t-test and wilcoxon test were used for comparing vitamin b12, homocysteine, and cbc parameters. nitrous oxide resulted in marked vitamin b12 and homocysteine level change. mean concentration of vitamin b12 reduced after exposure to n2o. this difference was significant when analyzed using paired sample t-test (p=0.0001). homocysteine concentration means decreased after exposure to n2o and this difference was statistically significant (p=0.0001). nitrous oxide will lead to a decrease in vitamin b12 and an increase in homocysteine and mean corpuscular volume in the early postoperative period. keywords: analgesic, homocysteine, nitrous oxide, vitamin b12 1. introduction nitrous oxide (n2o) is an inhaled anesthetic drug that uses during surgery and recently proposed as a treatment for mood disorder in a research setting. exposure to n2o during surgery can cause hematopoietic and neural side effects [1-3]. even exposure to nitrous oxide can induce ischemic stroke, aortic arch thrombosis, cortical vein thrombosis [4-6]. vitamin b12 plays a major role in the functioning of the central nervous system of all ages. its deficiency can be accompanied by disorders of the brain, the optic *mahmood abedinzade, phd department of physiology, guilan university of medical sciences, rasht, iran tel/fax: +98 13 42565058/+98 13 4256505 email: mahmood.abedinzade@gmail.com http://orcid.org/0000-0002-9216-7216 received: december, 08, 2021 accepted: february, 23, 2022 nerve, the spinal cord, and the peripheral nervous system [7]. vitamin b12 deficiency is common in both advanced and unprocessed countries [8] many factors, such as plant-based diet, malnutrition, congenital maladaptation, post-operative maladaptation, anti-neoplastic, and other digestive diseases (ileum cut, over-bacterial growth, and inflammatory bowel disease) can cause vitamin b12 deficiency [9]. plasma homocysteine is elevated after exposure to n2o and may have a role in nitrous oxide side effects including pregnancy complications. in © the author(s) 2022 https://jcbior.com/ https://jcbior.com/ azizi kolachahi et al. 2 several patients whose plasma homocysteine levels were compared after surgery, they all had vitamin b12 deficiency. so plasma homocysteine can be a good parameter for measuring folate-related disorders of the metabolism of cobalamin[10, 11]. in a study on 12 patients undergoing surgery, after surgery, the levels of folate and homocysteine increased and methionine was decreased. in some of these patients, even the suffering of cases has not returned to normal one week later, and it is worth noting that by determining the level of homocysteine, the effect of n2o on the deactivation of vitamin b12 can be estimated [12, 13]. so it is known that chronic exposure to nitrous oxide may have an impact on vitamin b12 and homocysteine but the effect of acute nitrous oxide exposure on vitamin b12 and homocysteine level and its impact on cbc parameters in the early postoperative period is unknown in orthopedic surgery patients. on the other hand, recent studies mostly explore the effect of recreational use of nitrous oxide but we studied acute single exposure to nitrous oxide. the purpose of this study was to the designation of serum cobalamin, homocysteine and cbc factors changes after exposure to nitrous oxide during orthopedic surgery. 2. materials and methods 2.1 study design this study has been done on 12 patients undergoing orthopedic operations from 2016 until 2018. the inclusion criteria were all patients undergoing orthopedic procedures with ages between 20 to 60 years. exclusion criteria were patients with abnormal cbc, low level of vitamin b12 or high level of homocysteine before exposure to nitrous oxide, n2o exposure less than 120 minutes, cardiac or renal disease, pregnant or lactating women, middle ear obstruction, high intracranial pressure, and pneumothorax and patients using vitamin b12 supplement. age, weight, height, and sex of patients were recorded. the whole procedure and aims of the study were explained to patients and written consent was obtained. this study was approved by the ethics committee of guilan university of medical sciences with the approval code ir.gums.rec.1395.102. 2.2 sampling and procedure up to 48 hours before surgery blood samples were collected from patients in two tubes. one tube contained edta and one without anticoagulant. blood with anticoagulant used to measure cbc parameters and clot samples used to measure homocysteine and vitamin b12 level. patients anesthetized by attending anesthesiologist using fentanyl 1-3 mic.kg-1, midazolam 1 mg, lidocaine 1 mg.kg-1, propofol 1.5-2.5 mg.kg-1 and atracurium 0.6 mg.kg-1 then after 3-minute patients intubated using cuffed tracheal tube. patients’ lungs are ventilated by 50 percent nitrous oxide and 50 percent oxygen. anesthesia maintained by isoflurane 1% and muscle paralysis provided with atracurium besylate intermittent doses. if any episode of hypoxemia occurred nitrous oxide exposure ceased and the patient was excluded from the study. at the end of surgery patients ventilated with 100% oxygen and all anesthetics ceased. after returning of patient’s spontaneous respiration remained effect of muscle relaxants reversed using neostigmine 0.04 mg/kg and atropine 0.02 mg/kg. after returning of patients' consciousness and adequacy of minute ventilation patients extubated and transferred to the postanesthesia care unit (pacu). after the entrance of patients to pacu up to 24 hours after surgery, we recollect blood samples from patients. all samples froze at -20°c immediately after collecting. then samples transferred to the university laboratory with regards to keeping a cold chain for analysis. all samples were analyzed in the paramedical sciences school’s laboratory. the serum was separated by centrifugation. life span bioscience quantitative competitive elisa kit (cat: ls-f27312) applied for determining the levels of serum vitamin b12. detection methods were colorimetric at 450 nm (tmb) with the range of 5-1000 pmol/l. the sensitivity of the kit was less than 1 pmol/l (intraassay cv (<9%); inter-assay cv (<10%)). both standard curves and samples ran in duplicate. 2.3 statistical analysis spss software version 16 was used for statistical analysis. paired t-test and wilcoxon test were used for comparing vitamin b12, homocysteine, and cbc parameters. a p-value less than 0.05 is considered significant. 3. results among patients, there were 6 females and 6 males. the patient's age was between 20 to 67. the mean age of patients was 47.25 year. nitrous oxide azizi kolachahi et al. 3 exposure time was between 120 minutes and 420 minutes. the mean nitrous oxide exposure time was 231.83 min and the standard deviation was 84.48. distribution of nitrous oxide exposure time was normal due to the kolmogorov smirnov test result. after analyzing data using the kolmogorov smirnov test it was revealed that data distribution was normal for cbc parameters including white blood cell count, hemoglobin, hematocrit, mean corpuscular hemoglobin concentration, homocysteine, and vitamin b12 before and after exposure to nitrous oxide. paired sample t-test was used to compare vitamin b12, homocysteine, white blood cell count, hemoglobin, hematocrit, and mean corpuscular hemoglobin concentration before and after exposure to nitrous oxide. the mean concentration of vitamin b12 before exposure to n2o was 527.75 (48.16) pg.ml1 and reduced to 139.17 (21.20) pg.ml-1 after exposure to n2o. the mean of differences with a 95 percent confidence interval was between 357.85 and 419.30. this difference was significant when analyzed using paired sample t-test (p<0.05). the effect size for this difference is -8.03. changes in vitamin b12 concentration showed in figure 1. homocysteine concentration mean was 10.48 (0.68) µmol. l-1 before and 19.94(1.27) µmol. l-1 after exposure to n2o and this difference was statistically significant (p<0.05). the effect size for the difference in homocysteine before and after exposure to nitrous oxide is 6.47. the mean of differences in homocysteine level before and after exposure to nitrous oxide with a 95 percent of the confidence interval was between 8.52 and 10.39. homocysteine concentration changes before and after surgery showed in figure 2. paired ttest analysis results are described in table 1. wilcoxon test was used to compare cbc parameters including red blood cell count, mean corpuscular volume, mean corpuscular hemoglobin, and platelet before and after exposure to n2o. there was a statistically significant difference for rbc and mcv before and after nitrous oxide exposure (p<0.05) mch and platelet count did not change before and after surgery significantly. these data are depicted in table 2. 4. discussion this study showed that nitrous oxide exposure during orthopedic surgery can affect vitamin b12 and homocysteine levels in the early postoperative period. the mean concentration of vitamin b12 before exposure to n2o was 527.75 (48.16) pg.ml-1 and reduced to 139.17 (21.20) pg.ml-1 after exposure to n2o. homocysteine concentration mean decreased after exposure to n2o and this difference was statistically significant (p<0.05). nitrous oxide prescribed to patients for more than a century and has favorable characteristics including low tissue solubility, analgesia, no or minimal cardiovascular depressant effect, and low cost. despite these advantages, nitrous oxide may cause nausea and vomiting, bowel distension, act as a greenhouse gas, and vitamin b12 deficiency. the most important clinical symptom after exposure to n2o is a deficiency in vitamin b12 level. n2o oxidizes vitamin b12 and converts it from active to an inactive form [14]. moreover, applies its harmful figure1. vitamin b12 level before and after exposure to n2o 0 200 400 600 1 2v it a m in b 1 2 l e v e l before n2o after n2o v i t a m i n b 1 2 b e f o r e a n d a f t e r n i t r o u s o x i d e e x p o s u r e figure 2. homocysteine level before and after exposure to n2o 0 5 10 15 20 25 1 2h o m o c y st e in e le v e l before n2o after n2o h o m o c y s t e i n e l e v e l b e f o r e a n d a f t e r e x p o s u r e t o n 2 o azizi kolachahi et al. 4 effects with the oxidation of cobalt ion in cobalamin and its conversion from the capacity (+1) to (+ 3) irreversibly. the oxidation of cobalt ion by nitrous oxide prevents methylcobalamin as a co-enzyme in the production of methionine and subsequently sadenosylmethionine, which is essential for the methylation of phospholipids in the myelin sheath and the result is reduced myelin building [15, 16]. in our study, we aimed to evaluate nitrous oxide exposure effect on vitamin b12, homocysteine, and cbc factors in orthopedic surgery patients. we found that exposure to nitrous oxide even for short time (between 120 to 420 minutes) will decrease vitamin b12 level significantly. there is a similar result in the study of glinj et al., lundin et al., and noh et al. [1719]; however, controversy was seen in a previous study [20]. vitamin b12 may be affected during anesthesia using nitrous oxide which may lead to granulocytopenia, megaloblastic anemia, thrombocytopenia, leaukopenia, and bone marrow suppression. when we evaluate the correlation between age and the effect of nitrous oxide on homocysteine level, we found a significant negative correlation between these parameters. previous study revealed that nitrous oxide would increase homocysteine in children [21]. our study cleared that increase in age increase this effect. on the other hand, powers et al, revealed that nitrous oxide would increase homocysteine in old rats but their study there was on rats and we evaluate nitrous oxide effect on humans [22]. nitrous oxide binds to vitamin b12 and oxidizes it irreversibly and therefore decreases vitamin b12 level. most studies emphasize on duration and concentration of nitrous oxide exposure as a determinant factor in vitamin b12's destructive effect [23]. but we found that time has no effect on nitrous toxicity which is consistent with patel et al., study [24]. studies revealed that vitamin b12 deficiency induced by nitrous oxide is severe and according to the latest studies vitamin b12 level lower than 200 pg/ml considered deficient which is consistent with our study results [25, 26]. the mean vitamin b12 value after exposure to nitrous oxide was 169.00 which is considered deficient. based on our finding it seems to short-time exposure to nitrous oxide will inactive vitamin b12 and decrease its blood level which may table 1. mean, standard deviation, and differences of measured parameters before and after exposure to nitrous oxide variable mean (sd) before exposure mean (sd) after exposure p-value* vitamin b12 527.75 (48.16) 139.17 (21.20) <0.001 homocysteine 10.48 (0.68) 19.94 (1.27) <0.001 white blood cell count 9766.70 (3802.47) 12292.0 (4522.96) 0.03 hemoglobin 11.51 (1.2) 10.63 (1.38) 0.02 hematocrit 34.10 (3.07) 31.85 (3.60) 0.04 mean corpuscular hemoglobin concentration 33.72 (1.88) 33.30 (1.77) 0.12 *paired sample t-test table 2. mean and standard deviation of rbc, mcv, mch, and platelet count variable mean (sd) before exposure mean (sd) after exposure mean rank z p-value red blood cell count 4.04 (0.60) 3.73 (0.65) negative rank 6.60 -2.12 0.03 positive rank 6.00 mean corpuscular volume 85.25 (9.85) 86.55 (9.69) negative rank 5.00 -2.27 0.02 positive rank 6.80 mean corpuscular hemoglobin 28.80 (4.01) 28.90 (4.06) negative rank 4.75 -0.40 0.68 positive rank 7.50 platelet count 247330.0 (106491.0) 260250.0 (134538.0) negative rank 6.20 -0.62 0.53 positive rank 6.71 azizi kolachahi et al. 5 lead to numerous complications including subacute combined degeneration of the spinal cord and even acute psychosis [27]. it is proposed that nitrous oxide used with more cautions in patients with known vitamin b12 deficiency diagnosed based on neurologic symptoms or laboratory investigations. we measured homocysteine level in addition to vitamin b12. results showed that there was increased homocysteine level in all cases exposed to nitrous oxide and there was a statistically significant difference in homocysteine level before and after exposure to nitrous oxide. our finding is consistent with lan et al., myles et al., and nagele et al., studies [28-30]. lan et al. found that exposure to nitrous oxide will increase homocysteine level and patients with increased homocysteine level experienced neurologic dysfunctions including numbness, paresthesia, weakness, ataxia, impaired cognition, and other neurologic symptoms [30]. pichardo et al., found that exposure to nitrous oxide for more than two hours in pediatric patients will increase homocysteine levels [21]. but in our study, we found that there is no relationship between nitrous oxide exposure time and homocysteine level. in our study, neither vitamin b12 level nor homocysteine did not get impressed by nitrous oxide exposure time. it was interesting that in the acute phase of nitrous oxide exposure time patients experienced an increase in mean corpuscular volume and this increase was statistically significant. our finding was against duma et al. [31], this difference may be due to the different age groups studied. it is known that lack of vitamin b12 may lead to megaloblastic anemia but the pace of this event was too much rapid. on the other hand, there was a significant reduction in hemoglobin in our patients and, predictably, this reduction may lead to microcytic anemia which probably attenuates the effect of nitrous oxide on mean corpuscular volume. intraoperative fluid therapy had been done with isotonic solutions thus increase in mcv can’t be related to fluid therapy. we don’t know whether this effect persists hours or days after surgery in adults and elderly populations or not. also, we don't know if patients get nitrous oxide for a long-time but without bleeding surgeries how would be mcv impacted. we propose future studies explore the presence and severity of this effect in days after exposure to nitrous oxide and surgeries without or with minimal bleeding. according to the vitamin b12 metabolism cycle, it is logical that decreased vitamin b12 leads to increased homocysteine. vitamin b12 is a key factor in converting homocysteine to methionine. thus when there is a lack of vitamin b12 or there is a nonfunctional form of this vitamin it will result in increased homocysteine [32]. homocysteine is a sulfur-containing amino acid that participates in methionine metabolism. high homocysteine level is an independent risk factor for cardiovascular disease including coronary artery disease, cerebrovascular accident and can lead to high blood pressure and left ventricular hypertrophy [33-35]. even a five micromoles increase in homocysteine level more than normal amounts can increase the risk of cardiovascular disease up to 20 percent. homocysteine has many effects on all three layers of the arterial wall including endothelium, tunica media, and tunica adventitia. homocysteine induces inflammation, apoptosis, atherosclerosis plaque formation, smooth muscle proliferation, collagen formation, and elastin degeneration in the arterial wall [35]. so, it can be concluded that homocysteine can act as both indicators of low vitamin b12 function and a predictor of cardiovascular events. so, it can be assumed that exposure to nitrous oxide may put patients at risk of cardiovascular events. a direct relationship between nitrous oxide use, homocysteine level, and cardiovascular events needs further investigations. some case reports showed myelin sheath defects due to nitrous oxide recreational use [30]. vitamin b12 is crucial in the central nervous system for synthase and maintenance of myelin sheath. it is clarified that lack of vitamin b12 can lead to subacute combined degeneration which is characterized by a defect in myelin sheath in the lateral and dorsal part of the cervical and thoracic spine, brain white matter demyelination, and peripheral neuropathy [24]. when we explored cbc parameters, we found a statistical difference in white blood cells, red blood cells, hemoglobin, hematocrit, and mean corpuscular volume before and after surgery. other parameters including mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration and platelet count did not change statistically before and after surgery and exposure to nitrous oxide. based on our finding it is rational to consider measurement of vitamin b12 and homocysteine after surgery in patients susceptible to vitamin b12 deficiency-like old azizi kolachahi et al. 6 patients, vegans, and patients with malabsorptive diseases are to undergo nitrous oxide anesthesia. it may help in the reduction of cardiovascular, neurologic, and hematologic complications. as a limitation, we did not assess neurologic and cardiovascular complications due to vitamin b12 deficiency and homocysteine increase. we propose a study on these complications in future studies. the most important limitation of this study was a small number of orthopedic patients anesthetized with nitrous oxide, which can authors’ contributions mkh and ma designed the study, aa, mkh and ab performed the study. ma and mkh do analysis and interpreting the data. all authors discussed the results and participated in the final version of the manuscript. conflict of interests authors declare that there is no conflict of interest. ethical declarations the whole procedure and aims of the study were explained to patients and written consent was obtained. this study design was approved by the ethics committee of guilan university of medical sciences with the approval code ir.gums.rec.1395.102. financial support this research supported by a grant from vice chancellor for research and technology of guilan university of medical sciences (grant no. 95040108). references 1. ilniczky s, jelencsik i, kenez j, szirmai i. mr findings in subacute combined degeneration of the spinal cord caused by nitrous oxide anaesthesia-two cases. eur j neurol. 2002; 9(1):1014. 2. keddie s, adams a, kelso ar, turner b, schmierer k, gnanapavan s, et al. no laughing 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invh protein: an in silico study behzad dehghani1,*, tayebeh hashempour1, zahra hasanshahia, iraj rasooli2,3 1 shiraz hiv/aids research center, institute of health, shiraz university of medical sciences, shiraz, iran 2 department of biology, shahed university, tehran, iran 3molecular microbiology research center, shahed university, tehran, iran abstract invh is an indispensable part of t3ss-i and has a significant role in spi-i mediated effector protein translocation. the invh mutations have significant effects including reduced secretory and inflammatory responses that result from preventing the normal secretion of several proteins. our team previous studies showed the capable ability of invh to induce the humoral immune system to prevent almost all salmonella strains infections. the current study aimed to determine all aspects of this protein using several bioinformatics tools and find the differences among all salmonella strains. this data could pave the way for further studies about invh protein and the production of an effective vaccine against salmonella infections. invh sequences for all salmonella strains were obtained from genbank and analyzed to determine physicochemical properties, b-cell and t-cell epitopes, and reliable structures. results showed some minimal differences among salmonella strains. b-cell and t-cell epitopes predicted by numerous software approved the ability of this protein to induce both humoral and cellular immune systems remarkably. this study provided a comprehensive data to determine all features of invh protein and our results showed the ability of this protein to design a capable vaccine and the effect of amino acid changes on structure and physicochemical properties, and epitopes. keywords: invh, salmonella, bioinformatics, spi-i, vaccine 1. introduction each year 1.3 million cases of salmonellosis occur globally. salmonella enterica (s. enterica) includes many foodborne pathogens causing salmonellosis in developing or developed countries. patterns of salmonellosis in humans include enteric fever, gastroenteritis, bacteremia, and chronic carries. typhoid fever is caused by salmonella enterica serovar typhi (s. typhi), and * corresponding author: behzad dehghani, msc 2nd floor, voluntary counseling and testing center, lavan ave, delavaran-e basij blvd, khatoun sq, shiraz, fars, iran tel/fax: +98 71 37386272 email: deghanibehzad@gmail.com https://orcid.org/0000-0002-4895-9419 received: may, 31, 2020 accepted: june, 10, 2020 paratyphoid fever is caused by salmonella paratyphi (s. paratyphi) a, b, and c. infection is spread by contaminated food or water. salmonella typhimurium (s. typhimurium) and salmonella enterica serovar enteritidis (s. enteritidis) cause gastroenteritis via contaminated food or water by animal waste. bacteremia is a serious infection associated with invasive serohttps://jcbior.com/ https://orcid.org/0000-0002-4895-9419 dehghani et al. 2 types, salmonella cholearaesuis (s. cholearaesuis), and salmonella dublin (s. dublin). chronic carriers are major factors to spread salmonellosis in the word [1]. many gram-negative bacteria have a type iii secretion system. the type-iii secretion system-i (t3ss-i) of s. enterica is necessary for invasion and translocation effectors proteins from bacteria into host cells. two salmonella pathogenicity islands of spi1 and spi2 encode structural components, effectors, and regulators of t3ss1 [2, 3]. invh, a main part of t3ss-i, is necessary for the effective performance of salmonella invasion of epithelial cells [4, 5]. deletion of invh gene is related to reduce the invasion efficiency of the bacterium compared to the wild-type [5]. genetic analysis has shown the presentation of invh in many salmonella strains [6]. immunization with recombinant invh against s. enteritidis and s. typhi in animal models has shown its potential as a vaccine candidate [7, 8]. many bioinformatics tools have been employed to analyze the properties and features of different bacterial and viral genes [913]. this study aimed to compare experimental and bioinformatics data to find epitopes, modification sites, and structure analysis that can provide much useful information on immunogenicity and antigenicity of invh protein. 2. material and methods 2.1. invh sequence analysis for bioinformatics analysis, 6 sequences (full length: 444 bp, 148 amino acids) were obtained from genbank (http://www.ncbi.nlm.nih.gov). the sequences included were accession number: cp009102.1 (s. typhimurium), accession number: aoyf01000046.1 (s. paratyphi b), accession number: al513382.1 (s. typhi), accession number: nc_006511.1 (s. paratyphi a), accession number: cp000857.1 (s. paratyphi c), and accession number: am933172.1 (s. enteritidis). 2.2. amino acid changing and phylogenetic trees all sequences were translated, edited, and aligned using clc sequence viewer version beta (qiagen). phylogenic trees were determined by neighbor-joining (nj) methods 1000 times to confirm the reliability of phylogenetic trees. signal peptide and signal peptide cleavage sites for gram-negative bacteria were determined using "signalp 4.1” at (http://www.cbs.dtu.dk/services/signalp/) [14], "predisi" (http://www.predisi.de/predisi/startprediction) [15], and "signalblast” (http://sigpep.services.came.sbg.ac.at/signalblast.html) [16]. 2.3. invh physicochemical properties "expasy’s protparam" (http://expasy.org/ tools/protparam.html) was used to determine theoretical isoelectric point (pi), instability index, aliphatic index, and grand average hydropathy (gravy) [17-19]. 2.4. immuno-informatic prediction "immuneepitope" (http://tools.immuneepitope.org/tools/bcell/iedb_input) was employed to determine b-cell epitopes positions. chou and fasman method was used to predict beta-turns, karplus and schulz for predicting the flexibility; emini method was used for surface accessibility prediction, and parker method for hydrophilicity evaluation and bepipred method to define b-cell epitopes using crystal structures. b-cell epitopes prediction was based on hydrophilic, flexibility, surface accessibility, b-turns, antigenic, exposed surface, and polarity scale methods using bcepred software at (http://www.imtech.res.in/raghava/bcepred) [20], and abcpred software (http://www.imtech.res.in/raghava/abcpred/) [21]. t-cell epitope prediction was done by "syfpeithi" (http://www.syfpeithi.de/bin/mhcserver.dll/epitopeprediction.htm) [22], and "immuneepitope" (http://tools.immuneepitope.org/main/tcell/) [23]. antigenicity probability was estimated (http://www.ddg-pharmfac.net/vaxijen/vaxijen/vaxijen.html) using vaxijen software [24]. prediction of ige epitopes was carried out by algpred at (http://www.imtech.res.in/raghava/algpred/submission.html) [25]. 2.5. disulfide bond prediction disulfide bonds position was predicted using dianna [26] (http://clavius.bc.edu/~clotelab/dianna) and scratch [27] (http://scratch.proteomics.ics.uci.edu/). 2.6. secondary structure prediction secondary structure was predicted and confirmed by sopma (http://npsa-pbil.ibcp.fr/cgibin/npsa_automat.pl?page=npsa_sopma.html), http://www.cbs.dtu.dk/services/signalp/ http://www.cbs.dtu.dk/services/signalp/ http://tools.immuneepitope.org/main/tcell/ http://scratch.proteomics.ics.uci.edu/ http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html dehghani et al. 3 phyre server (http://www.sbg.bio.ic.ac.uk/phyre) [28]. 2.7. prediction and validation of tertiary structure i-tasser (http://zhanglab.ccmb.med.umich.edu/itasser) [29-32], phyre2server (http://www.sbg.bio.ic.ac.uk/~phyre) [28], and (ps)2 server [33] (http://ps2v2.life.nctu.edu.tw) were employed to build 3d structures the stereochemistry and quality of the 3d structures models were evaluated by qmean [34, 35] (http://swissmodel.expasy.org/qmean/cgi/index.cgi) and ramachandran plots were mapped by rampage (http://mordred.bioc.cam.ac.uk/~rapper/rampage.php). 3. results 3.1. sequences analysis and phylogenetic trees the alignment of all sequences is shown in figure 1. phylogenetic tree for 6 sequences by nj method: two main clades were shown in the tree, upper clade is divided into two clusters; in the first cluster paratyphi a and typhi (with 95 bootstrap score); in the second cluster paratyphi c and typhimurium are very close (with 83 bootstrap score). a clade contains enteritidis and paratyphi b (figure 2). signal peptide prediction of each software was similar for all sequences. 3.2. amino acid changing a comparison of the sequences with s. typhi revealed only 4 amino acid residue changes. changes at positions 36 (k-q) and 139 (a-s) in s. paratyphi c, amino acid 67 (q-h) in s. typhimurium, position 146 (a-s) in s. enteritidis. 3.3. protparam results almost the results were similar for all sequences except for pi of s. paratyphi c (table 1). 3.4. immuno-informatics immuneepitopes by beta-turn methods determined three positions of 65-72, 70-76 and 126132 as b-cell epitopes regions. surface accessibility method showed two regions (87-92 and 109-114) and flexibility method showed three regions of 124-131, 87-95 and 106-114, antigenicity method indicated region 6-16, hydrophilicity method revealed a region (107-114), bepipred (threshold:1) method determined three regions. the regions were similar in all sequences. seven parameters of hydrophilicity, flexibility, surface accessibility, beta turs, surface exposure and polarity were almost similar according to bcepred results (table 2). abcpred results revealed a start position of 16 meric b-cell epitopes of 97, 46, 89, and 29 were similar for all sequences. prediction of protective antigens by "vaxijen" program was around 0.34 and was probable non-antigen for all sequences. prediction by mapping of ige epitopes using "algpred" software revealed as non-allergen for all sequences. hla-e and hla b27-5 t-cell epitopes results are shown in table 3. hla-e epitopes in all sequences were similar and hla-b27-5 epitope regions were similar in all sequences except for s. paratyphi c in which region 27 in hla-b*27:05 was absent. the 8-14 meric epitopes were similar in all sequences except for s. paratyphi c in which region 27 in hlab*27:05 was absent. 3.5. disulfide bond prediction three disulfide positions (7, 48, and 85) were predicted by "dianna" and 4 positions (7, 16, 48, and 85) by "scratch". positions 7, 16, 48, and 85 were conserved for disulfide bonds. 3.6. secondary and tertiary structures prediction percentages of secondary structures by "sopma" are given in table 4. the results were almost similar in all sequences. the secondary structures are shown in figure 3. we could not construct a valid structure for invh protein using all programs. 4. discussion cloning and molecular characterization of invh were performed in 1993 [6]. the role of this protein in adherence and invasion to epithelial cell culture was determined in previous studies [6]. invh is necessary for intestinal secretory and inflammatory responses, bovine macrophages lysis, and secretion of salmonella effecter proteins by type iii secretion system (ttss). invh is an important part of the needle complex of ttss and is required for efficient assembly of the organelle. invh is present in all salmonella strains except salmonella heidelberg (s. heidelberg) and salmonella enterica subspecies arizonae (s. arizonae). invh is highly conserved in all strains. http://www.sbg.bio.ic.ac.uk/phyre http://zhanglab.ccmb.med.umich.edu/i-tasser http://zhanglab.ccmb.med.umich.edu/i-tasser http://www.sbg.bio.ic.ac.uk/~phyre2/html http://swissmodel.expasy.org/qmean/cgi/index.cgi http://swissmodel.expasy.org/qmean/cgi/index.cgi http://mordred.bioc.cam.ac.uk/~rapper/rampage.php http://mordred.bioc.cam.ac.uk/~rapper/rampage.php dehghani et al. 4 figure 1. the alignment of all used sequences figure 2. phylogenic tree for all sequences by neighbor-joining method with 100 bootstrap score dehghani et al. 5 table 4. percentages of the secondary structures for all sequence sequences percentages of alpha helix, extended strand, beta turn, random coil typhimurium 50.34, 4.76, 2.72, 42.18 paratyphi b 51.02, 4.76, 2.72, 41.50 typhi 51.02, 4.76, 2.72, 41.50 paratyphi a 51.02, 4.76, 2.72, 41.50 paratyphi c 51.02, 4.76, 2.72, 41.50 enteritidis 51.02, 4.76, 2.72, 41.50 table 1. physico-chemical properties of the selected sequences. sequences theoretical pi halflife (hours) instability index instability classifies aliphatic index hydropathicity typhimurium 7.65 >10 59.76 unstable 78.37 -0.490 paratyphi b 7.63 >10 57.64 unstable 78.37 -0.493 typhi 7.63 >10 57.64 unstable 78.37 -0.493 paratyphi a 7.63 >10 57.64 unstable 78.37 -0.493 paratyphi c 6.72 >10 60.26 unstable 77.69 -0.507 enteritidis 7.63 >10 60.63 unstable 77.69 -0.510 table 2. bcepred analysis of all sequences sequences hydrophilic flexibility surface accessibility βturns antigenic exposed surface polarity typhimurium 33-35, 68-75, 89-92, 109-112 22-23, 60-62, 70-72, 106-109, 125-127, 29-30, 33-39, 58-65, 68-69, 72-75, 87-94, 102-115, 127128 65-71 5-13, 21-28, 50-55, 121-125, 134-135 1-2, 87-92, 106-112 86-92, 107115, 140-144 paratyphi b 33-35, 68-75, 89-92, 109-113 22-23, 60-62, 70-72, 106-109, 125-127, 29-30, 33-39, 58-65, 67-70, 72-75, 87-94, 102-115, 127128 66-71 5-13, 21-28, 50-55, 121-125, 134-135 1-2, 87-92, 106-112 86-92, 107115, 140-144 typhi 33-35, 68-75, 89-92, 109-114 22-23, 60-62, 70-72, 106-109, 125-127, 29-30, 33-39, 58-65, 67-70, 72-75, 87-94, 102-115, 127129 66-71 5-13, 21-28, 50-55, 121-125, 134-135 1-2, 87-92, 106-112 86-92, 107115, 140-144 paratyphi a 33-35, 68-75, 89-92, 109-115 22-23, 60-62, 70-72, 106-109, 125-127, 29-30, 33-39, 58-65, 67-70, 72-75, 87-94, 102-115, 127130 66-71 5-13, 21-28, 50-55, 121-125, 134-135 1-2, 87-92, 106-112 86-92, 107115, 140-144 paratyphi c 33-35, 68-75, 89-92, 109-116 22-23, 60-62, 70-72, 106-109, 125-127, 29-30, 33-35, 58-65, 67-70, 72-75, 87-94, 102-115, 127131 66-71 5-13, 21-28, 50-55, 121-125, 134-136 1-2, 87-92, 106-112 86-92, 107115, 140-144 enteritidis 33-35, 68-75, 89-92, 109-117 22-23, 60-62, 70-72, 106-109, 125-127, 29-30, 33-39, 58-65, 67-70, 72-75, 87-94, 102-115, 127130 66-71 5-13, 21-28, 50-55, 121-125, 134-135 1-2, 87-92, 106-112 86-92, 107115, 140-144 table 3. hla-e*01:01, hla-b*27:05 t-cell epitope regions in selected sequences hla start length sequences hla-e*01:01 35 8 qkeqlana hla-e*01:01 66 8 hpqymrsk hla-e*01:01 102 9 pvflligca hla-e*01:01 9 10 nansidecqs hla-e*01:01 41 10 ekykqtkeqa hla-e*01:01 86 10 hpqymrsked hla-e*01:01 102 11 hpqymrskede hla-e*01:01 102 12 qpgaqkeqlana hla-e*01:01 31 12 hpqymrskedee hla-e*01:01 102 13 nkslsnqnadnsa hla-e*01:01 61 13 hpqymrskedeeq hla-e*01:01 66 14 decqslpyvpsdla hla-e*01:01 102 8 qkeqlana hla-e*01:01 46 8 hpqymrsk hla-e*01:01 55 9 pvflligca hla-b*27:05 106 10 m r s k e d e e q l hla-b*27:05 2 10 k k f y s c l p v f hla-b*27:05 129 10 k n l s i y q t l l hla-b*27:05 12 10 l l i g c a q v p l hla-b*27:05 27 10 k p v q q p g a q k hla-b*27:05 89 10 k q t k e q a l t f hla-b*27:05 3 9 k f y s c l p v f hla-b*27:05 19 9 v p l p s s v s k hla-b*27:05 121 9 k v l l e p g s k dehghani et al. 6 three residual changes were determined between s. typhimurium and s. choleraesuis [6]. in this study, we found 4 residual changes and the phylogenic tree showed high similarity between 6 sequences because of the similarity in invh sequences. s. typhimurium, s. paratyphi a, s. paratyphi b, s. paratyphi c, s. typhi, and s. enteritidis are important in foodborne diseases. in our previous studies, the presentation of invh on bacterial surface and antibody interaction with this protein was validated. theoretical pi for all sequences was 7.63 except for s. paratyphi c, which was 6.72. this could be attributed to a change in acidic amino acid to a basic one (lysine to glutamine 36). instability index provided estimated protein stability of 77.7 to 78.3 in a test tube that showed this protein is an unstable protein (instability index of <40 is regarded). the aliphatic index was 78 for all sequences which is a positive factor for the thermostability of globular proteins and depends on aliphatic side chains (alanine, valine, isoleucine, and leucine). the aliphatic index of a protein is defined as the relative volume occupied by aliphatic side chains (alanine, valine, isoleucine, and leucine) and it may be regarded as a positive factor for the increase of the thermostability of globular proteins. the aliphatic index of a protein is calculated according to the following formula: aliphatic index = x(ala) + a * x(val) + b * ( x(ile) + x(leu) ) where x(ala), x(val), x(ile), and x(leu) are mole percent (100 x mole fraction) of alanine, valine, isoleucine, and leucine. the coefficients a and b are the relative volume of valine side chain (a = 2.9) and of leu/ile side chains (b = 3.9) to the side chain of alanine. hydropathicity index was around -0.6 for all sequences shows hydrophilicity of the protein. hydrophilicity of protein parts is a positive patent for immune proteins because these parts are almost exposed and have the potential of interaction with immunoglobulins. signal peptide cleavage sites and signal peptide region prediction by three programs shows three (19, 26, and 15) different sites. "predisi" results were more reliable and similar to experimental results. the mutation in nucleotide 106 (a to c) brought about amino acid change (k-q 36) in s. paratyphi c, nucleotide 199 (g to t) brought about amino acid change (q-h 67) in s. typhimurium. the mutation in nucleotide 415 (g to t) changed the amino acid 139 from a-s in s. paratyphi c, and that in nucleotide 436 (g to t) changed the amino acid 67 (q-h) in s. enteritidis. b-cell epitopes prediction by "immuneepitope" and six other methods showed some potent similar regions. a combination of the results of all methods showed four potent b-cell regions (65figure 3. secondary structures of all sequences predicted and validated by "sompa". blue: helix, red: strand, purple: coil and green: beta turn. a: typhimurium, b: paratyphi b, c: typhi, d: paratyphi a, e: paratyphi c, f: enteritidis dehghani et al. 7 76, 87-95, 106-114, and 124-132). b-cell epitopes by bcepred showed similar regions in all sequences. abcpred regions (29-45, 46-62, 89-105, and 97-113) were similar in all sequences. immuneepitope, bcepred, and abcpred showed the most potent b-cell epitope of 86-115 for invh protein. region 86-115 contains the best potential in terms of hydrophilicity, flexibility, surface accessibility, surface exposed position, polarity, and linear b-cell epitopes prediction. this region contains almost alpha helix and random coil structures. immunization of mice by recombinant invh showed a significant rise of igg and humoral response and protective immunization against s. typhi and s. enteritidis [7, 8] confirming our immune-informatics analysis. "vaxijen" showed invh protein as probable non-antigen. algpred determined invh as non-allergen. hla-e binds nonamer peptides derived from bacterial proteins and triggers cd8-mediated lysis and ifnproduction when exposed to infected targets, raising the possibility that this novel effector mechanism might contribute to host defense against intracellular bacterial infections. salmonella omp peptides binding to hlab*27:05 were identified to modulate the host-microbe interaction and hla-b27 confers a very strong genetic predisposition to the development of reactive arthritis after infection by bacteria such as s. typhimurium [36]. our results showed 9 hla-b*27:05 and 15 hla-e t-cell epitopes regions. we, however, found two 9 meric t-cell epitopes (102-111 and 55-64) that indicated a good potential of invh to stimulate cell-mediated immune system. disulfide bonds are important in determining the functional linkages and stability of proteins. ss bonds were analyzed using two reliable programs. four cysteine amino acids making two disulfide bonds in invh contributed to protein stability. amino acid 16, 48 were located on coil structure, 7 on strand structure, and 58 was on helix structure and any of them were not located on selected b-cell epitope region (86-115). secondary structure in invh protein contained almost alpha helix and random coil. results of all the sequences were similar except for s. typhimurium in which region 60-80 helix was changed to random coil because of the change in amino acid (q-h 67). we, however, could not achieve a proper 3d model for invh, in spite of using three popular and reliable programs. the limited number of sequences and the number of bioinformatics software employed are the main limitation of the present study. in addition, other salmonella strains can be added for further studies. to sum up, it can be concluded that the findings of the present study provided valuable data about invh protein which can be used to design a novel vaccine against all salmonella strains as well as all physicochemical features of this protein were defined which can be useful to express this protein in available hosts. acknowledgements the authors wish to thank farahnaz dehghani for her kind support. author contributions conception or design of the work: bd and ir; 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metformin, retrospective studies, meta-analyses, pertinent reviews, pre-print articles and consensus guidelines are reviewed. retrospective studies suggest that metformin use prior to hospital admission may be associated with reduction in mortality among patients with diabetes admitted to the hospital with covid-19. meanwhile, continuing metformin administration after hospital admission did not have significant impact on 28-day all-cause mortality. metformin use after hospitalization of patients with covid-19 was associated with approximately 4.6 times increase risk of lactic acidosis in patients with severe symptoms of covid-19, patients taking 2 gm/d of metformin or higher, and patients with estimated glomerular filtration rate (egfr) less than 60 ml/min/1.73 kg/m2. metformin intake in hospital was associated with significant decrease risk of heart failure and acute respiratory distress syndrome (ards). in patients with diabetes and covid-19 admitted to the hospital, metformin should not be used in presence of severe symptoms of covid-19, kidney dysfunction (egfr < 60 ml/min/1.73 m2), and with daily doses of 2 gm or more due to increased risk of lactic acidosis. keywords: covid-19, diabetes, metformin, safety, mortality, lactic acidosis 1. introduction the prevalence of diabetes in coronavirus disease 2019 (covid-19) patients ranges from 5.3% to 58% representing the second comorbidity after hypertension [1,2]. available data suggest that diabetes confers a poor prognosis in covid-19 patients admitted to the hospital. in a meta-analysis of 30 retrospective studies (n=6,452), huang et al. [3] showed that diabetes was associated with excess mortality: risk ratio (rr) 2.12 (95% ci 1.44-3.03, p<0.001, severe degree of covid-19: rr 2.45, 95% * corresponding author: dr. nasser mikhail, md endocrinology division, department of medicine, olive-view-ucla medical center, david-geffen school of medicine, ca, usa tel/fax: +1 747 210 3205 /+1 747 210 4573 email: nmikhail@dhs.lacounty.gov http://orcid.org/0000-0002-7269-9338 received: september, 18, 2020 accepted: september, 25, 2020 ci, 1.79-3.35, p < 0.001, ards: rr 4.64, 95% ci, 1.8611.58, p < 0.001, and disease progression: rr 3.31, 95% ci, 1.08-10.14, p= 0.04. hence, adequate glycemic control is necessary after patient admission. metformin is the most common anti-diabetic drug worldwide due to its well-established long-term overall high efficacy and safety profile and low cost [4]. it is still unclear whether to continue or stop metformin after admission of patients with covid-19. the american diabetes association (ada) generally recommends stopping all oral anti-diabetic agents in https://jcbior.com/ mikhail et al. 2 most patients after hospital admission admitted to the hospital due to due to lack of data, presence of adverse effects, and limited efficacy [5]. the ada recommends insulin as the standard therapy for hyperglycemia in hospital [5]. however, one possible exception is metformin due to emerging data showing several clinical benefits in patients with covid-19. this review discusses the safety profile of metformin in covid-19 in the hospital setting, with special emphasis on its impact on mortality. based on available data, the authors attempt to determine whether metformin should be continued or not in patients with covid-19 and diabetes after admission to the hospital. 2. effect of metformin on mortality in hospitalized patients with covid-19 2.1 effect of pre-admission use of metformin on mortality the most comprehensive data in this respect may be derived from the recent meta-analysis conducted by kow and hassan [6]. in this study, the authors analyzed data (up to august 8, 2020) of 5 studies including 8,121 patients with diabetes and covid-19 who were using metformin prior to hospital admission. their pooled analysis revealed a significantly reduced risk for mortality with the use of metformin prior to admission, pooled odds ratio (or) being 0.62 (95% ci, 0.43-0.89) compared to patients with diabetes who were non-users of metformin [6]. interestingly, the largest study included in this metaanalysis by bramante et al. [7] that contributed to 48.7% of the meta-analysis weight showed that preadmission metformin use was associated with decreased in-hospital mortality in women only (or 0.79 (95% ci 0.64-0.98), but not in men. another retrospective study from the university of alabama included in the previous meta-analysis [6] provided information on mortality in a diverse population of 604 patients (55% women, 51% african-american) with covid-19 and diabetes admitted to the hospital [8]. in the latter study, crouse et al. [8] reported that metformin treatment was independently associated with a significant reduction in mortality; or =0.33 (95% ci, 0.13-0.84; p = 0.02). the absolute reduction in mortality was substantial [8]. thus, whereas the mortality rate in patients with covid-19 and diabetes who were not taking metformin was 23%, this rate dropped to 11% in patients who were using metformin [8]. contrary to the study of bramante et al. [7] who found that metformin mortality reduction was limited to women, crouse et al. [8] did not observe a significant differential effect of metformin on mortality based on gender. it should be emphasized that while the results of the previous studies are encouraging, they should be considered preliminary due to the following limitations. first, all included studies were retrospective prone for confounding factors. second, it was not possible to know to what extent patients were adherent to metformin intake prior to hospital admission. third, the duration and dosage of metformin were not known. fourth, it was not clear in any of these studies whether patients continued to take metformin or discontinued it after admission to the hospital. 3. effect of continuing metformin intake during hospitalization to the best of authors’ knowledge, there is only one study that evaluated the effects of continuing metformin after admission on mortality in patients with diabetes and covid-19 [9]. thus, in this large (n=1,213) retrospective study from china, cheng et al. [9] have shown that metformin administration during hospitalization was not associated with increase in 28day all-cause mortality compared with metformin non-users; adjusted hazard ratio (hr) 0.87, 95% ci 0.36-2.12); p =0.75. these data, although based on a retrospective study, may provide reassuring information about metformin safety in the hospital setting among patients with covid-19. 4. adverse effects of metformin use during hospitalization of patients with covid-19 4.1 effect of metformin on lactic acidosis lactic acidosis is a rare, but potentially lethal adverse effect of metformin [10]. the large study of cheng et al. [9] allowed the evaluation of metformin safety in different subgroups of patients. thus, they found that lactic acidosis was increased in patients with covid-19 compared with metformin non-users, adjusted hr 4.66, 95% 1.45-14.99; p=0.01 [9]. however, this increased risk of lactic acidosis was limited to the following subgroups of patients: those with severe covid-19, patients using metformin in doses of 2 gm/d or higher, and in presence of kidney mikhail et al. 3 dysfunction defined as egfr <60 ml/min/1.73 kg/m2 [9]. 4.2 effect of metformin on heart failure and acute respiratory distress syndrome in the study of cheng et al. [9], metformin inhospital use was shown to be associated with decreased risk of heart failure adjusted hr 0.61 (95% ci 0.43-0.87; p=0.006), and ards, adjusted hr 0.66, 95% ci 0.46-0.96; p=0.03). there was no significant effect of metformin on acute kidney injury or disseminated intravascular coagulation [9]. 5. potential mechanisms underlying metformin clinical benefits metformin has been shown to improve the immune response and reduce inflammation [11]. indeed, cheng et al. [9] found that serum levels of proinflammatory cytokines known to mediate cytokine storm in covid-19 were increased to a lesser extent among metformin-users versus metformin non-users. such pro-inflammatory cytokines included: interleukin-6, interleukin-2, and tumor necrosis-alpha (tnf-α) [9]. likewise, chen et al. [12] found that levels of il-6 were lower in metformin users than non-users. moreover, cheng et al [9] recorded lower neutrophil count among metformin users compared with nonusers. however, it should be emphasized that metformin should not be used in presence of hypoxia, decreased tissue perfusion, sepsis, acute or chronic kidney disease, and acute heart and liver failure to avoid the risk of lactic acidosis [10]. 6. conclusions and future needs preliminary data suggest that metformin use prior to hospitalization of patients with diabetes and covid-19 might reduce mortality. meanwhile, continuing metformin after hospitalization did not affect mortality, but increased risk of lactic acidosis. the risk of lactic acidosis was evident only in patients with severe symptoms of covid-19, those with kidney dysfunction, and in patients taking 2 gm of metformin or more daily. accordingly, metformin should be discontinued in cases of severe covid-19 and kidney disease, and its daily doses should not exceed 2 gm. on the other hand, metformin in lower doses may be continued in mild cases, and in presence of normal kidney function in view of its possible benefits in reducing risk of heart failure and ards. table 1 summarizes the conditions in which metformin may be continued after hospital admission in patients with covid-19 and diabetes based on available data. unfortunately, all current data related to covid-19 and metformin are based on retrospective studies prone for multiple bias and confounding factors. randomized trials are urgently needed to determine safety and efficacy of metformin in hospitalized covid-19 patients. author contributions all authors contributed equally to this manuscript and approved the final version of manuscripts. conflict of interests the authors do not have any conflict of interest to disclose. ethical declarations not applicable. financial support none. t a b le 1 . c o n d it io n s in w h ic h m e tf o rm in m a y b e c o n ti n u e d a ft e r a d m is si o n o f p a ti e n ts w it h c o v id -1 9 a n d t y p e 2 d ia b e te s c o v id -1 9 o f m il d s e v e ri ty a b se n c e o f k id n e y d y sf u n c ti o n d e fi n e d a s e g f r⃰ < 6 0 m l/ m in /m 2 a b se n c e o f a n y t y p e o f a c id o si s, i n c lu d in g d ia b e ti c k e to a c id o si s a b se n c e o f h y p o x ia a b se n c e o f h e m o d y n a m ic s h o c k o r d e c re a se d t is su e p e rf u si o n a b se n c e o f c o n c o m it a n t in fe c ti o n m e tf o rm in d o se s sh o u ld b e l e ss t h a n 2 g m /d a y * e g f r = e st im a te d g lo m e ru la r fi lt ra ti o n r a te mikhail et al. 4 references 1. singh ak, gupta r, ghosh a, misra a. diabetes in covid-19: prevalence, pathophysiology, prognosis and practical considerations. diabetes metab syndr. 2020; 14(4):303-10. 2. guan w-j, ni z-y, hu y, liang w-h, ou c-q, he j-x, et al. clinical characteristics of coronavirus disease 2019 in china. new england journal of medicine. 2020; 382(18):1708-20. 3. huang i, lim ma, pranata r. diabetes mellitus is associated with increased mortality and severity of disease in covid-19 pneumonia a systematic review, meta-analysis, and metaregression. diabetes metab syndr. 2020; 14(4):395-403. 4. american diabetes association. pharmacologic approaches to glycemic treatment. standards of medical care in diabetes-2020; 43: s98-s110. 5. american diabetes association. diabetes care in the hospital: standards of medical care in diabetes-2020; 43: s193-s201. 6. kow cs, hasan ss. mortality of covid-19 with preadmission metformin use in patients with diabetes: a meta-analysis. j med virol. 2020. [in press]. doi: 10.1002/jmv.26498. 7. bramante c, ingraham n, murray t, marmor s, hoversten s, gronski j, et al. observational study of metformin and risk of mortality in patients hospitalized with covid-19. medrxiv. 2020. [preprint]. doi: 10.1101/2020.06.19.20135095. 8. crouse a, grimes t, li p, might m, ovalle f, shalev a. metformin use is associated with reduced mortality in a diverse population with covid-19 and diabetes. medrxiv. 2020. [preprint]. doi: 10.1101/2020.07.29.20164020. 9. cheng x, liu y-m, li h, zhang x, lei f, qin j-j, et al. metformin is associated with higher incidence of acidosis, but not mortality, in individuals with covid-19 and pre-existing type 2 diabetes. cell metabolism. 2020. 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https://pubmed.ncbi.nlm.nih.gov/27418629/ https://pubmed.ncbi.nlm.nih.gov/32409498/ https://pubmed.ncbi.nlm.nih.gov/32409498/ https://pubmed.ncbi.nlm.nih.gov/32409498/ https://pubmed.ncbi.nlm.nih.gov/32409498/ journal of current biomedical reports jcbior.com volume 3, number 1, 2022 eissn: 2717-1906 1 review the application of human wharton's jelly mesenchymal stem cells in wound healing: a narrative review reza rezaee1, javad verdi2, mahsa sadeghi3, mehran soleymanha4, mojtaba mirzaei5, mohammad reza mobayen3, arad kianoush3,* 1department of healthcare management, school of management and social sciences, north tehran branch, islamic azad university, tehran, iran 2department of applied cell sciences, school of advanced technologies in medicine, tehran university of medical sciences, tehran, iran 3burn and regenerative medicine research center, guilan university of medical sciences, rasht, iran 4orthopedic research center, poursina hospital, guilan university of medical sciences, rasht, iran 5yale new haven medical center, waterbury hospital, waterbury, connecticut, united states abstract management and treatment of chronic wounds remain a significant problem in clinical practice. stem cell therapies are an important and promising approach for regenerative medicine because of their selfrenewal and differentiation potential. mesenchymal stem cells (mscs), a major cellular source for regeneration, are present in almost all tissues. the use of embryonic stem cells is morally controversial because of the need to nurture and destroy embryonic cells. therefore, adult umbilical cord tissues are of particular importance as an alternative source of perinatal tissues. wharton jelly is a gelatinous connective tissue in the umbilical cord containing mscs that can differentiate into osteogenic, adipose, chondrogenic, and other lineages. these cells do not express the mhc-ii molecule and show immunomodulatory properties that make them viable for allogeneic and xenogenic transplants in cell therapy. therefore, the umbilical cord, especially the part named wharton's jelly, is an important and promising source of mesenchymal stem cells. keywords: stem cells, wharton's jelly, cell therapy, wound healing, regenerative medicine 1. introduction wounds are physical injuries caused by breakage of skin [1, 2]. immediate and appropriate wound healing is essential for the re-establishment of functional tissues and the maintenance of structure following injury [3]. this complex and dynamic phenomenon involves cell-matrix interactions that heal wounds in three different overlapping phases, including the inflammatory, the proliferation, and the regenerative phase [4]. the naturally slow healing of wounds, rising costs, and inconsistency in healing are *corresponding author: arad kianoush, md 3rd floor of velayat hospital, burn and regenerative medicine research center, namjoo street, rasht, guilan, iran tel/fax: +98 911 9295638/+98 13 33368540 email: arad.kianoush@gmail.com http://orcid.org/0000-0001-7184-054x received: july, 26, 2021 accepted: november, 20, 2021 the most critical problems of this treatment. these problems have led to the discovery of more advanced therapies such as tissue engineering, gene therapy, platelet-rich plasma (prp), the use of growth factors (gf) and stem cells (sc) [5]. numerous studies have been performed using stem cells in different fields of diseases with promising results [6]. cell therapy involves the replacement of stem cells or tissue made from stem cells for various disorders and injuries [7]. stem cells are undifferentiated multifunctional cells that can differentiate into various types of cells. these © the author(s) 2022 https://jcbior.com/ rezaee et al. 2 cells are of embryonic or adult origin, depending on the type of tissue they are derived from [8]. mesenchymal stem cells are a group of pluripotent, fibroblast-shaped mature stem cells that have the ability to self-renew, modulate the immune system, and differentiate into several cell lines [9, 10]. although adult bone marrow is the most common source of mesenchymal stem cell extraction for clinical use, these cells can now be obtained from various tissues, including skin, adipose tissue, peripheral blood, umbilical cord (blood/wharton jelly), endometrium, and tooth pulp [10-14]. in conditions of tissue damage, proinflammatory factors are usually produced by both innate and compatible immune responses. studies have shown that in pathological conditions such as tissue damage, mesenchymal stem cells are inherently mobilized to the site of injury and are activated in an inflammatory environment in close interaction with the immune system [15]. these cells then facilitate wound healing by secreting specific cytokines and growth factors, increasing angiogenesis, inhibiting inflammation, increasing fibroblast migration, and collagen production [16]. in fact, the interactions between mesenchymal stem cells (mscs) and inflammatory cells determine the outcome of tissue repair processes [15]. today, mesenchymal stem cells isolated from extra-embryonic tissues such as umbilical cord wharton's jelly have been considered as a suitable cellular source [14, 17]. the reason for using these cells is easy and unlimited access, low cost, non-invasive in tissue isolation, the ability to differentiate into different cells, non-tumorigenic and abundant sources of these cells [10, 18]. wharton's jelly is a mucous connective tissue surrounding umbilical arteries and veins covered by an amniotic epithelium. umbilical cords are considered hospital waste, so their clinical application in research and cell therapy has no ethical concerns [19, 20]. since the amount of wharton jelly cells is limited and the amount of extracellular matrix (ecm) compounds is very high, it seems that its cells produce high amounts of collagen and glycosaminoglycans under the induction of existing growth factors [21]. previous studies show that wj-mscs can be used for various diseases such as cancer, neurological disorders, kidney failure, and liver, lung, and orthopedic injuries. recent advances show that wj-mscs reinforced with microparticles and scaffolds can be used more effectively for various clinical applications [22-28]. the advantage of wj-derived mesenchymal stem cells over bone marrow-derived mesenchymal stem cells (bm-mscs) and adipose tissue is that they do not express mhc-ii. moreover, these cells have stronger immunomodulatory properties due to the release of large amounts of anti-inflammatory molecules such as tgfβ, il-10, ido, tsg-6 and pge2 [29-31]. although, to date, there have been no reports of the use of human wj-mscs in human skin lesions, paracrine effects of wj-msc appear to improve wound healing, at least in mice [32]. 2. wound healing as one of the largest organs of the human body, the skin has several vital roles, including a protective barrier against fluid loss, electrolyte imbalance, and microbial infections [33, 34]. skin lesions are defined as any mechanical, thermal, or chemical damage to the skin that interferes with its function or fails to maintain its integrity [35, 36]. wound healing is a complex, multi-step process that is often divided into three stages: 1. the inflammatory phase: in this phase, following the tissue injury, the process of hemostasis is initiated, and the resulting fibrin clots provide an extracellular matrix for the migration of the white blood cells (neutrophils and macrophages) and platelets. these elements play a pivotal role in wound healing. although platelets affect wound healing by secreting various growth factors such as plateletderived growth factor (pdgf), they are not essential for wound healing without bleeding. the next steps are the result of neutrophils' function against infections, foreign bodies, and pus. then, the transformation of monocytes into macrophages indicates the end of this phase and the beginning of the proliferation stage [34, 37]. 2. the proliferative phase: this phase is divided into several stages, including neoangiogenesis, fibroblast migration, epithelialization, granulation tissue formation and, contraction [34, 37, 38]. in short, at this stage, the number of macrophages decreases, and granulation tissue begins to form. migration of other cells, such as fibroblasts and keratinocytes, also begins. these cells secrete various growth factors and help the growth of granulation tissue. as more granulation tissue grows, more collagen is synthesized as a scaffold. the combination of these interactions rezaee et al. 3 causes the wound margin to close and eventually the wound to close. 3. extracellular matrix regeneration stage: in this stage, which is the longest stage, collagen is regenerated. all these stages overlap to some extent [18, 33, 34, 38, 39]. figure 1 shows phases of wound healing. limits vary within faded intervals, mainly by wound size and healing conditions, but the image does not include major impairments that cause chronic wounds [40]. wounds can be classified as acute wounds or chronic wounds based on the healing time. a wound is described as acute when the three phases mentioned above progress as is expected and the healing process reaches a healthy conclusion within four weeks, whereas a chronic wound is one in which the healing process is not complete. it has been claimed that recovery has stopped at one of these stages, and all evidence suggests that recovery has not been completed within four weeks [37]. many factors can trigger this delay in healing and increase the risk of turning an acute wound into a chronic wound. these factors can be divided into systemic and local reasons and include the items listed in table 1 [34, 41-44]. there are also factors that can facilitate healing such as a more profuse blood flow[45] [45], vitamin c [46], and more [34, 43, 47]. unfortunately, chronic wounds remain a challenge because there is no sign of complete healing of these wounds [38]. current treatments include both modern and traditional dressings [42, 48]. one interesting approach has been the use of hydrogel dressings and the conditioned media (stem cell secretome) of stem cells which showed promising results in both reducing scar figure 1. approximate times of the different phases of wound healing on a logarithmic scale, with faded intervals marking substantial variation, depending mainly on wound size and healing conditions, but the image does not include major impairments that cause chronic wounds. table 1. risk factors of delay in wound healing systemic local miscellaneous advanced age microbial infections excess inflammatory mediators obesity exudates some medications (eg. chemotherapeutic drugs, corticosteroids, nsaids) renal diseases ischemia and necrosis inappropriate keratinocytes proliferation malnutrition/poor nutrition hypoxia trauma diabetes mellitus vasculitis hypothermia rezaee et al. 4 formation, and the healing rate in studies [41, 49, 50]. some strategies to deal with chronic wounds challenge are antibiotics to prevent and treat infections, growth factors such as epidermal growth factors (egf) or pdgf, or fibroblast growth factors. these factors affect the proliferative phase of wound healing [41, 51, 52]. also, there are reports of immune-modulating molecules such as il-4 in wound healing [41, 52, 53]. despite all of these, stem cells offer a very bright horizon for future research and therapeutic applications [5, 6, 41, 51, 54-57]. some studies have claimed that mscs secrete vascular endothelial growth factor (vegf), which can help regenerate the skin. these findings can potentially change the management of chronic wounds and our view of wound management in general [41, 58]. stem cells are notable due to their vast potential in solving problems of current medicine, including wound healing [45, 5962], diseases of the nervous system [63-67], diabetes mellitus [68, 69], crohn's disease [70], chronic myeloid leukemia [71, 72], heart failure [73, 74], liver cirrhosis [75, 76] and others. 3. stem cells and their classification stem cells are undifferentiated cells with differing degrees of potential for differentiation, varying from unipotent (capable of differentiating into a single lineage) to totipotent (capable of differentiating into all cell types). these cells can "self-renew", which is defined as the generation of daughter cells utterly identical to the original cell. this ability can help preserve the limited pool of stem cells available at birth [77-83]. stem cells are classified into two main categories based on the source of origin and potential for differentiation. 3.1 classification of stem cells based on their origin embryonic: these cells are derived from the fetus’s internal cell mass and can form all three layers of the embryo [77]. adult: these stem cells are derived from different adult cells with a differentiation capacity of at least one lineage. recent studies show that some of them have can generate up to three lineages [81, 82, 84, 85]. 3.2 classification of stem cells based on the potential to differentiate unipotent: these are stem cells with the potential of differentiating into a single cell type that can produce a lineage, such as muscle stem cells that differentiate into adult muscle cells [77, 79, 86-89]. oligopotent: these stem cells can generate two or more lineages but are limited to a specific tissue. a good example is the hematopoietic stem cells because these cells can differentiate into both myeloid and lymphoid lineages [90-92]. multipotent: this type of stem cell is found in most tissues and can differentiate into cells from a single layer. mscs are the most recognized in this class of stem cells. they can be taken from various tissues such as bone marrow, adipose tissue, wharton's jelly, umbilical cord, and peripheral blood [93-96], and differentiated into all mesodermderived tissues such as adipose, bone, cartilage, and muscle [96-99]. there have also been reports of “transdifferentiation”, the differentiation of cells of one layer (mscs) into the tissue of another layer (such as ectoderm-derived neuronal tissue) [90, 100]. pluripotent: these cells have the potential to differentiate into cells from all three germ layers, ectoderm, endoderm, and mesoderm. embryonic stem cells (escs) are examples of these stem cells that were first extracted from the inner cell mass of the blastocyst [90, 101]. totipotent: totipotent or omnipotent stem cells are the most undifferentiated cells and are only seen in early development. fertilized oocytes and the daughter cells of the 1st and 2nd generation are examples of these cells that can differentiate into embryonic and extraembryonic tissues [90, 102]. 4. stem cells in wound healing one issue to consider is that, despite all the evidence, long-term aspects of stem cell therapy and research are unknown, and its potentially harmful effects can be far more devastating than they seem. however, with more and long-term research, these concerns will be reduced [103, 104]. while the exact mechanisms of action of stem cells in wound healing have not yet been discovered, some aspects have been investigated. various studies have shown that stem cells are involved in eliminating necrosis, neoangiogenesis, vascularization, reduction of scar formation, accelerated epithelialization and wound contraction [105-107]. these effects suggest that stem cell activity is beneficial for wound healing and reducing local inflammation. most of these cases can rezaee et al. 5 be attributed to signaling, which plays a crucial role in the stem cell effect during wound healing [32, 108, 109]. the studies have shown that transplanted mesenchymal stem cells release various growth factors and cytokines, which trigger the following two processes: 1. promotes the migration and activity of fibroblasts and keratinocytes, which cause angiogenesis and healing of skin wounds and modulate the migration of leukocytes to the site of injury [110]. 2. release of immunosuppressive and antiinflammatory agents, which reduce leukocyte proliferation and inflammation. excessive inflammatory mediators are one of the predisposing factors for chronic skin wounds [47, 106]. thus, mesenchymal stem cells enhance and improve the complex process of wound healing at all stages [111]. 5. mesenchymal stem cells (mscs) mscs are adult stem cells that originate in the embryonic mesoderm layer. these stem cells are derived from a wide range of different tissues such as bone marrow, adipose tissue, nerve tissue, cord blood, and wharton jelly [18, 32, 38, 77, 112-115]. these cells can self-renew, and despite years of research, a single specific marker has not yet been identified to identify and differentiate them. the best effort to achieve a uniform definition has been made by the mesenchymal stem cell committee and the international cell therapy society, which defined mesenchymal stem cells as follows: 1. plastic adhesive cells when stored in standard culture conditions. 2. cells that should express (> 95%) cd105, cd73, and cd90 and lack the expression (<2%) of surface molecules cd45, cd34, cd14, or cd11b, cd79α or cd19, and hla-dr. 3. cells that should be differentiated into osteoblasts, adipocytes, and chondroblasts in vitro. these are only a minimal set of standard criteria set up to facilitate data exchange amongst researchers and academia [116]. mscs remain at the site of the skin wound, even after the wound has been closed. these cells play a pivotal role in almost all of the processes of inflammation, fibrosis, tissue repair, angiogenesis, wound contraction, scar development, and granulation tissue formation [117-120]. the studies have shown that mesenchymal stem cells have immunomodulatory effects such as inhibition of proliferation and reduced function in various immune cells, including natural killer (nk) cells, dendritic cells (dc), and lymphocytes [121-123]. mesenchymal stem cells reduce the secretion of inflammatory cytokines [124] and secrete various anti-inflammatory cytokines such as tgfβ, ido, pge2, nitric oxide, il-6, semaphorin-3a, and the gal-1 and gal-9 of galactins [125-131]. some studies have used the mscs conditioned mediums. these studies showed significant contributions to tissue regeneration and wound healing [5, 119, 132-135]. interestingly, all mesenchymal stem cells appear to have some similarities, regardless of the tissue from which they are isolated. these similarities include nuclear cell markers, growth factors, and cytokines. in addition, there are differences, so when designing msc-based treatments, differences such as their degree of differentiation and proliferation should be considered [136-138]. 5.1 bone marrow-derived stem cells (bmcs) these stem cells are pluripotent and include mesenchymal stromal cells, hematopoietic stem cells, and even epithelial progenitor cells [139-141]. bmcs secrete various growth factors, cytokines, and exosomes [142, 143]. many studies on different diseases such as myocardial infarctions (mi) [144, 145], chronic kidney disease (ckd) [146, 147], spinal cord injuries [140], and uveitis [148] have been undertaken. these stem cells have angiogenesisinducing effects and a positive effect on microvasculature [139]. 5.2 adipose-derived stem cells (ascs) stem cells derived from adipose tissue are yet another source of multipotent adult stem cells with numerous advantages compared to other sources of adult mesenchymal stem cells, given the massive pool of available adipose tissue and the minimally invasive extraction methods used [149]. zuk et al. were the first to introduce this new source of mesenchymal stem cells around the turn of the century [150]. some of the characteristics of these ascs that have led to the interest taken in them are their anti-apoptotic, antiinflammatory, proangiogenic, immunomodulatory, and anti-scarring effects [151]. various studies have evaluated the therapeutic effects of ascs on soft tissue regeneration, myocardial infarctions, ischemic rezaee et al. 6 injuries, immune disorders such as diabetes mellitus, systemic lupus erythematosus, and many others [152]. 5.3 olfactory-ecto mesenchymal stem cells (oemscs) these are a relatively novel population of stem cells present in the olfactory lamina propria [153]. as a population of stem cells, these cells boast a high proliferation rate, the potential to differentiate into multiple various lineages, self-renewal, as well as impressive immunomodulatory effects [154, 155]. one study found that the immunoregulation was mainly through t cell response [156]. 5.4 neural stem cells (nscs) nscs are multipotent stem cells that have been the main target of interest in neural and spinal cord diseases [157, 158]. one major hurdle in their use, despite their beneficial secretions and differentiation into neurons, astrocytes, and oligodendrocytes, is the source from which they are derived, as well as the therapeutic approach that needs to be used be taken [159, 160]. 5.5 wharton's jelly-mesenchymal stem cells wharton jelly-mesenchymal stem cells (wjmscs) are mesenchymal stem cells isolated from the umbilical cord, especially connective tissue called wharton's jelly. what truly makes wj-mscs valuable is their immune-privileged status, high differentiation potential, easy isolation and collection, and minor moral issue. these stem cells have many features in common with embryonic stem cells, both phenotypically and genetically, although there are differences. some common features are high ex vivo expansion capacity and a shorter cell cycle [161]. wharton's jelly was first described in 1656 by thomas wharton as a mucosal connective tissue that separates the umbilical vessels and amniotic epithelium. then in 1991, mcelreavey et al. first isolated the wj-msc from the umbilical cord [162, 163]. many studies have been performed using stem cells in various fields, including oncology, pulmonology, nephrology, neurology, and orthopedics [22-25, 164]. due to the immunosuppressive and modulatory effect of mesenchymal stem cells, wj-mscs are very suitable candidates for use in allogeneic transplants for cell therapy. some immunomodulatory properties of wjmscs have been demonstrated in a study by zhang et al. [165]. they observed that wj-msc grafting in burn wounds significantly showed wound healing and reduction of inflammatory markers. these results meant that the wj-msc transplant helped repair the skin by suppressing the secondary inflammatory response. there are reports of several methods of administering wj-mscs during stem cell therapy. these include local injections [165-167], topical administration [168-170], and systemic injections [171, 172]. various methods have been used in studies in which topical administration has been investigated. in one study, pourfath et al. sprayed wj-msc at the wound site [168], while in two other studies by gholipour et al. [169, 170], mesenchymal stem cells were administered through seeding in tissueengineered scaffoldings. although topical application of wj-mscs to skin wounds may seem to be the least invasive and available method of application of these cells, the merits of other prescriptions such as systemic injections are so high that they cannot be ignored. one of these competencies is that in local injection, overcell dosing at the target site of skin ulcers is better controlled. on the other hand, the wj-mscs in the scaffold can help improve paracrine signaling and cell survival by keeping them out of the harsh environment after injection [173]. problems with systemic use are primarily immune responses that do not concern the immune-privileged status of wj-mscs, and secondly, whether injected mscs are present at the site of injury and participate in the healing process [173]. 6. therapeutic effects of wj-mscs in skin wound healing many studies have already been done on the clinical use of wj-mscs in wounds treatment. these studies [109, 174, 175] have been researching the healing properties of these stem cells on the rat, ovine, and sheep animal models. due to ethical issues related to the unknown long-term effects of stem cells, most research regarding the use of wj-mscs in wound healing in animal studies. however, as of september 12, 2021, there are 50 clinical trials registered that are attempting to assess the effects of wj-mscs on a wide range of diseases, including erectile dysfunction, osteoarthritis, diabetes mellitus type 1, systemic sclerosis (scleroderma), myocardial infarctions, and no-option critical limb ischemia (no-cli). in addition to the aforementioned clinical trials listed, there are also two clinical trials, in which the use of wharton’s rezaee et al. 7 jelly and umbilical cord are more directly assessed in the field of wound healing. the first one is clinical trial neox® cord 1k vs. standard of care in nonhealing diabetic foot ulcers (conduct i) (nct02166294), which the researchers on sponsor’s demand terminated. the second one is therapeutic potential of stem cell conditioned medium on chronic ulcer wounds (nct04134676) which despite being completed, has yet to post any results. furthermore, hashemi et al. conducted a randomized clinical trial in which [176], they used an acellular amniotic membrane seeded with wj-mscs to cover the wounds. they concluded that the use of these stem cells with the scaffolding had significantly decreased the wound size, and wound healing time. 7. clinical applications of wj-derived stem cells in wounds treatment wharton jelly-derived mscs have been widely studied as an unlimited, accessible, and promising source for skin wound healing. in 2014, arno et al. showed that administration of human wj-mscs repaired skin wounds in a mouse model by increasing epithelialization, angiogenesis, as well as fibroblast proliferation and migration [32]. in a study, zhang et al. examined the effect of subcutaneous injection of wj-mscs on an animal burn model. according to the results of this study, subcutaneous injection of wj‐ mscs suppresses secondary inflammation by reducing inflammatory cytokines and thus accelerates the skin repair process in burn models of mice [27]. recently, the results of a study showed that because of the potential for epidermal differentiation and lack of hla antigen expression, wj‐mscs are more suitable sources for bioengineered human skin replacement compared to mesenchymal stem cells derived from other tissues such as bone marrow. they are fat and tooth pulp [177]. according to several studies on the mouse skin wound model, the use of wj-mscs could accelerate the formation of the epithelial layer, increase wound contraction, neovascularization and increase collagen production [178-180]. it has also been shown that the use of extracellular vesicles and medium conditioning derived from wj-mscs at the wound site enhances the proliferation and migration of fibroblasts to the site of injury, epithelialization, angiogenesis, regeneration of sebaceous glands and hair follicles [32, 181-183]. application of wj-mscs combined with biocompatible scaffolds was also associated with reduced scar formation, wound healing time, and wound size [9, 176]. 8. concerns, dilemmas, ethical issues of the use of wharton jelly stem cells in wound healing ethical issues with stem cells remain a challenge, although the use of cells such as the wj-msc can significantly reduce this. the primary concern is that stem cell therapies, besides the limited number of human studies, are ethical [184]. one major point against the use of these cells, especially embryonic stem cells, has always been that to supply enough stem cells, many embryos would have to be raised and killed. while some argue that considering the benefits of stem cell research and therapy, it can be justified in part, but public opinion is still not interested in serious action. on the other hand, in recent years, public opinion on the therapeutic uses of stem cells seems to have greatly improved. this improvement of public opinion is largely achieved through general education about the extraordinary untapped benefits and potential of stem cells in therapeutic fields [184, 185]. furthermore, a specific study focusing on burn wounds showed that people fully accept stem cell therapy, especially autologous stem cells [186]. fortunately, clover et al. also showed that the general acceptance rate of allogeneic stem cells therapy is high and does not differ significantly for other diseases such as diabetes or parkinson's [186]. another concern that has diminished is immune system rejection and tumorigenesis that have been addressed in many articles [187-189]. in general, as research expands, our understanding of stem cells and the mechanism by which they affect living cells will improve, and more evidence will be discovered that stem cell therapy is safe [103, 104]. 9. conclusion and future perspectives in summary, according to what was discussed in our study, the future of the use of stem cells, especially mscs, in wound healing is auspicious. as science and technology advance, more innovative ways to meet the current challenges of stem cell therapy are being discovered. we believe that given the unique effects of wj-mscs on immunomodulation and its moral health, human studies and clinical trials should be conducted to provide further evidence of the safety and rezaee et al. 8 efficacy of these cells as a cornerstone of the future of reconstructive medicine. acknowledgments we would like to thank the burn and regenerative medicine research center and its personnel for their assistance in the numerous steps of this research. authors’ contributions all authors contributed equally in data collection and drafting of the manuscript. also, all authors approved the final version of the manuscript. conflict of interests the authors disclose no competing/conflicting 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https://pubmed.ncbi.nlm.nih.gov/28576253/ https://pubmed.ncbi.nlm.nih.gov/28576253/ https://pubmed.ncbi.nlm.nih.gov/28576253/ journal of current biomedical reports jcbior.com volume 1, number 2, 2020 eissn: 2717-1906 1 editorial letter the sound of getting rid of coronavirus by rna interference technology: rnai against covid-19 ali ahmadi1, hamed hekmatnezhad2,* 1faculty of biological sciences and technologies, sari branch, islamic azad university, sari, iran 2department of basic sciences, sari agricultural sciences and natural resources university, sari, iran keywords: rna interference, sirna, covid-19, sars-cov-2 coronaviruses are a large family of coronaviridae and a subfamily of coronavirinae and the order nidovirales that range from the common cold virus to the cause of more serious illnesses such as severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), and severe acute respiratory syndrome coronavirus 2 (sars-cov-2). spherical or pleomorphic enveloped particles containing singlestranded rna (ssrna) are associated with a nucleoprotein within a capsid comprised of matrix protein. the envelope bears club-shaped glycoprotein projections [1]. coronavirus disease 2019 (covid-19), reported on december 31, 2019, in wuhan, china, causes the acute respiratory syndrome. to date, thousands of people around the world are infected with the virus every day, while the number of people who die by covid-19 is also significant. covid-19 disease may manifest either as an asymptomatic infection or a mild to severe pneumonia [2]. due to the epidemic of covid-19 virus in the world, there is an urgent need for new antiviral [3]. to date, there is no known definitive cure for coronavirus, and no vaccine has been developed that is approved by the world health organization (who). research to date has shown that the sars-cov-2 and sars-cov genes are up to 79% identical sequence and their receptorbinding domain structure are very similar [4]. both *corresponding author: hamed hekmatnezhad, msc department of basic sciences, sari agricultural sciences and natural resources university, sari, iran tel/fax: +98 911 9915699 email: hamedhekmatnejad@yahoo.com https://orcid.org/0000-0002-5716-9240 received: november, 4, 2020 accepted: november, 5, 2020 viruses have an animal reservoir and have been transmitted from animal to human. sars was first reported in the guangdong province of southern china in 2002 [5]. as an effort to prevent and inhibit covid-19 virus replication, rna interference (rnai) technology can be considered. this technology has been previously used to turn off the expression of the virus gene in sars-cov, hiv, hcv, and hbv [6]. rna interference is a process in which double-stranded rna causes a specific sequence in homologous genes to be silenced. this natural mechanism for silencing specific sequence genes has given biologists hope in the treatment of many diseases, including viral diseases and cancer, and may have important practical applications in agriculture, functional genomics, and therapeutic interventions. historically, rnai has been identified with terms such as repressing or silencing genes after transcription. the rnai mechanism is evolutionarily conserved and was found in a wide range of eukaryotic organisms [7]. the mechanism of rnai can be accomplished in three ways, including synthetic short-interference rnas (sirnas; 19-27 long double-stranded nucleotide rnas) as well as in situ production of short hair rnas (shrnas) or pointed through plasmid dna-based expression vectors (pdna). the process of gene deactivation by https://jcbior.com/ https://orcid.org/0000-0002-2318-4397 ahmadi et al. 2 rnai mainly mediated through two small ribonucleic acid molecules called microrna (mirna) and small interfering rna (sirna). specific inhibition of cellular mrna in mammalian cells performed by the introduction of synthetic 21to 23-nucleotide duplexes of rna. thus, rnai specifically inhibits gene expression and viral genome replication in the host cell [8,9]. the rnai mechanism inhibits gene expression in many eukaryotes, including animals. in this process, when a double-stranded rna enters the cytoplasm, it is detected by the dicer enzyme, and fragments of 21 to 25 nucleotides, known as sirnas, are produced by the enzyme [10]. then, these short two-string pieces will follow different destinies. they may be attached to the rna-induced silencing complex (risc) set and become single-stranded, with one strand separated from the whole set by one of the subunits and then attached to complementary versions and cut by the risc set. cleaves transcripts will be degraded by cytoplasmic rnases [11]. good results have been obtained in the treatment of sars virus using rnai, which can be extended to other viruses of the coronavirus family. the rnai strategy has been proven to work well in inhibiting different types of virus infections such as rotavirus [12], influenza virus [13], and hiv-1 [14-15]. in recent years, many studies and researches have been done in the treatment of sars through sirna. the results of most of these studies show that all sirna duplexes specifically reduce the expression of the sars-cov gene to varying degrees compared to the control. also, it was well demonstrated that dna vector-based sirna can effectively and specifically inhibit the expression of the spike protein gene in sars-cov cells and inhibit virus pathogenicity [6]. these results suggest that rnai, as an effective antiviral strategy, may be used to inhibit covid-19 infection, as previously demonstrated in similar viral infections such as sars, mers, etc. since the coronavirus is very similar gnomically and structurally to the sars virus, it is to be hoped that rnai technology can be used to control the virus and prevent it from becoming pathogenic in humans, thus ending this bitter nightmare. various cellular functions, including control of gene expression, chromatin status change, and gene silencing, are controlled by rnai pathways. speed, accuracy, economy, targeted transmission, and minimal toxicity are some of the factors affecting the therapeutic properties of rnai. access to methods to identify, design, and proprietary pharmaceuticals in the process of rnai, can be considered for the next generation of drugs and can also be an effective treatment for emerging viral diseases such as covid19. author contributions all authors contributed equally to this manuscript, and approved the final version of manuscripts. conflict of interests the authors declare that they have no conflicts of interest. ethical declarations not applicable. financial support none. references 1. chen y, liu q, guo d. emerging coronaviruses: genome structure, replication, and pathogenesis. j med virol. 2020; 92(4):418-23. 2. lundstrom k. coronavirus pandemic-therapy and vaccines. biomedicines. 2020; 8(5). 3. lu r, zhao x, li j, niu p, yang b, wu h, et al. genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. lancet. 2020; 395(10224):565-74. 4. zhou p, yang xl, wang xg, hu b, zhang l, zhang w, et al. a pneumonia outbreak associated with a new coronavirus of probable bat origin. nature. 2020; 579(7798):270-3. 5. zhu h, wang l, fang c, peng s, zhang l, chang g, et al. clinical analysis of 10 neonates born to mothers with 2019-ncov pneumonia. transl pediatr. 2020; 9(1):51-60. 6. zhang y, li t, fu l, yu c, li y, xu x, et al. silencing sars-cov spike protein expression in cultured cells by rna interference. febs lett. 2004; 560(1-3):141-6. 7. kurreck j. rna interference: from basic research to therapeutic applications. angew chem int ed engl. 2009; 48(8):1378-98. 8. elbashir sm, lendeckel w, tuschl t. rna interference is mediated by 21and 22-nucleotide rnas. genes dev. 2001; 15(2):188-200. 9. zamore pd, tuschl t, sharp pa, bartel dp. rnai: doublestranded rna directs the atp-dependent cleavage of mrna at 21 to 23 nucleotide intervals. cell. 2000; 101(1):25-33. 10. taylor dw, ma e, shigematsu h, cianfrocco ma, noland cl, nagayama k, et al. substrate-specific structural rearrangements of human dicer. nat struct mol biol. 2013; 20(6):662-70. 11shi y. mammalian rnai for the masses. trends genet. 2003; 19(1):9-12. https://pubmed.ncbi.nlm.nih.gov/31967327/ https://pubmed.ncbi.nlm.nih.gov/31967327/ https://pubmed.ncbi.nlm.nih.gov/31967327/ https://pubmed.ncbi.nlm.nih.gov/32375268/ https://pubmed.ncbi.nlm.nih.gov/32375268/ https://pubmed.ncbi.nlm.nih.gov/32007145/ https://pubmed.ncbi.nlm.nih.gov/32007145/ https://pubmed.ncbi.nlm.nih.gov/32007145/ https://pubmed.ncbi.nlm.nih.gov/32007145/ https://pubmed.ncbi.nlm.nih.gov/32015507/ https://pubmed.ncbi.nlm.nih.gov/32015507/ https://pubmed.ncbi.nlm.nih.gov/32015507/ https://pubmed.ncbi.nlm.nih.gov/32154135/ https://pubmed.ncbi.nlm.nih.gov/32154135/ https://pubmed.ncbi.nlm.nih.gov/32154135/ https://pubmed.ncbi.nlm.nih.gov/32154135/ 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sci u s a. 2003; 100(5):271823. 14. coburn ga, cullen br. potent and specific inhibition of human immunodeficiency virus type 1 replication by rna interference. j virol. 2002; 76(18):9225-31. 15. martínez ma, clotet b, esté ja. rna interference of hiv replication. trends immunol. 2002; 23(12):559-61. https://pubmed.ncbi.nlm.nih.gov/12446562/ https://pubmed.ncbi.nlm.nih.gov/12446562/ https://pubmed.ncbi.nlm.nih.gov/12446562/ https://pubmed.ncbi.nlm.nih.gov/12594334/ https://pubmed.ncbi.nlm.nih.gov/12594334/ https://pubmed.ncbi.nlm.nih.gov/12594334/ https://pubmed.ncbi.nlm.nih.gov/12594334/ https://pubmed.ncbi.nlm.nih.gov/12594334/ https://pubmed.ncbi.nlm.nih.gov/12186906/ https://pubmed.ncbi.nlm.nih.gov/12186906/ https://pubmed.ncbi.nlm.nih.gov/12186906/ https://pubmed.ncbi.nlm.nih.gov/12464559/ https://pubmed.ncbi.nlm.nih.gov/12464559/ journal of current biomedical reports jcbior.com volume 1, number 1, 2020 1 original research in silico prediction of b cell epitopes of the hemolysisassociated protein 1 for vaccine design against leptospirosis sakineh poorhosein fookolaee1, somayyeh talebishelimaki1, mohammad taha saadati rad2,3, mostafa akbarian rokni4,* 1 department of nursing, faculty of nursing and midwifery, islamic azad university, sari, iran 2 young researchers and elite club, sari branch, islamic azad university, sari, iran 3 department of medicine, sari branch, islamic azad university, sari, iran 4 department of nursing, faculty of nursing & midwifery, babol university of medical sciences, babol, iran abstract leptospirosis is known as a zoonotic disease of global importance originated from infection with the spirochete bacterium leptospira. although several leptospirosis vaccines have been tested, the vaccination is relatively unsuccessful in clinical application despite decades of research. therefore, this study was conducted to predict b cell epitopes of the hemolysis-associated protein 1 (hap1) for vaccine design against leptospirosis. for prediction of linear epitopes, the sequence of extracellular region of hap1 was submitted to abcpred, bcpreds, bcepred, bepipred and ellipro servers. discotope 2.0 and b-pred servers were used for prediction of conformational epitopes from the entire pdb structure of hap1 that obtained from the homology modeling method. further analysis for solvent accessible areas and relative solvent accessibility of all the residues on the pdb structures using naccess program and netsurfp server defined that predicted conformational b cell epitopes had higher solvent accessible and their residues were exposed on the surface therefore, immunoinformatics analysis showed that hemolysis-associated protein 1 can properly stimulate the b cells immune responses. keywords: leptospirosis; hemolysis-associated protein 1; b cell epitope; in silico 1. introduction leptospirosis is an infection caused by corkscrewshaped bacteria called leptospira. signs and symptoms can range from none to mild such as headaches, muscle pains, and fevers to severe with bleeding from the lungs or meningitis [1]. if it causes jaundice, kidney failure, and bleeding, it is known as will's disease, and if it causes bleeding in the lungs, it is also known as severe pulmonary hemorrhage syndrome.[2]. leptospira can be transmitted by both wild and domestic animals, and up to 10 different * corresponding author: dr. mostafa akbarian rokni, ph.d department of nursing, faculty of nursing & midwifery, babol university of medical sciences, babol, iran tel/fax: +98 935 8462251 email: akbarianmostafa1357@gmail.com https://orcid.org/0000-0002-9198-6818 received: july, 28, 2020 accepted: august, 18, 2020 genetic types of it can cause disease in humans. the most common animals that spread the disease are rodents [3]. the identification of the common immunogenic proteins of leptospira would be a major step toward the development of purer, better-defined, and probably more-efficient vaccines. such vaccines would provide cross-protection against a wide range of pathogenic leptospira strains [4] outer membrane proteins such as lipl21 and lipl32, lipl41 are expressed only in pathogenic species of leptospira [5]. recent data indicated that hemolysis-associated https://jcbior.com/ poorhosein fookolaee et al. 2 protein 1 (hap1) (figure 1) could be a good candidate for developing a new generation of vaccines able to induce broad protection against leptospirosis disease [6]. it has been suggested that this protein is only produced by pathogenic leptospires and hap1 vaccination induces significant protection compared to similar ompl1 based vaccination [6]. active immunotherapy with the peptide vaccines which are designed to be chimeric with multi-epitopes of b cells and t helper cells can induce the generation of an adaptive immune response [7]. several experimental techniques are currently available for the selection of suitable b cell epitopes. the experimental approaches applied for detecting immunogenic regions are often laborious and resource-intensive. computational techniques for predicting b cell epitopes are fast, scalable, and cost-effective, focusing on experimental experiments and a better understanding of antigenantibody interaction [8]. recent researches have shown there are limitations to the current epitope prediction methods. therefore, enhancing the reliability of computational b cell epitope prediction methods remains a major challenge in computational vaccinology [9]. nevertheless, prediction results produced by multiple computational tools could be used to gain a consensus result. basically, the recognition of either small discrete t-cell epitopes or large conformational epitopes recognized by soluble antibodies and b cells is the key molecular event for the immune response to pathogens. b cell epitopes can be classified into two types: linear (continuous) and conformational (discontinuous). while linear epitopes comprise of continuous residues in the sequence, conformational epitopes are composed of amino acids that are not neighboring in primary sequence and are brought into close proximity in the folded protein structure [10]. localization of these epitopes is of clinical interest for the development of diagnostic tools, vaccines, and cancer immunotherapies. many attempts have been made for predicting the antigenic sites from certain features of proteins' primary structures. different parameters such as static accessibility, hydrophilicity, and mobility of the short segments in polypeptide chains have been associated with the position of continuous epitopes in proteins [11]. the present research aimed to predict new b cell epitopes for the hap1. combination approaches were used by combining results from the sequence and the structure-based methods and the solvent-accessible surface area calculating tools. furthermore, we used pepop 2.0 application to predict new conformational epitopes from pdb structure of hap1. 2. materials and methods 2.1. retrieval of protein sequences all sequences of hap1 were retrieved from uniprot (www.uniprot.org) in fasta format. 2.2. linear b cell epitopes prediction for the prediction of linear epitopes, the sequence of hap1was submitted to abcpred, bcpreds, bcepred, bepipred, and ellipro servers [9, 12]. the hidden markov model, thornton’s method, support vector machine classifiers, recurrent neural network, and physicochemical properties of amino acids were applied to predict linear b cell epitopes. only, the linear peptides which were predicted frequently by 3 or more servers were selected. 2.3. conformational b cell epitope prediction discotope 2.0 and b-pred servers were used for the prediction of conformational epitopes from the entire pdb structure of hap1 that obtained from the homology modeling method. the discotope method incorporates a new spatial neighborhood description and a half-sphere exposure as a surface measure based on the protein structure and epitope propensity scores and predicts residues that can be involved in b-cell epitopes. b-pred is a web-based platform for scoring and predicting b-cell epitopes based on the structures of the potential immunological proteins. the method scores the peptides set of a protein-based on the figure 1. tertiary structure of hap 1. in this figure the protein is shown in the third structure poorhosein fookolaee et al. 3 average solvent exposure, by a filter on filtering the average local model quality for each peptide [13]. 3. results the predicted linear b cell epitopes of the hap1 are shown in table 1. the predicted b cell epitopes were ranked according to their score obtained by a trained recurrent neural network. a higher score of peptide means a higher probability to be an epitope. as listed in table 1, peptides were predicted by 5 servers used in the current study. the peptide “tgeigepgdgdlvsda” had the highest score compared to other epitopes. these peptides reside in 37-210 regions of the hap1. some studies on the vaccine are shown in the table 2. discotope and b-pred predicted 167 b-cell epitope residues out of 3420 total residues corresponding to conformational epitopes, approximately located in the region of linear b cell epitopes (table 3). further analysis for solvent accessible areas and relative solvent accessibility of all the residues on the pdb structures using the naccess program and netsurfp server defined that predicted conformational b cell epitopes had higher solvent accessible and their residues were exposed on the surface. 4. discussion although the majority of b cell epitopes appear to be conformational, most of the computational methods focused on the prediction of sequential epitopes. linear epitope prediction approaches can be classified as propensity scale methods, improved propensity scale methods and machine learning methods [23]. if the tertiary structure of an antigen is known, there are improved methods for identifying conformational b cell epitopes. examples are discotope web server and pepop. these are based on features like amino acid propensity scales and solvent accessibility [24]. in this study, linear and conformational b cell epitopes of hap1 were predicted using both primary sequence and tertiary structure. based on combination approaches and considering lowest identity with the ir and frequently prediction using several tools, the best peptides were the linear b cell epitope tgeigepgdgdlvsda (table 1) and conformational b cell epitopes (table 2). in addition, pepop also predicted 167 new conformational peptides. table 1. predicted linear b cell epitopes using various servers. rank sequence start position score 1 tgeigepgdgdlvsda 101 0.96 2 gsvinyygyvkpgqap 54 0.94 3 yrisfttykpgevkgs 210 0.89 3 ddgddtykeerhnkyn 164 0.89 4 gqapdglvdgnkkayy 66 0.88 5 edtipgtnetvktllp 37 0.85 5 rikipnppksfddlkn 183 0.85 5 fdtwirvermsaimpd 131 0.85 6 peeksmphwfdtwirv 122 0.84 7 ygyvkpgqapdglvdg 60 0.83 7 netvktllpygsviny 44 0.83 7 rhnkynsltrikipnp 174 0.83 8 asvgllfppgipgvsp 228 0.81 9 aviaemgvrmisptge 88 0.80 9 akaakakpvqkldddd 149 0.80 10 qkqaiaaeeslkkaas 253 0.78 poorhosein fookolaee et al. 4 t ab le 2 . s o m e st u d ie s o n t h e v ac ci n e ar e sh o w n i n t h e ta b le b el o w v a c c in e a n ti g e n a d ju v a n t v e c to r r e si st a n c e m a r k e r p r o m o te r l e a d e r se q u e n c e a n im a l m o d e l im m u n iz a ti o n c h a ll e n g e s e r o v a r /d o se v a c c in a te d /c o n tr o l r e f. d o se r o u te t o ta l n o . % s u rv iv a l d n a h a p 1 n a p c d n a 3 .1 n e o m y c in h u m a n c y to m e g a lo v ir u s (c m v ) – g e rb il s 2 1 0 0 m g /i m c a n /1 0 7 1 5 /2 0 6 0 /3 5 [1 4 ] d n a o m p l 1 n a p c d n a 3 .1 n e o m y c in h u m a n c y to m e g a lo v ir u s (c m v ) – h a m st e rs 3 1 0 0 m g /i m p o m /1 0 3 6 /6 3 3 /0 [1 5 ] d n a l ip l 3 2 n a p t a r g e t a m p ic il li n h u m a n c y to m e g a lo v ir u s (c m v ) – m ic e 2 1 0 0 m g /i m n a 5 /5 n a [1 6 ] d n a l ip l 2 1 n a p c d n a 3 .1 n e o m y c in h u m a n c y to m e g a lo v ir u s (c m v ) – g u in e a p ig 2 1 0 0 m g /i m l a i 5 6 6 0 1 8 /8 1 0 0 /1 0 0 [1 7 ] d n a l ip l 3 2 n a p v a x 1 k a n a m y c in h u m a n c y to m e g a lo v ir u s (c m v ) – m ic e 3 5 0 m g /i m n a 1 0 /1 0 n a [1 8 ] d n a l ip l 3 2 – 4 1 o m p l 1 n a p v a x 1 k a n a m y c in h u m a n c y to m e g a lo v ir u s (c m v ) – m ic e 3 5 0 m g /i m n a 1 0 /1 0 n a [1 8 ] d n a l e m a a lh y d ro g e l p t a r g e t n e o m y c in h u m a n c y to m e g a lo v ir u s (c m v ) – h a m st e rs 2 1 0 0 m g /i m c o p /1 0 1 8 /6 6 2 .5 /0 [1 9 ] p b l e m a a lh y d ro g e l p t a r g e t n e o m y c in h u m a n c y to m e g a lo v ir u s (c m v ) – h a m st e rs 2 1 0 0 m g /i m c o p /1 0 1 8 /6 8 7 .5 /0 [1 9 ] d n a l ip l 3 2 p o ly i :c p v it r o 1 n e o m y c in e f – 1 a v z v g e si g n a l p e p ti d e m ic e 3 2 0 m g /i m o r e p n a 6 /6 n a [2 0 ] d n a o m p l 3 7 a lh y d ro g e l p t a r g e t n e o m y c in h u m a n c y to m e g a lo v ir u s (c m v ) – h a m st e r 2 1 0 0 m g /i m c o p /1 0 3 6 /6 0 /0 [2 1 ] p b o m p l 3 7 a lh y d ro g e l p t a r g e t n e o m y c in h u m a n c y to m e g a lo v ir u s (c m v ) – h a m st e r 2 1 0 0 m g /i m c o p /1 0 3 6 /6 2 5 /8 .3 [2 1 ] d n a l ig b re p a lh y d ro g e l p t a r g e t n e o m y c in h u m a n c y to m e g a lo v ir u s (c m v ) – h a m st e r 2 1 0 0 m g /i m c o p /1 0 1 5 /5 4 0 /0 [2 2 ] p b l ig b re p a lh y d ro g e l p t a r g e t n e o m y c in h u m a n c y to m e g a lo v ir u s (c m v ) – h a m st e r 2 1 0 0 m g /i m c o p /1 0 1 6 /5 8 3 .3 /0 [2 2 ] poorhosein fookolaee et al. 5 the predicted epitopes by pepop are widely distributed within the crr (cysteine‐rich region) domain and often partly overlapped, consistent with the view that pepop predicted segmented epitopes and the crr domain displayed a mosaic of overlapping epitopes. considering less identity with the ir and high rsa (relative solvent accessibility) score conformational epitopes can be suitable for further experimental tests. in the present work, new conformational epitope and a linear b cell epitope were predicted using various bioinformatics analyses. in conclusion, findings of the present work, using the bioinformatics analyses could be used in mabs (monoclonal antibodies) production, vaccine design and the diagnostic tools. in addition, the current in silico approaches are reducing time and minimizing the total number of necessary tests to find possible and proper epitopes. in the next step, synthesis of determined peptides in vitro and in vivo experimental studies are essential for assurance of the predicted epitopes. due to the prevalence of this disease in iran, especially in the northern regions of the country, which have paddy fields and agricultural lands and many people are at risk of contracting this disease, the preparation and design of vaccines seems necessary. in this research, we used immunoinformatics methods to predict appropriate vaccine against leptospira. selection of suitable epitopes of the hap1 as antigens, and utilizing them for raising mabs against the hap1, with ability of leptospira inhibition would be beneficial in treatment of leptospirosis. to the best of our knowledge, for the first time, in this study the linear and conformational b cell epitopes of the hap1 were predicted, screened and assessed using the well-known bioinformatics comprehensive analyses. all sequences were joined to each other by proper linkers. epitopes were evaluated as nonallergenic, antigenic, soluble, with safety and efficacy. these predicted epitopes might be used to design a vaccine against leptospira, could be validated in model hosts to verify their efficacy as vaccine. author contributions all authors contributed equally to this manuscript, and approved the final version of manuscripts. conflict of interests the authors declare that they have no conflicts 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reports jcbior.com volume 3, number 4, 2022 eissn: 2717-1906 1 original research demographic and clinical characteristics of patients with interstitial lung disease admitted to razi hospital, the north of iran heydar ali balou1, somaye ramezanpour1, banafsheh ghavidel-parsa2, alireza jafarinejad1, ali alavi foumani2,* 1inflammatory lung diseases research center, department of internal medicine, school of medicine, razi hospital, guilan university of medical sciences, rasht, iran 2rheumatology research center, razi hospital, school of medicine, guilan university of medical science, rasht, iran abstract respiratory disease is the third cause of death, which accounts for one-seventh of all deaths worldwide and millions of people suffer from chronic lung disease in the world. interstitial lung diseases (ilds) are one of the most important lung diseases. so, we aimed to evaluate demographic data and clinical characteristics of ild patients in our region. this cross-sectional descriptive study was conducted on patients diagnosed with ild who were referred to razi hospital, rasht, iran during 2013-2018. out of a total of 300 participants, 160 (53.3%) patients were non-occupational exposed. the most prevalent clinical symptoms and signs were coughs, dyspnea, and abnormal findings in lung auscultation. totally, 113 (37.7%) of patients mostly consumed methotrexate. among 158 patients with reported antinuclear antibody (ana) status, 29 (18.4%) cases were positive. in 186 (71.3%) cases with available pathological findings, alveolar wall thickening, fibroblast deposition, and honeycomb pattern was reported. also, granulomatous lung tissue and diffuse fibrosis were detected in 58 (22.2%) patients. ild patients with different clinical conditions may show regional differences in risk factors, patient exposure, access to medical facilities for diagnosis and treatment, and genetic differences, which require careful attention to these factors individually in each region. keywords: demographic data, clinical characteristics, radiological findings, interstitial lung disease 1. introduction respiratory disease is the third leading cause of death, which accounts for one-seventh of all deaths worldwide and millions of people suffer from chronic lung diseases around the world [1]. interstitial lung diseases (ilds) or diffuse parenchymal lung diseases (dplds) are among the most important lung diseases with a large and heterogeneous set of more than 200 lung diseases characterized by varying degrees of inflammation and fibrosis in the parenchyma, often *corresponding author: ali alavi foumani, md inflammatory lung disease research center, razi hospital, guilan university of medical sciences, rasht, iran tel/fax: +98 13 33542460 email: fomanii99@gmail.com http://orcid.org/0000-0002-7436-6617 received: february, 08, 2021 accepted: october, 26, 2022 classified as rare lung disease [2–4]. in ild, the lung elasticity is reduced due to collagen deposition and interstitial lung fibrosis (ilf), and more pressure difference is required to cause a certain volume change in the lung than the normal state. from the functional and physiological perspective, respiratory function is impaired and its effects are evident in the pulmonary function tests (pfts). from a pathological point of view, which is examined by a pathologist by evaluating a lung biopsy specimen in the laboratory, the lung © the author(s) 2022 https://jcbior.com/ balou et al. 2 parenchymal structural change and destruction could be seen. the last viewpoint is the radiological and anatomical aspect of the disease, in which a radiologist determines the ild patterns by examining the patient's lung images [1]. according to epidemiological studies conducted in different parts of the world, idiopathic pulmonary fibrosis (ipf) and sarcoidosis are the most common forms of ild, which account for 50% of cases [5]. ilds represent a large number of conditions that affect the lung parenchyma, including alveolar epithelium, capillary endothelium, and the spaces between these structures, as well as the perivascular and lymphatic tissues. this heterogeneous group of disorders is categorized together because of their similar clinical, radiographic, physiological, or pathological manifestations. each ild may have an acute phase but typically they represent a chronic illness. some ilds rarely recur and the disease is subclinical at recurrence intervals [6]. sarcoidosis, ipf, and pulmonary fibrosis with ctds are the most prevalent ilds of unknown etiology. the ilds with known etiologies include contact with occupational and environmental factors, in particular inhalation of inorganic and organic particles, various vapors and gases, infections, drugs, and radiation [5]. factors affecting ilds include age, gender [6,7], family history [6], smoking history [2,6], occupational and environmental history [3,6], and so on. the histopathological findings include varying degrees of inflammation with accumulation and immune cells often associated with abnormal extracellular matrix in the terminal airways, alveolar walls, and interstitium [8]. dyspnea is a common indicator complaint in patients with ild [6]. in special cases, serological tests, echocardiography, bronchoscopy, and surgical lung biopsy are also helpful in the specific disease diagnosis [9]. a metaanalysis study was performed to determine specific computed tomography (ct) patterns and clinical features to differentiate between nonspecific interstitial pneumonia (nsip) and usual interstitial pneumonia (uip) and showed significantly lower levels of the honeycomb pattern with less peripheral predominance and more subpleural sparing cases. overall, the honeycomb pattern with peripheral predominance was significantly associated with uip diagnosis [10]. according to the importance of accurate diagnosis and treatment of ild, and also due to the few conducted study on this topic in guilan province, iran, we aimed to conduct this study to investigate demographic data and clinical characteristics of ild patients in razi hospital, rasht, iran. 2. materials and methods this cross-sectional descriptive study was conducted on patients diagnosed with ild who were referred to razi hospital, rasht, iran during 20132018. inclusion criteria were definitive diagnosis of ild by a specialist based on history and clinical signs, chest x-ray (cxr), lung high resolution computed tomography (hrct), and pathology. exclusion criteria were incomplete file information, including age, gender, clinical signs, and results of imaging. this study was approved by the research ethics committee of guilan university of medical sciences, rasht, iran with the number code ir.gums.rec.1398.078. the informed consent and consent to publication were taken from patients. information in files and variables, such as age, gender, heredity, level of education, occupation, and smoking history; laboratory findings including white blood cell (wbc), red blood cell (rbc), hemoglobin (hb), blood urea nitrogen (bun), creatinine (cr), aspartate transaminase (ast), alanine transaminase (alt), alkaline phosphatase (alp), and calcium (ca); serological factors such as (anti-nuclear antibody (ana), rheumatoid factor (rf), cyclic citrullinated peptide antibody (anticcp), autoantibodies against topoisomerase i (anti-scl-70), anti–sjögren'ssyndrome-related antigen a autoantibodies (antiro/ssa), anti-sjogren's syndrome b autoantibodies to ss-b/la, serum (anti-la/ssb), native doublestranded dna antibody (anti-ds dna), perinuclear anti-neutrophil cytoplasmic antibodies (p-anca), antineutrophil cytoplasmic autoantibody, cytoplasmic (c-anca), anti-smith, antinuclear ribonucleoprotein (anti-rnp), and angiotensin-converting enzyme (ace); echocardiography, comorbidities, used medications, radiographic patterns [septal thickening, ground-glass attenuation (gga), honeycomb change, centrilobular nodules, mediastinal lymphadenopathy, emphysema, and reticulation], spirometry parameters, and presence/absence of pathology reporting ild were recorded in the information form. collected data were analyzed by spss software (version 18) (ibm, armonk, ny). quantitative variables with normal distribution were described balou et al. 3 using mean ± standard deviation (95% confidence interval), and median and interquartile ranges were used for quantitative variables with non-normal distribution. qualitative variables were also described based on the number and percentage. the normal distribution of quantitative variables was verified using the kolmogorov-smirnov test. 3. results according to our results, 170 (56.7%) patients were male and the rest were female. the mean age of the patients was 56.95 ± 14.78 years with a median of 56.50 years (in the range of 15-93 years). most of the patients were married and 158 (52.7%) individuals were illiterate or without high school diplomas. all demographical data of participants are illustrated in table 1. according to the occupational exposure status of patients, smoking was the most frequent one by 160 (53.3%) patients included. based on the patients' spirometry results, obstructive and restrictive patterns were reported in 103 (34.3%) and 197 (65.7%) patients, respectively. in the studied patients, the most prevalent clinical symptoms were cough (66%), dyspnea (57.7%), and abnormal findings in lung auscultation (25.3%) (table 2). also, no cases were reported with symptoms of tachypnea and tachycardia. the hereditary background was positive in 68 cases of patients based on family history. furthermore, 110 ild patients had normal left ventricular ejection fraction (ef) on echocardiography. medication use was detected in 113 (37.7%) studied patients (table 3), with methotrexate used most frequently (86.7%). sarcoidosis, scleroderma, and churg-strauss syndrome were the most frequent underlying diseases in ild patients (table 3). the laboratory findings are illustrated in tables 4 and 5. according to the serological results, 29 (18.4%) patients were positive for ana among 158 reported cases. also, rf test was positive in 27 (14.8%) cases. no information was available on the status of antiro/ssa, anti-la/ssb, anti-smith, ace, and antirnp serological tests. the radiological findings of patients represented that septal thickening, honeycomb change, mediastinal lymphadenopathy, and gga were the most frequent in patients. according to histopathological results, alveolar wall thickening (awt), fibroblast deposition, honeycomb pattern, and interstitial lung fibrosis pattern were reported in 186 (71.3%) patients, and granulomatous lung tissue and diffuse cellular fibrosis were reported in 58 patients (22.2%) (table 6). 4. discussion due to the result of the current study, middle-aged males were predominant. in a study, it has been reported that the mean age of autoimmune-featuredild (aif-ild) patients were 66 ± 10 years at the time of referral, which was similar to the ipf group and higher than that of ctd-ild patients (54 ± 14.6 years) [11]. alavi foumani et al., reported that the mean age of evaluated sarcoidosis patients was 42.8 ± 9.8 years with the most frequency in females (63.5%) [12]. table 1. demographic characteristics of patients diagnosed with ilds variables subgroup mean ± sd age (year) 56.95 ± 14.78 number (%) sex female 130 (43.3) male 170 (56.7) marital status single 12 (4) married 288 (96) level of education illiterate 98 (32.7) without high school diplomas 60 (20) diplomas 99 (33) graduate 43 (14.3) occupational status housewife 143 (47.7) farmer 59 (19.7) self-employment 30 (10) construction worker 24 (8) staff 19 (6.3) driver 9 (3) unemployed 16 (5.3) balou et al. 4 table 2. occupational exposure status and clinical symptoms of ild patients variable subgroup number (%) non-job exposure yes 160 (53.3) no 140 (46.7) types of non-job exposure smoking 160 (53.3) secondhand smoke 46 (28.7) opium 32 (20) hookah 3 (1.9) fireplace 10 (6.3) oven old 9 (5.6) rice-crop residue burning smoke 1 (0.6) chemicals 5 (3.1) plaster dust 1 (0.6) clinical signs shortness of breath yes 173 (57.7) no 127 (43.3) cough yes 198 (66) no 102 (34) hemoptysis yes 22 (7.3 no 278 (92.7) fever yes 25 (8.3) no 275 (91.7) clubbing yes 11 (3.7) no 289 (96.3) abnormal findings in the lungs yes 76 (25.3) no 224 (74.7) joint pain yes 17 (5.7) no 283 (94.3) skin lesions yes 6 (2) no 294 (98) *since some patients were exposed to more than one type of noe, the total percentage is more than 100 table 3. characterization of medications and underlying disease in patients with a diagnosis of ild variables subgroup number (%) taking medicines yes 113 (37.7) no 187 (62.3) types of drug methotrexate 98 (86.7) cyclophosphamide 13 (11.5) amiodarone 2 (1.8) sarcoidosis yes 94 (31.3) no 206 (68.7) comorbidity scleroderma yes 48 (16) no 252 (84) churg-strauss yes 18 (6) no 282 (94) dermatomycosis yes 6 (2) no 294 (98) rheumatoid arthritis yes 5 (1.7) no 295 (98.3) lupus yes 3 (1) no 297 (99) balou et al. 5 table 3. characterization of medications and underlying disease in patients with a diagnosis of ild (continued) variables subgroup number (%) comorbidity hypersensitivity pneumonitis yes 3 (1) no 297 (99) sjögren yes 3 (1) no 297 (99) pneumoconiosis yes 3 (1) no 297 (99) undetermined lung fibrosis yes 3 (1) no 297 (99) wegener yes 2 (0.7) no 298 (99.3) table 4. the laboratory data of the studied patients variable available cases mean ±sd wbc (cumm) 297 8638/2212 ± 77.5 rbc (x106miu/l) 255 3 ± 91.55 hb (g/dl) 299 11.1 ± 40.33 platelets (u/l) 294 262955/2212 ± 78.5 bun (mg/dl) 287 19.9 ± 83.8 cr (mg/dl) 284 1 ± 11.9 ast (u/l) 103 49.2 ± 75.6 alt (u/l) 104 45.2 ± 29.2 alk-p (u/l) 83 393.121 ± 66.3 ca (mg/dl) 72 10.1 ± 53.3 abbreviations: wbc (white blood cell), rbc (red blood cell), hb (hemoglobin), bun (blood urea nitrogen), cr (creatinine), ast (aspartate transaminase), alt (alanine transaminase), alp (alkaline phosphatase), ca (calcium) table 5. characterization of serological tests of patients with ild diagnosis variables available cases number (%) ana positive 158 29 (18.4) negative 129 (81.6) rf positive 183 27 (14.8) negative 156 (85.2) anti-ccp positive 1 1 (100) negative 0 anti-scl-70 positive 28 19 (67.9) negative 9 (32.1) anti-ds dna positive 18 4 (22.2) negative 14 (77.8) c-anca positive 1 1 (100) negative 0 p-anca positive 17 17 (100) negative 0 abbreviations: ana (anti-nuclear antibody), rf (rheumatoid factor), cpc (cyclic citrullinated peptide), scl-70 (topoisomerase i), ds-dna (native double-stranded dna), c-anca (antineutrophil cytoplasmic autoantibody, cytoplasmic), p-anca (perinuclear anti-neutrophil cytoplasmic antibodies) balou et al. 6 in the united states during 2004-2010, a study on people aged 18-64 years indicated that the rise of the age and male gender were two important and influential factors in increasing the incidence of ipf, and individuals under 65 years of age were less likely to develop ilds in this country [13]. according to another study, smoking, increasing age, and male gender significantly increased the incidence of ilds, which was 6.9 times higher in patients over 70 years [14]. alavi foumani et al., observed that only 3.8% and 1.5% of sarcoidosis patients were active and inactive smokers, respectively [12]. another study on histological test results of an 81-year-old patient illustrated asbestos accumulation in the lungs due to indirect exposure to domestic asbestos dust by washing asbestos-contaminated industrial clothes of the husband who worked in a textile factory. it can be conducted that exposure to domestic asbestos dust could be considered a risk factor for fatal pulmonary fibrosis [15]. in this present study, occupational history has been identified as an important cause of ild development. gysbrechts et al. suggested that exposure to talc in people over 40 years of age is a reason for the development of pulmonary fibrosis [16]. also, kim et al. reported ild development in several construction workers who worked in the outside yard of a company producing hydrofluoric products [17]. according to our results, obstructive and restrictive spirometric patterns were the most reported ones. the most common clinical symptoms and signs were cough, dyspnea. alavi foumani et al. also found that the most common signs and symptoms in their sarcoidosis patients were related to the respiratory system, in which 63.3% of patients showed symptoms such as dyspnea and cough, and systemic symptoms (e.g. weight loss and fever) as secondary signs [12]. dhooria et al. examined the range of ilds on 803 patients in a specialty center and reported the most common symptoms to be cough (86.1%), dyspnea (76.1%), weight loss (30.9%), anorexia (24.2%), joint pain (23.9%), and fatigue (17.9%), respectively [4]. a review of 4086 studies conducted from 1996 to 2015 demonstrated that the most prevalent symptoms in fibrotic ild were dyspnea (54-98%) and cough (59100%), followed by heartburn (25-65%) and depression (49-49%) [18]. muscle pain, chills, fever, headache, cough, sputum, nausea, vomiting, bloating, arthritis, and decreased total lung capacity have been table 6. the radiological symptoms and pathological evidence of patients variable number (%) radiological indication septal thickening 128 (42.7) honeycomb change 76 (25.3) mediastinal lymphadenopathy 72 (24) ground glass attenuation 61 (20.3) emphysema 19 (6.3) reticulation 17 (5.7) centrilobular nodules 8 (2.7) pathological evidence thickening of alveolar septa + fibroblast accumulation + honeycomb pattern + interstitial lung fibrosis yes 186 (71.3) no 75 (28.7) granuloma in lung tissue + diffuse fibrosis yes 58 (22.2) no 203 (77.8) eosinophilia in lung tissue + alveolar dense fibrosis yes 10 (3.8) no 251 (96.2) rheumatoid nodule + alveolar dense fibrosis yes 1 (0.4) no 260 (99.6) diffuse histiocytic in lung tissue + diffuse fibrosis yes 6 (2.3) no 255 (97.7) lymphocytes in lung tissue yes 1 (0.4) no 260 (99.6) *due to the fact that some patients had more than one finding, the overall percentage has increased to more than 100 https://www.google.com/search?sxsrf=alekk028oh2icbw1w6p2ykoqtntowbducw:1620214366680&q=honeycomb+pattern&spell=1&sa=x&ved=2ahukewixuatuullwahvpq0eahyyabuoqkeeckab6bagbec4 https://www.google.com/search?sxsrf=alekk028oh2icbw1w6p2ykoqtntowbducw:1620214366680&q=honeycomb+pattern&spell=1&sa=x&ved=2ahukewixuatuullwahvpq0eahyyabuoqkeeckab6bagbec4 https://www.google.com/search?sxsrf=alekk028oh2icbw1w6p2ykoqtntowbducw:1620214366680&q=honeycomb+pattern&spell=1&sa=x&ved=2ahukewixuatuullwahvpq0eahyyabuoqkeeckab6bagbec4 https://www.google.com/search?sxsrf=alekk028oh2icbw1w6p2ykoqtntowbducw:1620214366680&q=honeycomb+pattern&spell=1&sa=x&ved=2ahukewixuatuullwahvpq0eahyyabuoqkeeckab6bagbec4 balou et al. 7 reported in patients with ild [19]. a study by sheikh et al. on the prevalence of pulmonary disorders in patients with scleroderma represented six (11.5%) patients without dyspnea, 22 (42.3%) without cough complaints, 20 (38.4%) with no crackle in the lungs, and 26 (50%) with no clubbing on examination, but patients represented an abnormal hrct pattern [20]. in another study, 41 out of 288 patients with proven ctd-ild initially presented pulmonary symptoms, and the remaining 247 (86%) patients developed extrapulmonary symptoms as the primary manifestation [21]. in our study, sarcoidosis, scleroderma, and churgstrauss syndrome were reported in a higher percentage of patients diagnosed with ild. in a study by zubairi et al., the most common ild was ipf in 217 (40.4%) patients, followed by nonspecific interstitial pneumonia, sarcoidosis, and connective tissue disease (ctd)-related ild in 106 (19.7%), 82 (15.3%), and 56 (10.4%) patients, respectively [22]. duchman et al. stated that sarcoidosis (42.6%), ctd-ild (16%), ipf (11.6%), and occupational ilds (5%) were the most common diagnoses in 848 cases of ild [23]. the incidence of ilds in denmark was reported to be 4.1 cases per 100,000 people per year. ipf was the most common diagnosis with an annual incidence of 1.3 cases per 100,000 patients (28%), followed by ctdild (14%) and hp (7%) [24]. the result of a study showed that out of 2678 patients who were diagnosed with ild, ctd-ild and ipf were detected in 1798 (67%) and 299 (11.2%) patients, respectively [21]. also, in another study, it was reported that out of 381 patients, 325 (85.1%) individuals were classified in the ild-ctd group (ra 31%, systemic sclerosis 29%, and dermatomyositis 15%), 36 (9.5%) patients in the group of interstitial pneumonia with autoimmune features (ipaf), and 13 (3.5%) patients in the antineutrophil cytoplasmic antibodies (anca)-positive ild group [25]. an investigation demonstrated that hp, ctdild, and ipf accounted for 47.3%, 13.9%, and 13.7%, respectively, among 1084 patients with ild [26]. saghafi et al. performed research on 100 patients with acute sarcoidosis in rheumatology clinics of mashhad during 2010-13, they found that acute sarcoidosis was more prevalent in females. the average age of patients was 40-50 years and 93% of patients represented the acute phase and 7% of patients were in the chronic phase. among 91 cases of acute arthritis, 64 were diagnosed with lofgren's syndrome and out of 93 patients with acute sarcoidosis, the onset of erythema nodosum, uveitis, and general symptoms (fatiguefevernight sweetly) were reported in 67 (72%), 10 (10.8%), and 41 (44.1%) cases [10]. in this present study, the serological results were incomplete for the majority of patients. positive ana result was reported in 18.4% out of 158 patients and positive rf test result was reported in 14.8% of cases. an investigation illustrated that american indians accounted for 27% of patients with systemic lupus erythematosus (sle) with a concomitant diagnosis of pulmonary fibrosis, or ild. african americans were also divided equally and had high anti-dsdna (52%) and 23% in controls. an increasing trend was observed in anti-la (43%), anti-sm (43%), and rnp (53%) compared to the control sample. among 517 patients, only two cases were anti-jo1 positive, both of whom were african-american women and lay among significant cases with ild that also had cytoplasmic ana patterns and were positive for anti-dsdna [27]. according to the result of a study in shanghai lung hospital, out of 1044 individuals with interstitial lung disease, 332 (32%) did not receive an accurate diagnosis at early admission, 195 (18.7%) had continuously negative autoantibody tests, and 262 (25.1%) patients initially had negative autoantibodies but became positive in subsequent follow-up courses [21]. it has been reported that the presence of a high ana titer or rf was attributed to individuals with ctild [28]. regarding the radiological abnormality results in our study, septal thickening, honeycomb change, mediastinal lymphadenopathy, and gga were reported in a higher percentage of patients. awt, fibroblast deposition, honeycomb pattern, and interstitial lung fibrosis were the most frequent reported among patients. in a study by sheikh et al. the normal hrct, nsip, and uip were reported in 21 (40.4%), 19 (36.5%), and 12 (23.1%) patients, respectively. normal, mild reticular, clear reticular, and honeycomb cxr were reported in 19 (36.5%), 15 (28.8%), 14 (26.9%), and four (7.7%) patients, respectively (20). in another study, abnormal serology was observed in 114 (82%) out of 139 patients diagnosed with ipf, 53 (38%) cases had greater values of ≥2, 99 (71%) subjects showed an abnormal ana titer ≥1:80, and the most common ana positive was 1: balou et al. 8 160. mean values of rf positive, esr, and crp were 43 (normal < 14 iu/ml), 48 (normal < 41 mm/h), and 22 (normal < 5 mg/l), respectively. in this study, positive cases were found for rf (18, 13%), anti-ssa (6, 4%), ck (3, 2%), anca, anti-dsdna, anti-smith, and accp (1, 0.01 %), and anti ssb (2, 1%), but the results were negative for anti-rnp [11]. in this study, males and older age represented a higher number of ild that was similar to many other studies. most of the patients were married which could be due to the high prevalence of diseases in aged people, the high percentage of illiterate or the lower level of high school diploma in the studied patients may indicate various unprotected respiratory exposures in this group that probably referred to their difficult and high-risk jobs. the results showed that more than 50% of patients were exposed to noe, among whom smoking, exposure to shs, and opium were higher than other noes. other studies, however, have shown deleterious effects of chemicals and occupational exposures in lung injury and fibrosis, which may indicate differences in geographic risk factors in each particular area that should be considered in related studies. based on the spirometric parameters of patients in the patients’ data record, the obstructive pattern was detected in 34.3% of patients and the rest of the cases presented restrictive patterns. the significant proportion of obstructive disease among our ild patients may be due to the presence of smoking and other similar exposure in the significant number of patients. ipf is one of the most common clinical, radiologist, and pathologic diagnoses of ild. besides genetic and environmental exposure differences, visiting ipf patients, and disorganized diagnostic approaches to ipf pattern recognition hrct and pathologic reports may contribute to the small percentage of ipf in this study. 5. conclusion in summary, the variety of clinical, radiological, and laboratory findings in ilds can demonstrate regional differences in risk factors, patients' exposure, access to medical facilities for diagnosis and treatment, genetic differences, and also necessitate careful examination of these factors individually in each region, which therefore explains the epidemiological differences in various parts of guilan province, iran. authors' 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reports jcbior.com volume 1, number 2, 2020 eissn: 2717-1906 1 case report case reports in the use of vitamin c based regimen in prophylaxis and management of covid-19 among nigerians goodluck a. k. ohanube1, uchejeso m. obeta2,*, chinaza r. ikeagwulonu3 1 department of biomedical sciences, university of applied sciences, bonn-rhein-sieg, bonn, germany 2 department of medical laboratory management, federal school of medical laboratory science, jos, nigeria 3 department of medical laboratory services, alex ekwueme federal university teaching hospital, abakaliki-nigeria abstract this case study was based on the novel opinion that daily intake of vitamin c can serve as a prophylactic and curative intervention for the coronavirus disease of 2019 (covid-19) via homeostatic and epigenetic mechanisms. to substantiate this hypothesis, some nigerians who used vitamin c based regimen consented to share their experience in the prevention and management of the coronavirus irrespective of their country of abode. participants all agreed to the use of their information for the public, albeit anonymous, via appending their signatures on an informed consent form. the documented cases took place from march 15 to september 1, 2020, this was to allow for more time to be able to observe the participants. the study provided evidence that regular use of ascorbic acid tablets and vitamin c enriched plants could improve the immunity needed against the infection of severe acute respiratory syndrome coronavirus-2 (sars-cov-2). keywords: covid-19, ascorbic acid, vitamin c, epigenetics, homeostasis, coronavirus 1. introduction since 1747, citrus fruits which have a high content of vitamin c have been used to treat the disease called scurvy by the british naval surgeon, james lind [1]. vitamin c was coined by casmir funk in 1912, isolated in 1928 by albert szentgyörgyi, and christened ascorbic acid by walter haworth in 1932 [2]. it is a six-carbon lactone electron donor (reducing agent or antioxidant), synthesized from glucose by many animals. the liver of some mammals and the kidney of aves and reptiles synthesizes it. however, it is not synthesized in primates, due to the absence of the terminal enzyme, l-gluconolactone oxidase in the biosynthetic pathway of ascorbic acid. this situation came to be because the gene encoding for this enzyme has undergone substantial mutation such that it can no longer produce the needed protein [3]. * corresponding author: uchejeso m. obeta, msc.,mpa department of medical laboratory management, federal school of medical laboratory science, jos, nigeria tel/fax: +2348039664892 email: uchejesoobeta@gmail.com http://orcid.org/0000-0002-1382-6034 received: october, 20, 2020 accepted: october, 26, 2020 severe acute respiratory syndrome coronavirus2 (sars-cov-2), the rna virus that causes the novel disease called coronavirus disease of 2019 (covid19), was discovered in wuhan, china, in the latter part of 2019. the virus sars-cov-2 is known to be airborne, hence can be transmitted to an unsuspecting person, especially within the range of 1 meter. studies have shown it can stay in the air or on inanimate objects for hours or days, partly explaining its propensity for widespread and transmissibility. this viral agent has a predilection for the tissues of the lungs causing pulmonary edema, hyperplastic pneumocytes, and often, pneumonia; the combination of these manifests as the typical signs and symptoms such as pyrexia, cough, dyspnoea, myalgia, anosmia, and anorexia seen in covid-19. diagnosis is via these symptoms, https://jcbior.com/ http://orcid.org/0000-0002-1382-6034 ohanube et al. 2 travel history, and real-time polymerase chain reaction [4]. currently, obeying the safety measures as enunciated by the world health organisation (who) and administering a cocktail of drugs to patients has been the norm; irrespective of the cost and possible side effects associated with this cocktail. this is because there is no known standard cure for covid-19 at the moment; despite the myriad of studies recommending vitamin c enriched plants such as bitter kola, ginger, garlic, citrus, turmeric, and giloy for the prophylaxis and management of covid-19 [5-7]. these case reports are intended to serve reference purposes. it is a documentation of information shared by nigerians who used vitamin c based regimen. it is based on the novel opinion that daily intake of vitamin c can serve as a prophylactic and curative intervention for covid-19 via homeostatic and epigenetic mechanisms. 2. case presentation case 1 a physiotherapist in her mid-thirties, who resides in abakaliki, ebonyi state, nigeria; she started her dose of ascorbic acid tablets (100 mg/day) on march 18. she upgraded and maintained 1000 mg/day from april 1 till september 1. a friend of hers, whom she fortuitously spent about 1 hour with, after a religious service on sunday, june 28, later tested positive and died of covid-19 on saturday, 4th of july. she was not conscious of wearing her mask and using hand sanitizers during this visit as she never anticipated that her friend could be infected. she was advised to continue with vitamin c rich foods and ascorbic acid tablets daily. she never contracted covid-19 even as at the time of concluding this report. case 2 an aviation analyst in his fifties, who lives in ikotun, lagos, nigeria; was told by a friend to take ascorbic acid tablets (1000 mg/day) for prevention. he started the routine of taking ascorbic acid on march 25. during the second week of april, he and his friend voluntarily went to test themselves for sars-cov-2 at the nigeria centre for disease control (ncdc) in lagos. he was negative, while the friend was positive. here is what he had to say on may 6: "...i inadvertently drove someone with covid-19 around lagos. we talked for long hours in my car, even with the air condition switched on. i've gone for repeat tests to check if i'm positive for covid-19, surprisingly, i'm not”. he has not fallen ill to covid-19 as at the time of concluding this report on september 1, despite not using the mask inside the car whist the ac was on. case 3 a businesswoman in her fifties, living in ejigbo, lagos state, nigeria; started her routine of ascorbic acid tablets (500 mg/day) as at march 18, though she skipped her dose sometimes. she was not also diligent in her use of the mask and hand sanitizer as she felt there was no need for it, since she was often indoors during this period. she tested positive to covid-19 on april 24, according to the call she received from the ncdc located in lagos. she noted that the test was conducted about 11 days before this call. symptoms she manifested were cough and myalgia. she contacted her caregiver, who advised her to place her family members on the dose of 1000 mg/day, while she takes 2000 mg/day for seven days after which she can taper it. her treatment with 2000 mg/day of ascorbic acid commenced on april 25; on may 2, she tapered her dose to 1000 mg/day. the significant side effect seen after tapering this dose was that her habit of defaecating increased from once a day to four times; an effect which stopped in 24 hours. she was encouraged to go for a follow-up test on may 12. the result of the follow-up test came out about a week after, and it still read positive, even though the typical signs and symptoms of covid-19 were gone within three days of her taking ascorbic acid. the repeat test that read positive could imply that the antibodies developed against sars-cov-2 were still present, even after two weeks of commencement of treatment. she also drank warm water containing a blend of plants with a high content of vitamin c such as garlic, ginger, lime, lemon, and others. she maintained a prophylactic dose of ascorbic acid tablets (1000 mg/day) until september 1. it is pertinent to note that during the period she was sick, she was never isolated nor quarantined from her family, and none of them contracted the disease, as they have been on ascorbic acid before she fell sick. she and the entire members of her family are currently very healthy. speaking with her on june 20, she said: “…africans are surviving the onslaught of covid-19, due to the organic content of our foods, which is highly fortified with vitamin c…”. case 4 a nigerian in his late thirties who currently works as a caregiver and lives in wolfsburg, ohanube et al. 3 germany; he might have contracted the disease because he was not consistent with his ritual of ascorbic acid tablets after the advice was given to him as at march 15. he manifested symptoms such as pyrexia, cough, dyspnoea, myalgia, anosmia, and anorexia sometimes in april, despite his use of mask and hand sanitizer as a caregiver. he tested positive on april 14 and subsequently infected his octogenarian mum, pentagenerian sister, and nephew within this period. they used ascorbic acid tablets (1000 mg/day), steam inhalation, and plants with a high content of vitamin c such as garlic, ginger, and lime, and recuperated faster than when other drugs were used. he tested negative on may 4 alongside these relatives of his. they have prophylactically maintained the tradition of vitamin c fortified fruits and ascorbic acid tablets (480 mg/day) till the time of compiling this report as of september 1. 3. discussion for ages, vitamin c (ascorbic acid) continues to be an antioxidant, boosting immunity; repairing worn-out tissues; enhancing wound healing; and replenishing extracellular matrix. it also quells inflammation, cardiovascular diseases, and cancer. all these attributes make it invaluable in the control of covid-19 [8-11]. natural sources of vitamin c in nigeria include garcinia kola, moringa, guava, pineapple, paw-paw, citrus fruits, garlic, ginger and some others. in case 1, the participant started her vitamin c tablets (100 mg/day) on march 18 and upgraded to 1000 mg/day from april 1, which she maintained until september 1. case 2 shows a participant who took vitamin c tablets (1000 mg/day) for prevention from march 25, 2020. both cases remains negative for sars-cov-2, indicates the potential protective effects of ascorbic acid. this situation agrees with previous reports who opined that vitamins could boost immunity against covid-19 [1,5,12,13]. in the case 3, the participant used vitamin c tablets (500 mg/day) but skipped some days and later tested positive to covid-19 on april 24 and remained at home to manage herself with 2000 mg of vitamin c tablets while the family members maintained 1000 mg/day. one can say that the use of vitamin c should be a continuous one. notably, there were no debilitating symptoms of covid-19 observed in the subject, thereby showing that earlier doses of vitamin c could have improved the immunity in agreement with those studies that showed vitamin c (tablet or infusion) serve as a potent prophylactic and curative intervention in some viral infections [1,12-14]. in the case 4 we saw where a nigerian living in wolfsburg, germany was not keen with the regular use of vitamin c tablets as at march 15 and tested positive on april 14 and subsequently infected his octogenarian mum, pentagenerian sister, and nephew. this case confirms that a combination of ascorbic acid tablets and a hot pulp of vitamin c enriched plants, either drank or inhaled, made a faster recovery than when other means were used. this agrees with previous reports [5,6,15], with regards to vitamin c enriched plants in the management of covid-19. the sine qua non of containing this pandemic as observed among africans, especially nigerians is via the prophylactic use of vitamin c fortified plants or ascorbic acid tablets and to an extent proper use of the masks and hand sanitizers. observations made during this period show that doses lower than 500 mg/day can also be used by adults to maintain a steady-state needed to ward off the virus, however for optimal efficiency, the given range is preferred. due to the need for standardization and scarcity of vitamin c fortified fruits; it was advised that pupils below 11 years, take ascorbic acid tablets (100 mg-20 0mg) depending on their body weight and age. while 300 mg to 400 mg per day is sufficient for students under 18; 500 mg/day is for adults; essential workers survive on 1000 mg/day [13]. this modality is vital, especially during an unpremeditated violation of social distancing due to overpopulation in major cities, just like it is epochal in remote areas with a looming paucity of face masks and the dearth of hand sanitizers. these case reports agree with stories found on the internet, which talked about the testimonies of recovery from covid-19 given by governor seyi makinde and professor otegbayo j.a.o; testimonies which confirm the efficacy of ascorbic acid and medicinal plants in nigeria [16-18]. it could be part of the factors which led to the low number of cases (53,021) seen in nigeria [19] as at august 26, while her german counterpart was having 239,000 cases [20]. however, the inability for nigeria to test to full capacity might have also contributed to this situation, posing a significant limitation to this work. 4. conclusion encouraging the use of ascorbic acid alongside the face mask and hand sanitizers by local health authorities could help to break the chain of transmission, de-escalates the number of hospital ohanube et al. 4 visits, and circumvents the debilitation associated with covid-19, averting many cases of in-patient care. this feat amortizes the humongous amounts budgeted on health by nations; brings to the barest minimum, the stress on every healthcare facilities around the world; revitalizes the ailing economies of countries; expedite the opening of schools, and saves the caregivers from prolonged exposure and death. this case report support the use of ascorbic acid or vitamin c loaded plants as an important supportive medicine for preventing the infection by sars-cov2, or in the management of covid-19. acknowledgments the authors declare that the study was selffunded and appreciates the essential workers and others who volunteered the information used in compiling these case reports. ethical declarations this study was performed in accordance with the declaration of helsinki. consent for publication written informed consent was obtained from the patients for publication of this case report. a copy of the written consent is available for review by the editor-in-chief of this journal. financial support self-funded. conflict of interests the authors declare no competing interests. authors contribution all authors contributed equally and approved the final version of manuscript. references 1. carr ac. vitamin c in pneumonia and sepsis. in: chen q, vissers m, editors. vitamin c: new biochemical and functional insights. oxidative stress and disease. boca raton, fl crc press/taylor & francis; 2020. p. 115-35. 2. hemilä h. a brief history of vitamin c and its 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https://www.vanguardngr.com/2020/04/breaking-uch-chief-prof-otegbayo-tests-negative-for-coronavirus/ https://www.vanguardngr.com/2020/04/breaking-uch-chief-prof-otegbayo-tests-negative-for-coronavirus/ https://www.worldometers.info/coronavirus/country/nigeria https://www.worldometers.info/coronavirus/country/germany/ https://www.worldometers.info/coronavirus/country/germany/ journal of current biomedical reports jcbior.com volume 1, number 2, 2020 eissn: 2717-1906 1 brief report the occurrence of antibiotic resistance, esbls, mbl and ndm-1 in uropathogenic escherichia coli in central part of iran farshad kakian1, javad fathi1, fatemeh alvandi2, mahnaz moumivand2, abbas rabei gholami3, abolfazl gholipour4, mohamad hossein rezaei5, behnam zamanzad6,* 1department of bacteriology and virology, school of medicine, shiraz university of medical sciences, shiraz, iran 2department microbiology, hamadan university of medical sciences, hamadan, iran 3cellular and molecular research center, shahrekord university of medical sciences, shahrekord, iran 4cellular and molecular research center, basic health sciences institute, shahrekord university of medical sciences, shahrekord, iran 5cellular and molecular research center, shahrekord university of medical sciences, shahrekord, iran 6department of microbiology and immunology, school of medicine, shahrekord university of medical sciences, shahrekord, iran abstract extended spectrum β-lactamases (esbls) are enzymes that capable of destroying the antibiotics of βlactam, and cephalosporin, and metallo-β-lactamase enzymes (mbl) can also deactivate all β-lactams and carbapenems. this study aimed to determine esbls and mbls enzymes and the frequency of ndm-1 gene. in this study, 200 escherichia coli isolates of women with urinary tract infection were collected (100 inpatients and 100 outpatients). minimum inhibitory concentration (mic) for ceftazidime and meropenem was determined by e-test. a phenotypic confirmation test was used to detect esbl enzymes. mbls production was performed with modified hodge test (mht) and edta disk synergy (eds) test. pcr was used for detecting the presence of ndm-1 gene. from 200 isolates, 93 isolates produce esbl enzymes. overall, 97 isolates were resistant to ceftazidime, and 38 isolates resistant to meropenem. the results of the mht and eds positive tests were 41 and 16 isolates, respectively. ndm-1 was not found in any of the patients. the prevalence of e. coli isolates producing both esbls and mbls enzymes, is a serious threat to clinical infections. accordingly, for the control and treatment of these strains, rapid and accurate identification can be greatly helpful. keywords: escherichia coli, mic, esbl, ndm-1 1. introduction one of the most important infections that affect parts of the urinary tract is urinary tract infection (uti). bacteria are the main reason for this infection. the most common bacterial cause for uti is escherichia coli. utis report about 150 million cases each year and are more common in women than men [1-3]. up to 10% of women have a uti in a given year, * corresponding author: dr. behnam zamanzad, md, ph.d department of microbiology and immunology, school of medicine, shahrekord university of medical sciences, shahrekord, iran tel/fax: +98 38 33346732; +98 38 3334911 email: bzamanzad22@gmail.com http://orcid.org/0000-0001-8348-7827 received: november, 5, 2020 accepted: november, 30, 2020 and half of them have at minimum one infection at some point in their lifetime [4]. there is a rising worry for multidrug-resistant bacteria that produce extended-spectrum β-lactamases (esbls). e. coli becomes progressively resistant to expandedspectrum cephalosporins by the production of esbls [5,6]. class d β-lactamases or oxacillinases (oxa) are commonly distributed between clinically relate gramhttps://jcbior.com/ https://en.wikipedia.org/wiki/escherichia_coli kakian et al. 2 negative bacteria [7,8]. carbapenem antibiotics are now used to treat infections caused by β-lactam resistant bacteria [2,9]. this antibiotic is used to treat infections caused by penicillin or cephalosporinresistant gram-negative bacilli because this antibiotic, in addition to a broad spectrum of action, has acceptable stability against most β-lactamase [2,10,11]. resistance to carbapenem is mainly created with metallo-β-lactamases (mbl) such as ndm (new delhi metallo-β-lactamase), a class b type of βlactamases that know bivalent metal ions [12,13]. recently, a new mbl named ndm-1 appeared in e. coli [14,15]. this gene causes resistance to penicillins, cephalosporins, and carbapenems [16]. this study aimed to determine esbl, and mbl production ability and determination of the frequency of ndm gene in e. coli strains isolated from uti in women. 2. materials and methods 2.1 bacterial strain collection and identification in this descriptive-analytical study, 200 e. coli isolates with urinary tract infection were collected from women with uti in two groups of inpatients (n=100) and outpatients (n=100) in teaching hospitals from shahrekord, iran from 2016 to 2017. in this study, patients who were diagnosed with uti 48-72 hours after hospitalization were considered as inpatient samples. patients who have not been hospitalized in the past month were also used as outpatient samples. isolates were detected using standard tests including gram stain, microscopy analysis and culture in emb media, macconkey agar, and blood agar (merck, germany), and other biochemical tests. 2.2 determination of mic and esbl production the minimum inhibitory concentration (mic) test was done by the epsilon test (e-test) of liofilchem company (italy) with a concentration range of 0.00232 μg/ml to meropenem and ceftazidime. evaluation and determination of inhibition zones were performed after culturing and incubating the plates at 37 °c overnight, using the clsi guidelines. the phenotypic combined disc method was also used to determine esbl enzymes [8]. e. coli atcc 25922 and klebsiella pneumonia atcc 1705 were considered as negative and positive controls. 2.3 mht and eds test in this study modified hodge test (mht) and edta disk synergy test (eds) were performed to detect mbls enzymes. further, e. coli atcc 25922 and klebsiella pneumonia atcc1705 were considered as negative and positive controls for running mht, respectively [17,18]. 2.4 dna extraction and pcr at this stage, after culturing samples on media, dna was extracted using the boiling method [8]. the single-pcr technique was also used to detect ndm-1 and 16s rrna genes. primers for this study are shown in table 1 [19-21]. the presence of the 16srrna gene (internal gene for e. coli) was investigated to approve the phenotypic tests for e. coli detection. 2.5 statistical analysis the statistical analysis was performed using spss, version 23.0 (spss ibm, ny, usa). the results are presented as descriptive statistics in terms of relative frequency 3. results in this study, the age range of patients was 2-88 years. the maximum and minimum prevalence of isolates were allocated to the internal wards and intensive care units (icu) respectively. in this study, the result of resistance to ceftazidime was 73% for inpatients and 25% for outpatients. resistance to meropenem was 28% for inpatients and 10% for outpatients. from 100 isolates from inpatients, 70 isolates (70%), and from 100 isolates from outpatients, 23 (23%) isolates produce esbls. the results of the mht and eds tests were positive in 21% and 20% of inpatient samples and 8% and 8% of outpatient samples, respectively. after the electrophoresis of pcr products, ndm-1 gene was not found in any of the pcr. 4. discussion table 1. primers used in this study target primer sequence (5' to 3') size (bp) ndm-f ggtttggcgatctggttttc 624 bp ndm-r cggaatggctcatcacgatc 16s rrna-f aggccttcgggttgtaaagt 420 bp 16s rrna-r acctccaagtcgacatcgtt kakian et al. 3 resistant e. coli is considered a healththreatening pathogen and causes many diseases in humans, especially those with underlying diseases and immune deficiencies. the infections owing to e. coli are treated with broad-spectrum antibiotics [22-24]. antibiotic resistance in these bacteria is increasing and is spreading all over the world [2]. carbapenem and βlactam antibiotics are drugs of choice for e. coli infections. it is very valuable to early detection of esbl and mbl in bacteria to control and prevent the spread of resistance strain in society and hospitals. these results and information can lead to a proper prognosis and selection of the appropriate medication by the physician [25,26]. we observed that 93 isolates produce esbl enzymes. also, 97 isolates resistance to ceftazidime and 38 isolates resistance to meropenem. ndm gene was not funded in the patients. in a similar study, in pakistan, it was observed that among 116 e. coli isolates from utis, 98 isolates were resistant to meropenem and imipenem and a total of 66 isolates were found to be esbl producers [27]. a study in india showed that imipenem and ceftazidime resistance in clinical isolates of e. coli were 9%, and 62.1%, respectively. they also showed 61.1% of e. coli isolates were esbls [28]. jamil et al. from pakistan reported that among 25 carbapenem-resistant e. coli, 16% contained mbl by phenotypic test [29]. in another study by qamar et al. of 100 e. coli isolates, 81% were mbl producers and 18.4% were ndm-1 positive [30]. in a similar study by koraei et al. from southwest of iran, in 376 e. coli isolates, ndm-1gene was not found in any isolates [31]. in this study, we had some limitations. the main limitations were the study of two medical centers and the small sample size. the emergence and spread of antibiotic resistance by esbl and mbl producer isolates have been a global problem that indicates the excessive use of antibiotics. these types of antibiotic resistance are a serious problem in clinical therapeutic selections in the hospital as well as in the community. so, it is necessary to report esbl/mbl production along with the daily sensitivity reporting, which will help the clinicians in determining correct antibiotics. acknowledgments we thanks all the people and staffs who assisted us in conducting this work. author contributions fk, ag, bz: design of study; fk, ag, bz, mhr: acquisition of data; fa, ar, mm and jf: evaluation of data, preparation of the manuscript; fk, bz, ag and jf: assessment of data. all authors have read and approved the manuscript. conflict of interests we have not competing interests. ethical declarations the study protocol was obtained by the ethics code of ir.skums.rec.1395.215 from shahrekord university of medical sciences. however, we only used leftovers from clinical specimens. financial support this research was supported by the shahrekord university of medical sciences, shahrekord, iran (grant no. 2540). references 1. flores-mireles al, walker jn, caparon m, hultgren sj. urinary tract infections: 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https://pubmed.ncbi.nlm.nih.gov/27790433/ https://pubmed.ncbi.nlm.nih.gov/27790433/ https://pubmed.ncbi.nlm.nih.gov/30410154/ https://pubmed.ncbi.nlm.nih.gov/30410154/ https://pubmed.ncbi.nlm.nih.gov/30410154/ https://pubmed.ncbi.nlm.nih.gov/30410154/ https://pubmed.ncbi.nlm.nih.gov/31894059/ https://pubmed.ncbi.nlm.nih.gov/31894059/ https://pubmed.ncbi.nlm.nih.gov/31894059/ https://pubmed.ncbi.nlm.nih.gov/31894059/ journal of current biomedical reports jcbior.com volume 2, number 1, 2021 eissn: 2717-1906 1 original research evaluation of clinical and laboratory causes of burns in preschool children mohammadreza mobayen1, siamak rimaz1, arghavan malekshahi1,* 1burn and regenerative medicine research center, guilan university of medical sciences, rasht, iran abstract we investigate characteristics of burns in children aged up to seven years and hospitalized at velayat burn center unit for two years to evaluate clinical and laboratory causes of burns in pre-school children. in a retrospective study, all patients under 6 years who were admitted to velayat hospital due to burn injuries between 20 march 2016 and 20 march 2017 participated in this study. baseline data and information collected from hospital his system and patient records. the burn-injured children were 283 patients, of which 54.1% were male and 45.9% were female. these injuries mostly were caused by hot liquid (78.7%). the greatest number of burn victims were boys between one and two years old. almost all the accidents occurred in the home environment (90.1%). a significant correlation was observed between contact burns, serum albumin level, and serum c-reactive protein (crp) levels with burn severity. our data demonstrate the importance of developing a program for the prevention of pediatric scalds with the education of family members to be aware of the danger. the most important finding in our study was the hypothesis of estimating albumin 1b peptides (a1b) and crp laboratory factors during the initial week of hospitalization in the prognosis of children and their constant relation with the severity level of the burns. keywords: burn injuries, pediatric, epidemiologic, laboratory 1. introduction burn injuries continue to be one of the leading causes of injuries worldwide [1]. in addition, a considerable level of hospital care is attributed to burns annually [2]. according to reports, 95% of burnrelated deaths occur in lowand middle-income countries. in fact, half of the death cases are observed in south and southeast asia [2, 3]. therefore, burns are predictable injuries in children [4], which, at the same time, could lead to numerous destructive physical, mental and social issues [5]. in addition to their acute impacts, burns can cause chronic disorders in the lives of children [6]. children are highly vulnerable to burn-related injuries. while * corresponding author: arghavan malekshahi, md burn and regenerative medicine research center, poursina hospital, namjoo street, rasht, iran tel/fax: +98 13 333311473 email: arghavan.malekshahi.am@gmail.com https://orcid.org/0000-0002-7102-0653 received: january, 09, 2020 accepted: february, 16, 2020 infants and pre-school children are more at risk of burn injuries, they are more neglected, compared to other age groups [7, 8]. moreover, studies have shown that more than 50% of burns occur in children aged three-six years [9, 10]. given the high incidence rate of burns in children aged one to six years and the high rate of associated complications and mortality rate in children. we aimed to evaluate clinical and laboratory causes of burns in pre-school children. 2. materials and methods the research was performed on patients hospitalized from march 2016 to march 2017 due to different burn-related injuries with various https://jcbior.com/ mobayen et al. 2 mechanisms. burn mechanisms are divided into three thermal (heat-related), chemical, electrical categories. thermal burns include flame, hot liquids, and contact with hot surfaces. in this regard, not only flame burns are the most common causes of burn-related hospitalizations, but also, they have the highest mortality rate, which is primarily due to their association with building fires and respiratory impairments or carbon monoxide (co) poisoning. the burned area is estimated using the rule of nines; in this respect, 18% for each anterior and posterior trunk, 9% for each upper limb, 18% for each lower limb, 9% for head and neck, and 1% for perineum are estimated. based on depth, burns are classified into first-degree (superficial), second-degree (partial thickness), thirddegree (full thickness), and fourth-degree burns, which go through both layers of the skin and underlying tissue as well as deeper tissue. the inclusion criteria were patients experiencing burns with different mechanisms and the age range of zero to six years. on the other hand, the exclusion criterion was incomplete medical records. using a predesigned questionnaire, we collected the data related to demographic characteristics of the subjects, including age, gender, cause of the burn, the season of burn, the anatomical site of the burn, burn site, mortality, duration of hospitalization, burn severity (percentage), burn degree, crp serum level, and albumin (in the first seven days of hospitalization). the required information was extracted from patients' records and their history and the data were entered into spss software ver. 22. it should be noted that patients who have been hospitalized more than once were considered as one patient and the information about several hospitalizations was combined and analyzed as one patient. ordered logistic regression was used to investigate the relationship between different variables and burn severity. the poisson regression model was also used to investigate the relationship between burn percentage and different variables. 3. results a total of 283 patients entered the study aiming to examine the epidemiology of annual cases of burns in preschool children in velayat hospital. out of the 283 studied patients, 153 (54.1%) patients were male and 129 (45.9%) were female. the average age of the patients was 2.1±1.6 years (the youngest patient being an infant and the oldest being 6 years old). the study of the time of incident indicated that most of the cases of burns had occurred in july with 32 cases (11.3%) and the lowest number of cases was observed in september with 14 cases (4.9%); however, the largest number of cases had occurred during winter (35.4%). the study of patients’ place of residence divided by urban and rural areas indicated that out of the studied population, 219 people (77.4%) lived in the city while 64 patients (22.6%) lived in rural areas. evaluation of the reasons for burning indicated that over half of the patients (78.7%) had burned themselves with hot liquids including hot tea and water, stew, or soup. also, one patient had burned due to a gas explosion, six patients had burned due to contact with fire and alcohol, and 87 patients (30.7%) had suffered from other sources of burning such as contact with heaters, and hot objects. investigation of the burn site revealed that 99 patients (35%) had burns on several areas including the upper body and limbs. also, 82 patients had burns on their lower body, and 65 patients had burns on their upper body. besides, 23 patients suffered from burns on their torso, and 13 patients had burns on their face and head. investigation of the degree of burns indicated that 58 patients had suffered from first-degree burns, 173 patients suffered from second-degree burns, and four patients suffered from third-degree burns. besides, 48 patients suffered from moderate burns ranking between second-degree and third-degree burns. examination of patients’ burn percentage revealed that 32 patients with 5% burns, 27 patients with 6% burns, and 19 patients with 20% burns had the most frequent burn percentage among the study population. examination of the hospitalization period indicated that over half (53%) of patients were treated on an outpatient basis and discharged. besides, 54 patients were hospitalized for one day and 22 patients were hospitalized for two days. the study of burning consequences revealed that only one patient had died due to burns and other patients had been discharged. the study of wound culture in patients indicated that eight cultures (2.8%) tested positive and the rest tested negative. table 1 demonstrates the studied patient’s serum albumin levels on days zero, three, and seven, and serum crp levels on days zero and seven. table 2 demonstrates study results based the relationship between burn intensity and various order logistic variables used in the study. given that burn mobayen et al. 3 severity indicated in this table is an interval variable, sequential logistic regression model has been used. therefore, the coefficients in this table are odds ratio. according to this table, a significant correlation was observed between contact burns, serum albumin level (in all three days), and serum crp levels (in both examinations) with burn severity. table1. serum albumin levels on days 0, 3 and 7 and serum crp levels on days 0 and 7 variable minimum maximum mean std. deviation albumin baseline (gr/dl) 1.50 4.50 2.64 0.80 albumin day 3 (gr/dl) 1.80 4.00 2.73 0.54 albumin day 7 (gr/dl) 2.00 4.50 3.01 0.68 crp baseline (mgr/dl) 0 125.00 13.50 24.21 crp day 7 (mgr/dl) 0 150.00 10.03 19.21 table 2. examines the relationship between burn severity and other variables burn variable odds ratio standard error p-value low limit high limit age 1.061 0.087 0.473 0.903 1.246 (girl to boy) sex 0.579 0.162 0.51 0.335 1.003 season summer 2.028 1.295 0.268 0.580 7.087 fall 1.240 0.785 0.734 0.358 4.290 winter 1.137 0.722 0.840 0.328 3.947 reference: spring cause of burns explosion 0.855 0.918 0.990 <0.001 2.125 fire 0.855 0.785 0.865 0.142 5.168 contact burns 0.025 0.011 <0.001 0.011 0.058 reference: electric burn anatomical location of the burn body 1.441 1.154 0.648 0.300 6.920 upper limb 2.419 1.683 0.204 0.619 9.458 lower limbs 1.585 1.090 0.503 0.412 6.099 several limbs together 2.316 1.596 0.223 0.600 9.941 other anatomical places 0.352 0.863 0.670 0.003 42.932 reference: face burn site bathroom 0.373 0.653 0.573 0.012 11.537 school <0.001 <0.001 0.983 <0.001 1.203 nature 2.279 1.322 0.155 0.731 7.104 bedroom 1.824 1.310 0.403 0.446 7.455 reference: kitchen laboratory parameters serum albumin baseline 0.014 0.01 <0.001 0.01 0.048 serum albumin day 3 0.01 0.012 <0.001 0.02 0.045 serum albumin level day 7 0.012 0.08 <0.001 0.01 0.041 serum crp level baseline 0.01 0.01 <0.001 0.011 0.035 serum crp level 7 0.013 0.01 <0.001 0.012 0.033 mobayen et al. 4 4. discussion burning injuries are considered to be one of the most common types of injuries in the entire world [1113], besides, injuries in children being a significant percentage and a major part of hospital care annually [14, 15]. during the studies of the precise burn percentage of burnt patients which was conducted on the population who were referring to our hospital, it was discovered that the majority of these patients had approximately 5% burning injury overall, which is significantly lower than the burn percentage of similar studies conducted previously in turkey and india [6, 16]. in the study of the outcome of burn patients who referred to our hospital, it was discovered that only one patient’s burning injuries were fatal and the rest were all discharged from the hospital, which was considerably less than the number of fatally injured and burnt patients found in other parts of iran such as, isfahan and mashhad. in isfahan, it was discovered that 5.6% of burns resulted in death, and in mashhad, the mortality rate was about 3.85% [1, 17]. evaluation on the hospitalization period shows that the duration of hospitalization of burn patients who were referring to our hospital is approximately one or two days on average, which is significantly less than the results obtained from a study conducted in turkey [6]. in another study conducted under quite similar circumstances in turkey, the average number of days burnt patients were hospitalized was approximately 16 days [4]. in another study conducted in iran (isfahan), patients averaged about less than three days in hospital until released [1]. in the process of studying the wound cultures of our hospital burn patients, it was discovered that the wound culture samples of eight of these patients were positive and the rest all turned out negative, which was lower than other studies [6, 17]. based on the studies done so far, it can be concluded that most of these burn injuries occur in the home, especially in the kitchen, also, most of these fires occur due to the absence of parents and children being left alone [18], which can lead to many possible physical and psychological consequences, including dissatisfaction with body shape, post-accident trauma, and stress disorder, as well as a sleep disorder, wound and scar issues and on the other hand. it can also impose great costs both financially as well as mentally for the surrounding community and relatives of the individual in terms of fixing the deformities created, in addition to treating and compensating all the psychological issues in the aftermath of the burns. therefore, the prevention of burns is always preferred and much better than being cured, fortunately, there have been many positive reports recently about the reduction of burns and mortality rate due to the population following the fire prevention programs [19]. as a consequence, having either a proper parental awareness program through the mass media and tv or properly educating parents about the level of care and cautiousness the children under the age of six need can lead to a reduction in fire occurrences and all the unpleasant consequences that may follow afterward. in our study, we discovered that the precise serum albumin levels during the first week as well as crp in this group of children can be a factor accompanying the severity level of burns and it is for this purpose that these two criteria can be used as a useful predictor for the prognosis of burns. although we were not able to investigate any further due to the existence of only one case of mortality in our study, therefore, it is recommended to examine this particular hypothesis in upcoming future studies. the main limitations of the present study were due to incomplete files and incomplete entry of findings, some samples were excluded from the study. also, our results are limited only to two years which can limit the strength of our conclusion. according to the various study results obtained based on the high frequency of burn injuries in children under the age of six which usually seems to take place in their homes and kitchens and can potentially lead to death, scar issues and depression, etc. this can only indicate the extreme level of importance taking proper care of children can have, as well as the constant presence of the parents required who are prepared and aware of the potential dangers to prevent such disastrous incidents. however, the most important and major finding in our study so far was the hypothesis of estimating a1b and crp laboratory factors during the initial week of hospitalization in the prognosis of children and their constant relation with the severity level of the burns. mobayen et al. 5 authors contribution all authors contributed equally in data collection and drafting of the manuscript. also, all authors approved the final version of the manuscript. conflict of interests the authors do not have any conflict of interest to disclose. ethical declarations the study deign was approved by the ethics committee of guilan university of medical sciences (ir.gums.rec.1397.165). financial support none. references 1. ahmadabadi a, tavousi sh, sedaghat a, khadem rezaeyan m, yaghoubi moghaddam z, lalavi z. pattern of burn injuries in preschool children. saf promot inj prev. 2016; 4(4):225-30. 2. seifert j. incidence and economic burden of injuries in the united states. bmj publishing group ltd; 2007. 3. khadem-rezaiyan m, aghajani h, ahmadabadi a, zanganeh m, tavousi sh, sedaghat a, et al. epidemiology of severe burns in north-east of iran: how is the burn size different in a developing country from developed ones? burns open. 2020; 4(1):4-9. 4. anlatici r, ozerdem or, dalay c, kesiktaş e, acartürk s, seydaoğlu g. a retrospective analysis of 1083 turkish patients with serious burns. part 2: burn care, survival and mortality. burns. 2002; 28(3):239-43. 5. de young ac, kenardy ja, cobham ve, kimble r. prevalence, comorbidity and course of trauma reactions in young burn-injured children. j child psychol psychiatry. 2012; 53(1):56-63. 6. balseven-odabaşi a, tümer ar, keten a, yorganci k. burn injuries among children aged up to seven years. turk j pediatr. 2009; 51(4):328-35. 7. shields bj, comstock rd, fernandez sa, xiang h, smith ga. healthcare resource utilization and epidemiology of pediatric burn-associated hospitalizations, united states, 2000. j burn care res. 2007; 28(6):811-26. 8. carlsson a, udén g, håkansson a, karlsson ed. burn injuries in small children, a population-based study in sweden. j clin nurs. 2006; 15(2):129-34. 9. libber sm, stayton dj. childhood burns reconsidered: the child, the family, and the burn injury. j trauma. 1984; 24(3):24552. 10. kent l, king h, cochrane r. maternal and child psychological sequelae in paediatric burn injuries. burns. 2000; 26(4):317-22. 11. ghosh a, bharat r. domestic burns prevention and first aid awareness in and around jamshedpur, india: strategies and impact. burns. 2000; 26(7):605-8. 12. wiechman sa, ptacek jt, patterson dr, gibran ns, engrav le, heimbach dm. rates, trends, and severity of depression after burn injuries. j burn care rehabil. 2001; 22(6):417-24. 13. forjuoh sn. burns in lowand middle-income countries: a review of available literature on descriptive epidemiology, risk factors, treatment, and prevention. burns. 2006; 32(5):529-37. 14. kazemzadeh j, vaghardoost r, dahmardehei m, rabiepoor s, farzan r, asghar kheiri a, et al. retrospective epidemiological study of burn injuries in 1717 pediatric patients: 10 years analysis of hospital data in iran. iran j public health. 2018; 47(4):584-90. 15. kao cc, garner wl. acute burns. plast reconstr surg. 2000; 101(7):2482-93. 16. verma s, srinivasan s, vartak a. an epidemiological study of 500 paediatric burn patients in mumbai, india. indian j plast surg. 2007; 40(2):153-7. 17. rafii mh, saberi hr, hosseinpour m, fakharian e, mohammadzadeh m. epidemiology of pediatric burn injuries in isfahan, iran. arch trauma res. 2012; 1(1):27-30. 18. joseph ke, adams cd, goldfarb iw, slater h. parental correlates of unintentional burn injuries in infancy and early childhood. burns. 2002; 28(5):455-63. 19. van der merwe ae, steenkamp wc. prevention of burns in developing countries. ann burns fire disasters. 2012; 25(4):18891. https://doi.org/10.1016/j.burnso.2019.11.005 https://doi.org/10.1016/j.burnso.2019.11.005 https://doi.org/10.1016/j.burnso.2019.11.005 https://doi.org/10.1016/j.burnso.2019.11.005 https://pubmed.ncbi.nlm.nih.gov/11996854/ https://pubmed.ncbi.nlm.nih.gov/11996854/ https://pubmed.ncbi.nlm.nih.gov/11996854/ https://pubmed.ncbi.nlm.nih.gov/11996854/ https://pubmed.ncbi.nlm.nih.gov/21671940/ https://pubmed.ncbi.nlm.nih.gov/21671940/ https://pubmed.ncbi.nlm.nih.gov/21671940/ https://pubmed.ncbi.nlm.nih.gov/19950839/ https://pubmed.ncbi.nlm.nih.gov/19950839/ https://pubmed.ncbi.nlm.nih.gov/19950839/ https://pubmed.ncbi.nlm.nih.gov/20375698/ https://pubmed.ncbi.nlm.nih.gov/20375698/ https://pubmed.ncbi.nlm.nih.gov/20375698/ https://pubmed.ncbi.nlm.nih.gov/20375698/ https://pubmed.ncbi.nlm.nih.gov/16422729/ https://pubmed.ncbi.nlm.nih.gov/16422729/ 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https://pubmed.ncbi.nlm.nih.gov/24719838/ https://pubmed.ncbi.nlm.nih.gov/12163285/ https://pubmed.ncbi.nlm.nih.gov/12163285/ https://pubmed.ncbi.nlm.nih.gov/12163285/ https://pubmed.ncbi.nlm.nih.gov/23766751/ https://pubmed.ncbi.nlm.nih.gov/23766751/ https://pubmed.ncbi.nlm.nih.gov/23766751/ journal of current biomedical reports jcbior.com volume 2, number 1, 2021 eissn: 2717-1906 1 systematic review effects of black cumin (nigella sativa l.) on patients with cancer and tumor: a systematic review mohammad asadul habib1,2,*, akibul islam chowdhury1, monia afroze1, tanjina rahman1 1department of food technology and nutrition science, noakhali science and technology university, naokhali-3814, bangladesh 2department of nutrition, world vision international, cox’s bazar, bangladesh abstract experimental studies demonstrated a positive effect of administration of nigella sativa l. (black cumin) and its active chemical components on cancer and tumor through the antioxidant and anticancer activity. this study aimed to determine the beneficial effect of the use of black cumin in patients with cancer and tumor. this systematic review includes 4 randomized controlled trials that investigated the effect of the administration of black cumin in human cancer. articles were searched in pubmed, cochrane library, semantic scholar and directory of open access journal (doaj), open grey and grey literature report databases for studies from 1983 to 2020 before may using the following keywords, nigella sativa, black cumin, cancer, tumor, etc. the results examined that n. sativa is much effective in treating breast cancer, gastrointestinal cancer, brain tumor, and acute myeloid leukemia. according to the limited evidence from the study, black cumin may have favorable effects on cancer and tumor. however, more research is needed on different types of cancer to confirm and establish the above findings. keywords: nigella sativa l., cancer, tumors, black cumin 1. introduction nigella sativa l. or black cumin has become one of those popular medicinal plants that have been used for centuries for health benefits. nigella sativa l. belongs to the ranunculaceae family and is an annual herb with 8–12-inch-high pinnate, stratified leaves [1]. new products are also advocated from natural sources, as it is estimated that more than 300,000 herbal species exist globally among them only 15% have been explored for their pharmacological potential [2]. n. sativa, along with many medicinal plants (ranunculaceae) has been considered as one of the most appreciated nutrient-rich herbs in the world's history, and numerous scientific studies are under its way to justify the typically claimed use of this species' * corresponding author: mohammad asadul habib, msc department of food technology and nutrition science, noakhali science and technology university, bangladesh tel/fax: +8801847-102071 email: asadulhabib698@gmail.com http://orcid.org/0000-0003-2331-7871 received: november, 24, 2020 accepted: december, 04, 2020 small seed [3,4]. n. sativa is an herbaceous plant with wonderful therapeutic effects, such as antihypertensive, gastro-protective, nephroprotective, antioxidant, antimicrobial, geno protective, neuroprotective, immunomodulatory, anti-inflammatory, hypoglycemic, hypolipidemic, anti-carcinogenic and hepato-protective [5,6]. n. sativa seeds with some active components such as essential oil, thymoquinone, p-cymene, and thymol are very rich in the fixed oil, essential fatty acids, alkaloids, phytosterols, glycolipids and phospholipids, saponins, and essential oil components. thymoquinone (tq) is a cytotoxic agent in many human tumor cell lines that seem to be immune to particular multi drugs [7]. there is evidence that most https://jcbior.com/ habib et al. 2 of n. sativa's therapeutic effects are due to the influence of tq which has been the sativa's greatest bioactive component. crude and tq against noncommunicable diseases (cardiovascular disease, obesity, hypertension, diabetes, cancer, etc.) are beneficial from their seeds and oil, and communicable diseases (malaria, aids, hepatitis c virus, fungal, viral, parasitic infections, etc.) [8]. tq had already observed pleiotropic anti-cancer effects, including chemo potentiation, anti-inflammation, immunomodulation, as well as radiosensitization [9]. in addition to inducing apoptosis across several different cancers, tq has been shown to inhibit numerous tumorigenic signaling nodes, such as those involved in cell proliferation, epithelial to mesenchymal shift, invasive cell migration, and metastasis [10]. in this study, it was aimed to show the positive effects of n. sativa against human cancer. 2. materials and methods 2.1 literature search the reporting of this review follows the preferred reporting items for systematic reviews and metaanalyses (prisma) statement. we searched pubmed, cochrane library, semantic scholar, and directory of open access journal (doaj) databases for studies from 1983 to 2020 before may. for gray articles, we searched open grey (www.opengrey.eu) and grey literature reports (www.greylit.org). the keywords used for searching (nigella sativa or black cumin or black seed or kalonji) and (cancer or tumor or malignancy) to identify intervention studies investigating the effect of black cumin on cancer. 2.2 study selection and data extraction in this systematic review, we included published intervention studies (these include randomized controlled trials, clinical trials, and placebo-controlled trials), reporting the effect of black cumin on cancer and published in english). trials were excluded if they did not meet the criteria above and were involved in animal studies or in vitro studies, did not use the interests of intervention or outcome. two independent reviewers (mah and aic) screened the titles and abstracts of the initially identified studies to determine whether they would satisfy the selection criteria. any disagreements about selection were resolved through consensus or consultation with a third author (tr). the data collection form included questions on the year of publication, geographic origin, types of intervention, doses, patients’ type and characteristics (sample size, age, etc.). figure 1 summarizes the study selection process. 2.3 quality assessment the quality of each study was assessed by following the cochrane format [11] reported in table 1. the quality assessed for each study are (1) quality of randomization, (2) quality of blinding, and (3) quality of the description of withdrawals and dropouts. 3. results we selected four human studies (table 2) that described the effect of n. sativa on human cancer [1215]. in three days, behnamfar et al. analyzed the incidence and magnitude of phlebitis, applying n. sativa by a catheter upon the peripheral vein. in their research, they reported that the prevalence of phlebitis in the control group was optimum and that the occurrence of phlebitis in the intervention group was lowest during the 72 hours of the test, assuming that n. sativa oil seemed to have a massive effect on the intervention group [12]. hussain et al. demonstrated the effect of applying n. sativa oil on acute myeloid leukemia. the primary result of this research concluded that at the end of 28 days, the severity of figure 1: flow diagram of study selection records identified through database searching (n =222); databases: cochrane library (9), pubmed (14), doaj (11), semantic library (188) s c r e e n in g in c lu d e d e li g ib il it y id e n ti fi c a ti o n records after duplicates removed (n =203) records screened (n = 203) records excluded (n = 187) not relevant outcome (171) literature review (11) full-text articles assessed for eligibility (n = 16) full-text articles excluded, with reasons (n = 12) not reported the effect of black cumin on cancer (9) not use randomized controlled trial (3) studies included in qualitative synthesis (n = 4) http://www.opengrey.eu/ http://www.greylit.org/ habib et al. 3 table 1. assessment of study quality for selected studies authors randomization blinding outcome measured loss to follow up funding source conflict of interest behnamfar et al., 2019 adequate double blind adequate (6 times at 12 hours interval) none not provided none hussain et al., 2019 adequate open label both primary and secondary treatment: 7.4% not provided none mousa et al., 2017 adequate unclear incidence of febrile neutropenia and length of hospital stay none not provided none rafati et al., 2019 adequate single blind skin toxicity, worst experienced pain treatment: 6.5% control: 9.7% university grant none table 2. characteristics of studies included for systematic review examining effect of n. sativa on cancer authors country study design subjects measures dose intake main result comment ref behnamfar et al., 2019 iran randomized single blind control trial 60 patients having gastrointestinal cancers, 30 in treated group, 30 in control group n. sativa oil 5 drops of n. sativa oil applied 2 times a day there is no significant difference between the groups in terms of phlebitis incidence and severity result analyzed by chi-square test, fisher’s exact test and mann-whitney test 12 hussain et al., 2019 iraq randomized open-label controlled study 54 patients having acute myeloid leukemia, 27 in treated group, 27 in control group n. sativa oil 10 ml/6 hours oral mucositis assessment scale (omas) score was significantly lower in n. sativa oil group compared with control group result analyzed by chi-square test, fisher’s exact test, mann–whitney test, and kruskal–wallis test 13 mousa et al., 2017 egypt randomized pretest-post-test control group study 80 patients having primary brain tumors undergoing chemotherapy n. sativa seeds 5 g/day the incidence febrile neutropenia significantly decreased (p<0.001) in n. sativa seed group compared with control group result analyzed by two-way anova using bonferroni correction, montecarlo exact test (mcp) and fisher’s exact test (fep) 14 rafati et al., 2019 iran randomized double blind control trial 62 patients having breast cancer, 31 in intervention group, 31 in control group n. sativa gel 5% n. sativa gel applied twice daily the development of acute radiation dermatitis and moist desquamation were lower in n. sativa gel compared with those who used the placebo result analysed by the kaplan-meier method with the log-rank test, the generalized estimating equations (gee) method 15 habib et al. 4 oral mucositis was significantly lower in the n. sativa oil group compared with the control group. indeed, the incidence of erythematic and ulceration among the n. sativa oil community was lower. at the end of 12 days, pain severity among the n. sativa oil group dropped considerably compared with the control group. the secondary outcome of this study mentioned that salivary tnf-a was substantially lowered by the use of n. sativa oil mouth rinse on day 18 and day 28 compared to the control group but salivary il-6 was not considerably different in the n. sativa oil control samples compared to the corresponding group (p>0.05) [13]. mousa et al. in their study found that maximum children took n. sativa for 3-9 months (average of 6 months) and loss of body weight was less severe in the intervention group than the control group (p=0.048). the intervention group of children experienced less febrile neutropenia (2.2%) than the control group (19.3%). the percentage of febrile neutropenia among medulloblastoma (mb) and children with primitive neuroectodermal tumors were higher among the control group than the intervention group. in the case of hematological toxic effects, there have been statistically significant disparities in the occurrence of neutropenia (p=0.001) and febrile neutropenia (p=0.001) between both the intervention group and the control group, but the incidence of anemia, thrombocytopenia, and leucopenia was not significantly different in both groups [14]. rafati et al. established how acute radiation dermatitis might have been eliminated in breast cancer by applying n. sativa gel. the incidence of skin toxicity in the n. sativa gel group was significantly lower at weeks 3, 4, and 5 compared to the control group even though there were significant differences between the two groups in the size and severity of moist desquamation. the worst perception of pain among the control group was substantially higher than that of the n. sativa gel group (p<0.05). there was no dramatic difference in the skin-related quality of life between the two classes [15]. 4. discussion the present review showed the effects of black cumin on different types of cancer. anticancer activity of n. sativa was described in a study where certain tq (a chemical component of black cumin) had been used to manipulate phosphatase and tensin homolog (pten) expression and induce apoptosis in human breast cancer cells. in this study, it was found that tq significantly inhibits the proliferation of dox cells. it, therefore, triggered apoptosis and protein p53, and even some up-regulated pten by inhibiting the akt pathway [16]. another mouse study showed that tq induced cytotoxicity and apoptosis, and also inhibited tumor growth [17]. tq induces apoptosis and disrupts mitochondrial membrane potentials. it also activates the caspases 3, 8, and 9 in hl-60 cells to treat myeloblastic leukemia, thus showing its effect on myeloblastic leukemia [18]. tq indicates significant cytotoxicity for cancer of such bladder. it prevents the rapid multiplication of cancer cells, and by activating caspases induces apoptosis. tq's anticancer effect resulted in endoplasmic reticulum stress pathway activation and mitochondrial dysfunctions. it also boosted the bcl-2 anti-apoptotic protein and blocked cytochrome c entrance [19]. tq inhibited renal cell cancer (rcc) via cell migration, invasion, and epithelial-mesenchymal shift. this also increased the expression of e-cadherin and the level of hepatic kinase b1 phosphorylation was considerably upregulated following thymoquinone therapy [20, 21]. tq protects against the development of prostate cancer. it was found to inhibit the growth of du145 and pc3 cells which caused prostate cancer by treating with tq. tq dramatically inhibits the du145 and pc3 cell migration (p<0.05). the expression of epithelialmesenchymal transition (emt) markers in prostate cancer cells have also been repressed, and tgf-ß, smad2, and smad3 expression have also been minimized with tq [22]. the article has some strengths and limitations. using proper guidelines of the systematic review and quality assessment of the selected studies are the strengths of the articles. some limitations of the article are only used article published in english and had fulltext availability. n. sativa component potential anticancer activities have been identified thousands of years ago but appropriate scientific research with this essential traditional medicine is a very recent story. according to the limited evidence from the study, black cumin may have favorable effects on human cancer and tumor. it is worth emphasizing further research work behind this because it is a safe and effective anticancer agent. in particular, researchers should evaluate the active compounds more broadly, as very few authentic habib et al. 5 reports are available on the chemical composition of seeds or n. sativa exists. also, more importance should be placed to investigate the appropriate molecular pathways of tq and other components on different cancers as the current understandings are largely unclear. regarding the potential benefits of n. sativa on cancer, the anti-carcinogenic effects of n. sativa remained assessed through restricted clinical trials lacking a placebo community. for ongoing studies, it was recommended 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https://pubmed.ncbi.nlm.nih.gov/29039572/ https://pubmed.ncbi.nlm.nih.gov/29039572/ journal of current biomedical reports jcbior.com volume 2, number 1, 2021 eissn: 2717-1906 1 original research identification of bacterial isolates from nasolacrimal duct infection in children with congenital nasolacrimal duct obstruction from feiz teaching hospital, isfahan helma ebneali1, fereshteh saffari2, alireza zandi3, jamshid faghri1,* 1department of microbiology, school of medicine, isfahan university of medical sciences, isfahan, iran 2department of microbiology and virology, school of medicine, kerman university of medical sciences, kerman, iran 3department of ophthalmology, isfahan eye research center, isfahan university of medical sciences, isfahan, iran abstract dacryocystitis is a lacrimal sac and duct inflammation. it can be inherited or congenital. two primary forms are acquired dacryocystitis, acute and chronic. the goal of this study is to recognize common bacteria causing nasolacrimal duct infection in children with congenital nasolacrimal duct obstruction and to determine their antimicrobial susceptibility profiles. this cross-sectional research was conducted from january to february 2017 in the department of ophthalmology affiliated to isfahan university of medical sciences (center of iran). using phenotypic and genotypic approaches, identification of specimens was performed. disc diffusion method was used for checking antibiotic susceptibility. all of the 59 isolates from the culture of specimens belonged to grampositive cocci. staphylococcus epidermidis was the predominant species (n= 44, 74.6%) followed by staphylococcus aureus (n= 11, 18.6%), staphylococcus haemolyticus (n= 2, 3.4%) and each of staphylococcus saprophyticus, and streptococcus pneumoniae (n= 1, 1.7%). totally, the highest resistance was found against erythromycin and tetracycline while, chloramphenicol, and ciprofloxacin showed the highest susceptibility. the current research is useful in evaluating the suitable antibiotic in our area for the systemic treatment of dacryocystitis. the most effective agents against the most common isolates were chloramphenicol and ciprofloxacin. since the bacteriology of nasolacrimal duct infections varies from region to region, it is recommended that further studies in other areas of our country can be detected the etiology of bacterial pathogens involved in acute infections. keywords: dacryocystitis, antibiotic resistance, congenital nasolacrimal duct obstruction, bacteriology 1. introduction dacryocystitis is an inflammation of the lacrimal sac, usually followed by a blockage of the nasolacrimal duct [1]. dacryocystitis can be seen both in acute and chronic forms. acute type of dacryocystitis is an acute inflammation of the lacrimal sac, the most important clinical symptoms of which include discomfort, redness and swelling, and can be seen in 23 percent of cases of lacrimal abscess [2, 3]. the chronic form of * corresponding author: jamshid faghri, ph.d department of microbiology, school of medicine, isfahan university of medical sciences, isfahan, iran tel/fax: +98 31 37929038/+98 31 6688597 email: faghri@med.mui.ac.ir http://orcid.org/0000-0001-7500-168x received: october, 24, 2020 accepted: december, 17, 2020 dacryocystitis is more common than the acute form and is frequently associated with conjunctivitis [4]. approximately 60-90% of all cases of lacrimal sac infection are related to bacterial dacryocystitis [5]. the microbial spectrum of the dacryocyst depends on its acute or chronic form. in most cases, gram-positive bacteria are separated from acute dacryocystitis, while in the chronic form, gram-negative bacteria are predominant. the most important species isolated https://jcbior.com/ http://orcid.org/0000-0001-7500-168x ebneali et al. 2 from children include staphylococcus aureus, staphylococcus haemolyticus, streptococcus pneumoniae, and haemophilus influenzae, while staphylococcus epidermidis, s. aureus, s. pneumoniae, and pseudomonas aeruginosa are the most causative agents in adults [6]. oral antibiotics, anti-inflammatory drugs and local hot compresses were used in acute dacryocystitis therapy methods, whereas definitive treatment of chronic dacryocystitis is done with dacryocystorhinostomy [7]. the study of the status of drug resistance in dacryocystitis is important in two aspects: first, according to studies, approximately one-third of bacteria isolated are resistant to antibiotics, and on the other hand, because in most cases, the treatment of patients is empirically and without culture data if treatment failure results in consequences such as cellulitis, meningitis, abscess, and even lifethreatening conditions. in addition, the pattern of drug resistance varies in different regions [8]. early identification of microbial agents and awareness of the drug susceptibility pattern is important for effective treatment. the choice of antibiotic therapy for dacryocystitis usually depends on the age of the patient, the condition of the patient (acute or chronic), the type of microorganism and drug present, and the drug resistance pattern. therefore, epidemiologic studies are necessary for identifying and managing cases of bacterial dacryocystitis [5, 6]. since the range of bacteriology may differ by geographical region, and very few studies have been recorded from central iran on lacrimal sac bacteriology, the present study was aimed at identifying bacteria involved in acute dacryocystitis in a given population and investigating the trend of antibiotic susceptibility in the province of isfahan. 2. materials and methods 2.1 study design, period, and area a cross-sectional study was conducted from january to february 2017 among dacryocystitis diagnosed patients attending at ophthalmology outpatient of feiz teaching hospital, center of iran. 2.2 inclusion/exclusion criteria all children aged 6 years with a history of dacryocystitis were referred to the selected clinic of ophthalmology for sampling and 59 dacryocystitis cases were eligible for microbiological analysis. also, children with severe lid irritation due to persistent discharge were included in this study. however, people over 6 years old, the patients with the above symptoms who had received either topical or systemic antibiotics for the past week and all cases of canalicular obstruction were excluded from the study. 2.3 sampling after aseptically cleaning the surrounding area, specimens for microbiological analysis were obtained by sterile dacron swabs from the lacrimal sac, by applying pressure over the lacrimal sac and allowing the purulent material to reflux through the lacrimal punctum. the specimens were collected with the help of an ophthalmologist and sent for microbiological analysis. 2.4 microbiological analysis and bacterial identification specimens were inoculated on bhi broth, chocolate agar, and blood agar (oxiod, hampshire, uk). then, the inoculated media were incubated at 3537 ºc for 24 to 48 hours. in addition, in the presence of 5-10 % carbon dioxide, chocolate plates were incubated at 37 ºc for 24 to 48 hours. identification of the isolates was performed using different biochemical as well as standard microbiological tests. to identify gram-positive isolates standard biochemical and microbiological tests were included in order (catalase, coagulase, pyr, optochin susceptibility, etc.). the oxidase, novobiocin, and bacitracin tests were used for the identification of coagulase-negative staphylococci. novobiocin disc is used to differentiate staphylococcus saprophyticus from other coagulasenegative staphylococci. 2.5 antibiotic susceptibility pattern antibiotic susceptibility pattern was performed by the disc diffusion method based on the clinical and laboratory standards institute (clsi) recommendation [9]. the following antibiotic disks were used; ampicillin (10 μg), chloramphenicol (30 μg), gentamicin (10 μg), tetracycline (30 μg), amoxicillin (20 μg), ciprofloxacin (30 μg), ceftriaxone (30 μg), erythromycin (15 μg). also, escherichia coli (atcc 25922) and s. aureus (atcc 25923) were used as control strains. 2.6 dna extraction and polymerase chain reaction (pcr) ebneali et al. 3 the dna template was prepared as per the method of dilhari et al. with slight alteration [10]. 200 μl of phosphorus buffer saline (pbs) was taken in microcentrifuge tubes and a loopful of each isolate was mixed with the nuclease-free water thoroughly in each microcentrifuge tube. the suspended isolates in microcentrifuge tubes were then treated with boiling water for 15 minutes. after heat treatment, centrifuge tubes were centrifuged at 10,000 rpm for 10 minutes. 100 μl of the supernatant containing genomic dna transfer in a new tube and it was used for subsequent pcr amplification. in our study, isolates were screened for the presence of the 16s rrna, fema, and se705 genes. the sequences of primers as shown in table 1. the pcr reactions were conducted in a total volume of 25 μl containing the following: 12.5 μl of master mix (ampliqon, denmark), 11 μl distilled water, 1 μl template dna and 0.5μl primers. pcr assay was performed in a dna thermal cycler 480 (applied biosystems, usa). for amplification fema, and se705 genes, the pcr program was set at: denaturation for at 5 minute 94 °c, 29 cycles of 94 °c for 30 seconds, 53 °c for 30 seconds, 72 °c for 30 seconds and a final extension step of 72 °c for 5 minutes. amplification of 16s rrna according to the following program: initial activation of 94 °c for 5 min, 35 cycles of 94 °c for1 min, 56 °c for1 min and 72 °c for 1 min and a final extension of 72 °c/7 min. each amplification reaction included a negative control (nodna template control). five microliters of the amplified dna products were run on 1% agarose gel with 1 x tae (tris/acetate/edta) buffer, stained with safe stain load dye (cinnagen co., iran) and visualized under ultraviolet illumination. 2.7 data analysis the data were entered and analyzed using spss version 16 statistical software program. a p-value <0.05 was considered as a significant association between the variables which were tested. 3. results 3.1 patients’ demography in the present study, 59 clinically diagnosed patients of dacryocystitis in children and both sexes were studied over a period of two months. the average age of the patients was studied 3 years (range <1–6 years), with a males’ predominance 57.6% (n=34) compared to female 42.4% (n=25) with male and female ratio 34:25. although, a high frequency of dacryocystitis was reported in males than females, a significant difference between males and females (p= 0.36) was not observed. the most common age groups were the group under 1-2 years (33.9%) and the lowest age group was 5 years (5.1%). 3.2 bacterial isolates from 59 positive samples, five different species were isolated. as shown in table 2, s. epidermidis was the most commonly isolated organism followed by s. aureus, then s. hemolyticous. s. saprophyticus, and s. pneumoniae only detected one isolate respectively. 3.3 antimicrobial susceptibility test the most effective antibiotics against all organisms were chloramphenicol, and ciprofloxacin, each showing affectivity of 100% (table 3), followed by cefoxitin and gentamycin with 61% and 49.7% while the highest rate of resistance seen was to erythromycin, amikacin, and tetracycline, with at least one resistant organism present in 40 of 59 (67.8%) cultures and 30 of 59 (50.8%), and 30 of 59 (50.8%), respectively. 3.4 sequencing staphylococcus hominis strain efs 16s ribosomal rna gene, partial sequence gene with accession number “mg786536.1” were added in the database. discussion this prospective study of 59 patients was done to determine the type of bacteria involved in patients with acute dacryocystitis. the spectrum and incidence of pathogens, as well as our study, were so obvious that we could isolate five different species of bacteria. microbial ocular surface flora consists predominantly of gram-positive microorganisms, namely staphylococci and diphteroids [12]. the occurrence and severity of dacryocystitis depend on various factors, such as the geography of the area and the type of microbial agent [10]. badhu et al. (2006) revealed that in nepal the most common microorganism was s. pneumoniae, while in some countries like saudi arabia, china, ebneali et al. 4 austria, and australia, s. epidermidis and s. aureus are the most frequent species isolated from dacrysistis [13-17]. in the current study, the most common organism isolated was s. epidermidis and s. aureus (74.6% and 18.6%, respectively) this result is comparable with other studies [1, 6, 17, 18]. however, this finding was much lower than the prevalence of gram-positive bacteria in dacryocystitis recorded in saudi arabia (79.1 %), finland (62 %), usa (68.8 %), australia (54.4%), and in a previous study in iran (68.8 %) [1923]. in general, the most significant factors for differentiating the diversity of bacteria in different studies include variations in the number of patients, differences in the social status of patients, access to ophthalmology, and public health knowledge among individuals [6]. operational intervention is the main treatment of nasolacrimal duct obstruction. despite surgical procedures, the risk of infection of soft tissue increases fivefold without the use of antibiotics, suggesting the importance of antibiotics in the treatment of dacryocystitis [23]. on the other hand, resistance against antibiotics is a problem that affects the treatment of dacryocystitis. in recent decades, drug resistance has spread exponentially, which may be due to overuse and abuse of these medications [24]. in our study, gram-positive organisms exhibited a high rate of sensitivity to chloramphenicol, vancomycin, and ciprofloxacin. this is in correlation with the studies of kuchar et al. (2000), kebede et al. (2010), chung et al. (2019) also have documented effectiveness of chloramphenicol, and ciprofloxacin against grampositive bacteria [8, 23, 25, 26]. while s. epidermidis and s. aureus displayed the highest susceptibility to chloramphenicol and vancomycin, it should be noted that between 32% and 39% of antibiotic resistance to these two drugs has been recorded. therefore the determination of bacterial species and the determination of drug sensitivity in patients with dacryocystitis appear to be significant [6]. in a study by assefa et al. (2015) in northwest ethiopia, they found that the most susceptible antibiotics were nalidixic acid (87.1%), erythromycin (84.2%), ceftriaxone (95.3%), and gentamicin (83.3%) [18]. briscoe et al. (2005) showed obtained bacteria have more sensitivity to ceftazidime (95%), table 2. frequency of isolated microorganisms in patients with dacryocystitis isolate type frequency (%) (n=59) staphylococcus epidermidis 44 (74.6) staphylococcus aureus 11 (18.6) staphylococcus haemolyticus 2 (3.4) staphylococcus saprophyticus 1 (1.7) streptococcus pneumoniae 1 (1.7) table 3. distribution of antibiotic resistance of isolated bacteria in dacryocystitis antibiotics susceptible intermediate -resistant resistant chloramphenicol 59 (100) 0 0 ciprofloxacin 59 (100) 0 0 amikacin 29 (49.2) 0 30 (50.8) cefoxitin 36 (61) 0 23 (39) gentamycin 24 (40.7) 17 (28.8) 18 (30.5) tetracycline 29 (49.2) 0 30 (50.8) erythromycin 19 (32.2) 0 40 (67.8) table 1. sequences of primers used for the pcr primer sequence (5'-3') amplicon size (bp) annealing (◦c) references 16s rrna agagtttgatcctggctcag 1500 57 (30) tacggytaccttgttacgactt 55 se705 atcaaaaagttggcgaaccttttca 125 60.6 (31) caaaagagcgtggagaaaagtatca 59.8 fema cgatccatatttaccatatca 451 50 (32) atcacgctcttcgtttagtt 55 ebneali et al. 5 ciprofloxacin (86%), and cefuroxime (50%), respectively [27]. in the study by shah et al. (2011) norfloxacin was the most effective antibiotic while penicillin showed the most resistant antibiotic [28]. the disparity between studies shows that because of the regional pathogens, there are obvious differences in the pattern of antibiotic resistance in the geographic areas [29]. the small sample size, and short period, were the limitations of this research. a larger research group with a longer study duration may provide a better result. based on the results obtained, we have offered to direct alternatives for the selection of effective antibiotics for clinicians who take care of cases controlled for acute dacryocystitis diagnosis. the most frequent bacteria isolated from acute dacryocystitis were s. epidermidis and s. aureus in our area. the most effective antibiotics against all isolated microorganisms from acute dacryocystitis include ciprofloxacin and chloramphenicol. these 'regional' results have significant public health consequences in this area of iran for the treatment and prevention of dacryocystitis. acknowledgment we are grateful to the study participants and ophthalmology department and hospital laboratory staffs of feiz hospital for their help during data collection and sample processing. author contributions he, and jf, study designed. az, and he, carried out the experiments. he, wrote the manuscript. fs, analyzed the results. jf, supervised the project. he, fs, and, az, edited manuscript, submitted to the journal, and response to reviewers. all authors read and approved the final manuscript. conflict of interest none declared. ethical declarations this study has the formal approval of the research ethics committee of isfahan university of medical sciences, isfahan, iran (approval number: ir.mui.rec.1396.3.203). financial support this work was supported by the isfahan university of medical sciences, isfahan, iran (thesis code: 396203). references 1. bharathi mj, ramakrishnan r, maneksha v, shivakumar c, nithya v, mittal s. comparative bacteriology of acute and chronic dacryocystitis. eye. 2008; 22(7):953-60. 2. ali mj, joshi sd, naik mn, honavar sg. clinical profile and management outcome of acute dacryocystitis: two decades of experience in a tertiary eye care center. semin ophthalmol. 2015; 30(2):118-23. 3. ali mj, motukupally sr, joshi sd, naik mn. the microbiological profile of lacrimal abscess: two decades of experience from a tertiary eye care center. j ophthalmic inflamm infect. 2013; 3(1):57. 4. huber e, steinkogler fj, huber-spitzy v. a new antibiotic in the treatment of dacryocystitis. orbit. 1991; 10(1):33-5. 5. chen l, fu t, gu h, jie y, sun z, jiang d, et al. trends in dacryocystitis in china: a strobe-compliant article. medicine. 2018; 97(26):e11318. 6. eslami f, ghasemi basir hr, moradi a, heidari farah s. microbiological study of dacryocystitis in northwest of iran. clin ophthalmol. 2018; 12:1859-64. 7. negm s, aboelnour a, saleh t, yasser m, hassanin o. clinicobacteriological study of chronic dacryocystitis in egypt. bull natl res cent. 2019; 43(1):35. 8. chung sy, rafailov l, turbin re, langer pd. the microbiologic profile of dacryocystitis. orbit. 2019; 38(1):72-8. 9. clinical and laboratory standards institute (clsi). performance standards for antimicrobial susceptibility testing; 27th ed. clsi supplement m100. wayne, pa: clinical and laboratory standards institute; 2017. 10. dilhari a, sampath a, gunasekara c, fernando n, weerasekara d, sissons c, et al. evaluation of the impact of six different dna extraction methods for the representation of the microbial community associated with human chronic wound infections using a gel-based dna profiling method. amb express. 2017; 7(1):179. 11. brook i. ocular infections due to anaerobic bacteria in children. j pediatr ophthalmol strabismus. 2008; 45(2):78-84. 12. chaudhry ia, shamsi fa, al-rashed w. bacteriology of chronic dacryocystitis in a tertiary eye care center. ophthalmic plast reconstr surg. 2005; 21(3):207-10. 13. badhu bp, karki bs, khanal b, dulal s, das h. microbiological patterns of chronic dacryocystitis. ophthalmology. 2006; 113(12):2377.e1-2. 14. sun x, liang q, luo s, wang z, li r, jin x. microbiological analysis of chronic dacryocystitis. ophthalmic physiol opt. 2005; 25(3):261-3. 15. huber-spitzy v, steinkogler fj, haselberger c. 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https://pubmed.ncbi.nlm.nih.gov/15583325/ https://pubmed.ncbi.nlm.nih.gov/15583325/ https://pubmed.ncbi.nlm.nih.gov/19476638/ https://pubmed.ncbi.nlm.nih.gov/19476638/ https://pubmed.ncbi.nlm.nih.gov/19476638/ https://pubmed.ncbi.nlm.nih.gov/19476638/ journal of current biomedical reports jcbior.com volume 2, number 2, 2021 eissn: 2717-1906 1 brief report decrease of microbial contamination by application of routinely glycerol and phenol solution on human cadavers masoumeh faghani langroudi1, ali farzanegan2,* 1department of anatomical sciences, school of medicine, guilan university of medical science, guilan, iran 2department of microbiology, guilan university of medical science, anzali international campus, guilan, iran abstract human cadaver is imperative learning material for medical students. the cadavers expose to microbial contamination every time anatomists work with them. the microorganisms found in preparations’ cadavers could be pathogenic. although the cadaver has supported a preservation method but does not inhibit microbial germination. the purposes of this study were evaluation the use of the routine solution to decrease potential contamination. samples were collected air dissection hall and cadavers' sections after drying the cadavers, and after use of routinely glycerin and phenol solution (1.5 g phenol in 100 cc glycerol), inoculated on eosin methylene blue agar (emb), blood agar (ba), sabouraud dextrose agar (sda), and sda with the addition of chloramphenicol (50 mg/l in ethanol solution) (sc), in a petri dish and incubated at 25 °c and 37 °c. after 35 days, the morphology was studied macroscopically and microscopically. we found sporothrix schenckii; streptomyces, bacillus subtilis and aspergillus flavus, geotrichum candidum, on the samples of dryness cadavers and air dissection hall, respectively. but fungus and bacteria contamination was decreased after the use of glycerin and phenol solution. based on the results of this study, it is suggested that glycerol and phenol solution can be used at least once a week to reduce microbial contamination and prevent the spread of the disease from the cadavers to students and professors. keywords: cadaver, microbial contamination, glycerol, phenol 1. introduction cadavers are the main studying materials of anatomists. cadavers are a teaching tool for teachers and students but may infect persons who work with them. all of those are potentially in danger of exposure to pathogenic microorganisms like fungi, bacteria, and viruses. the infectious agent in the cadavers includes mycobacterium tuberculosis, hepatitis b and c viruses, human immunodeficiency virus, aspergillus, candida, penicillium, etc. specific care protections are necessary to avoid accidental disease transmission from cadavers before and during dissection [1]. therefore, the human cadavers in medical universities usually were protected for a very long time in a fixative solution, which contains formalin, alcohol, phenol, water, and glycerin. many of the laboratories in the departments of human anatomy ignore the damaging effect of air dryness on * corresponding author: ali farzanegan, msc thirty metery blvd. anzali, guilan, iran. zip code: 4314637758 tel/fax: +98 911 982 9233 email: alifarzanegan1349@gmail.com https://orcid.org/0000-0002-2425-8634 received: february, 04, 2021 accepted:, february, 28, 2021 the cadaveric sections [2]. airborne fungi are the most important microorganism in the air, which causes contamination problems in the environment. generally, saprophytic’ fungi in many ecosystems can cause respiratory system infections, especially in immunosuppressed people [3]. drying and hardening cadaveric tissue and fungi contamination is one of the problems predictable that to occur due to a long time of exposure cadaver to air drying, but this problem probably resolves with processed cadaveric with routinely use glycerin and phenol solution. due to the activity of bacteria and fungi on cadavers that are drying, probably many cases of destruction will be seen in cadavers. in addition to economic damage and trouble in teaching, this subject can be cases of contamination in the dissection hall and tools, and subsequently infection the staff, students, and professors [4]. © the author(s) 2021 https://jcbior.com/ faghani langroudi et al. 2 this study aimed to evaluate the use of routinely phenol and glycerin solution to decrease the fungal contamination in the university dissection hall. 2. materials and methods 2.1 study design the cadavers used for this study were obtained from the department of human anatomy, in the dissection hall of guilan university of medical sciences, anzali international campus. the dissection hall was 2.7 meters in height, 10.8 meters in length, and 10 meters in width. the room has a single door and five windows with 1.4 by 1.5 meters. the floor and the walls had been tile up to the ceiling. the room has two autopsy tables made of stainless steel. the air conditioning dissection hall is administered by a simple fan system on the south wall. the dissection hall has three radiator heating systems and they were on at the time of the experiment (figure 1). 2.2 sample collection all samples were taken from different surfaces of cadavers by the sterile cotton swap, after drying the cadavers, and one week after the use of routinely phenol and glycerin solution (1.5 g phenol in 100 cc glycerol). in addition, petri dishes with a diameter of 8 cm were put at dissection hall for taken airborne microorganisms for 24 h [5, 6]. 2.3 culture after collection, each sample was used to inoculate four different media: eosin methylene blue agar (emb) to suppress gram-positive bacterial growth, blood agar (ba), sabouraud dextrose agar (sda), sda with the addition of chloramphenicol (50mg/l in ethanol solution) (sc) to suppress bacterial growth. agar samples were incubated at 25 °c and 37 °c and colony growth was checked daily. 2.4 samples staining all fungi isolate were stained by lactophenol cotton blue and bacterial isolate were stained by gram stain and acid-fast stain. all samples were identified by comparing the microscopic characteristics of fungi and bacterial standard produedures. table 1. the culture results of samples from the surface of the cadavers and the air-dissection hall sample source fungi growth in sc bacterial growth in ba sample of head sample of neck sample of hand sample of foot sample of visceral sample of head sample of neck sample of hand sample of foot sample of visceral cadaver 1 no growth no growth no growth sporothrix schenckii no growth no growth no growth no growth no growth no growth cadaver 2 no growth no growth sporothrix schenckii sporothrix schenckii no growth no growth no growth no growth bacillus subtilis streptomyces spp. fungi growth in sc and sda south windows west windows eastern door north wall floor airdissection hall aspergillus flavus sporothrix schenckii geotrichum candidum geotrichum candidum aspergillus flavus emb: eosin methylene blue; ba: blood agar; sda: sabouraud dextrose agar; sc: sabouraud dextrose agar with the addition of chloramphenicol (50 mg/l in ethanol solution) figure 1. the plan of the autopsy room in the dissection hall of the university faghani langroudi et al. 3 3. results all the samples were culture in the department of microbiology in anzali international campus. all of the sample results from the air-dissected hall were positive for fungal growth, also five samples from different surfaces of the preserved cadaver were positive for fungal and bacterial growth (table 1). two different bacterial colonies were observed after 72 h on ba and three different fungi colonies were observed after 3-5 days on sda and sc media (table 2). all samples culture from the cadaver surface took after using glycerin and phenol solution were negative. also, the samples culture from the dissection hall was taken after cleaning were still positive, but they were reduced. table 2. colonial and microscopic morphology and biochemical identification of organisms organism colonial morphology and characteristics biochemical identification microscopic characteristics ba emb sc and sda bacillus subtilis brown color, mucoid surface, wrinkled shaped, hemolytic effect inhibition catalase (+), indole reaction (-), lactose (-) gram-positive, rod shape streptomyces spp. red-pink color, mucoid surface, soil smell inhibition indole reaction (-), sh2 (-), motility (+), gas (-) , citrate (+), catalase (+) acid from lactose(-), acid from glucose (+), urea hydrolysis(+) gram-variable, motile, filamentous, non acidfast aspergillus flavus granular, flat, with radial grooves, white at first but quickly becoming yellow to dark yellow-green with age typically radiate, loose columns, biseriate or uniseriate, directly on the vesicle (uniseriate). conidiophores are hyaline and roughened, often more visible near the vesicle. conidia are spherical, pale green, and echinulate geotrichum candidum fast-growing, flat, white to cream, dry, suede-like with no reverse pigment hyaline hyphae with septate branched and break up into chains of hyaline, smooth, one-celled, and cylindrical arthroconidia sporothrix schenckii slow-growing, compact and suedelike or floccose, dry, flat, brownish-black, grey color, dark-brown diffusible pigment hyaline and produce mostly simple awl-shaped erect phialides with inconspicuous collarettes. conidia are hyaline or pigmented, spherical to cylindrical, one-celled, and typically aggregated in heads at the apex of each phialide. chlamydospores may be present. emb: eosin methylene blue; ba: blood agar; sda: sabouraud dextrose agar, sc: sabouraud dextrose agar with the addition of chloramphenicol (50 mg/l in ethanol solution) faghani langroudi et al. 4 4. discussion the human cadavers in medical colleges usually were preserved in a fixative solution, which contains formalin, alcohol, phenol, water, and glycerin but after staying in the dissection hall for a long time, they will dry out. in this research study, not only the surfaces of the cadavers were examined for the presence of viable microorganisms but also the airdissection hall was examined for dangerous infectious agents. three different fungal colonies identified as sporothrix spp. (from the surface of the cadavers), aspergillus spp. and geotrichum (from the air-dissection hall) and two bacteria colonies identified as streptomyces and bacillus (from the surface of the cadavers), the source of contamination. those are microorganisms that transmitted by air and cause opportunistic disease [7-11]. this is a matter of concern because students and anatomists may be exposed to the infectious disease every time they work with a cadaver [1]. normally, 10% formalin or 4% formaldehyde in a water solution is used to prevent the contamination of microorganisms. furthermore, formalin-fixed human cadavers are routinely used for study and research in hospitals and universities. airborne fungi have become a serious problem in dissection hall. therefore, the presence of airborne fungi on embalmed human cadavers could be perilous for teachers and students and necessary measures must be taken to reduce fungi contamination. one report has suggested that formalin concentration for inhibiting fungal growth should not be less than 4% (5‒7.5 percentage) [12]. previously, it was showed that 15% formalin was very effective in the prevention of growth of bacteria, fungi, and also decay and discoloration [3]. the main factors leading to fungal contamination in the dissection hall are room humidity, the concentration of airborne fungal spores, and the duration of time to fungal inoculation. all instructors use routinely glycerin and phenol solution in the dissection hall to prevent drying and decrease microbial contamination of human cadavers after work with cadavers. glycerol (glycerin) is a simple polyol complex that uses in the dissection hall. it is colorless, odorless, sweet-tasting, and non-toxic. multiple journals describe the antimicrobial and antiviral effects of glycerol [13], as well as, glycerol catalyzes spore germination at about 64–70% humidity for microbial life. the germination of fungal xerophiles can occur at 60% moisture but for the most dangerous fungal xerophiles, metabolic activity and cell division typically stop between 64–70% humidity. these findings describe an inconsistency. on the one hand, glycerol can be particularly stressful and prevent cellular development. on the other hand, may be essential for cells (microbiota) to function at the water-activity limit for life, and used routinely in cadaver dissection hall [14]. phenol derivatives isolated from the plants showed good antifungal activity that uses in the dissection room for the elimination of microbiota contaminations [15]. phenol, or carbolic acid, is a colorless or white crystalline solid with a partly low melting point. it has been shown that phenol becomes bactericidal/fungicidal at concentrations of 1.0–1.5% and destroys cell walls. phenol is an excellent fungicide and bactericide but it denatures proteins, with resultant drying and discoloration of tissues, and has a nasty odor. phenol is harmful to the esophagus, causing vomiting, and headache, and faintness, loss of blood pressure, pulmonary edema, and cyanosis [16]. despite the use of phenol for decreased microbiota contamination, and the use of glycerin to decrease resulting phenol's dryness, we cannot prevent the spread of bacteria and fungi in the indoor air and cadavers' contaminations, even by closing the windows and routinely use of glycerin and phenol solution and installing a proper ventilation system, but only we can decrease contaminations. on the other hand, the phenol has side effects, nevertheless is still used in universities to disinfect cadavers. the use thymol for moistening the cadavers at the end of every dissection course, at least once a week. thymol (2-isopropyl-5-methyl phenol) is naturally occurring and found in thyme oil. a thymol-ethanol solution as a humidifying solution is keeping at room temperature. thymol has no carcinogenic or other serious effects on health but it has bactericidal and fungicidal effects and it is better than phenol [16, 17]. suitable preservation is critical to the effective use of cadavers in educational settings. everybody, those works in the anatomy department must ensure the safety of the cadaver from harm, decay, or disintegration [18]. in the present study, three different types of fungus (sporothrix, aspergillus, and geotrichum) and two bacteria (bacillus and streptomyces) were identified. the potential infectious hazard from human cadavers is one of the main hazards associated with the anatomy department. safe working conditions for handling cadavers can be provided through proper education, use of protective clothing, and practice of hygienic measures. embalming decreases the risk of transmission of faghani langroudi et al. 5 potentially infectious microbial agents, but it is not possible to prevent the cadavers from drying out in the cold season due to the heating system are work. the use of glycerin for moistening and prevent drying of the cadavers and use of thymol (instead of phenol), because it is not toxic and side effects, externally at the end of every dissection course or at least once a week, is necessary and recommended. acknowledgments this work was supported by the department of anatomy and examined at the microbiology laboratory, anzali international campus, guilan university of medical science (gums). finally, we also thank the dean of anzali international university, dr. seyed mahdi zia zibari for his generosity in this study. author contributions all authors contributed equally to this manuscript and approved the final version of the manuscript. conflict of interests the authors declare that they have no conflicts of interest. ethical 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https://pubmed.ncbi.nlm.nih.gov/24438435/ https://pubmed.ncbi.nlm.nih.gov/22434588/ https://pubmed.ncbi.nlm.nih.gov/22434588/ https://pubmed.ncbi.nlm.nih.gov/22434588/ https://pubmed.ncbi.nlm.nih.gov/22434588/ https://pubmed.ncbi.nlm.nih.gov/2760229/ https://pubmed.ncbi.nlm.nih.gov/2760229/ https://pubmed.ncbi.nlm.nih.gov/2760229/ journal of current biomedical reports jcbior.com volume 1, number 2, 2020 eissn: 2717-1906 1 review coronavirus disease 2019 (covid-19): a new pandemic and its challenges davood mansury1, sharareh moghim1,* 1department of microbiology, school of medicine, isfahan university of medical sciences, isfahan, iran abstract emerging of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has caused a worldwide outbreak and a major public health problem. the present review was conducted to provide brief information about the origin, symptoms, transmission, pathogenesis, diagnosis, and treatment of the virus. a search was performed in the databases of pubmed, scopus, science direct, and google scholar with english keywords including 2019-ncov, coronavirus disease 2019 (covid-19), sars-cov-2, and novel coronavirus from december 2019 to 15 march 2020, and the search results were evaluated. selected studies have shown that the virus may have originated from the bat. it has also been shown that the virus receptor is angiotensin-converting enzyme 2 (ace2) which is also the sars virus receptor and is expressed in most human tissues. the most common way of virus transmission was suggested through respiratory droplets and close contact. it is also transmitted by asymptomatic patients, but vertical transmission from mother to fetus has not been confirmed. real-time reverse transcriptase (rt)-pcr is the gold standard for sars-cov-2 detection, but chest computed tomography (ct) can be more sensitive to detect positive cases. since no effective vaccine or drug for prevention and treatment of this disease has not yet been identified and also because of the high incubation and infection period, easy transmission, and the lack of complete recognition of the characteristics and stability in different environments, the best way to control of covid-19 is to prevent the spread of the infection in different ways and take seriously personal and public hygiene. keywords: coronavirus, covid-19, sars-cov-2, emerging viruses, pathogenesis 1. introduction coronaviruses are one of the most important pathogens in the human respiratory system. severe respiratory syndrome (sars) and middle east respiratory syndrome (mers) have previously been identified as risk factors for general health [1]. in late december 2019, a report was published on pneumonia in a group of patients with unknown cause, possibly related to contact with the seafood market in wuhan, china [2]. in january 2020, the world health organization (who) temporarily * corresponding author: dr. sharareh moghim, ph.d department of microbiology, school of medicine, isfahan university of medical sciences, isfahan, iran tel/fax: +98 31 37929038 email: moghim@med.mui.ac.ir http://orcid.org/0000-0002-7134-3145 received: september, 30, 2020 accepted: november, 11, 2020 named this virus the 2019 novel coronavirus (2019ncov). later, in february 2020, they officially named it severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [3, 4]. the coronaviruses are a class of coated single-stranded rna viruses that are genetically and serologically subdivided into four subfamilies, including alpha, beta, gamma, and delta. in this regard, most human infections are caused by alpha and beta subfamilies. phylogenetic analyses show that the etiology of coronavirus disease 2019 (covid-19), like sars and mers, belongs to the https://jcbior.com/ mansury et al. 2 beta-coronavirus and is now known as sars-cov2 due to its high genetic similarity to sars [1, 5, 6]. reports suggest that the virus has spread to most countries in the world (table 1) [7-9], and the world health organization (who) considers it a pandemic [10]. so far, no evidence of mutation has been reported for sars-cov-2, probably due to the compatibility of the virus with humans, which binds to its receptor angiotensin-converting enzyme 2 (ace2) with very high affinity [11]. it is more likely that the sars virus also mutated during the 2002-2004 epidemics to better bind and replicate to its cellular receptor and increased its pathogenicity [12]. in this regard, it is necessary to investigate clinically isolated viruses with more time and geographical diversity to develop and use appropriate prevention and treatment methods with full knowledge of the characteristics of the virus. the present study was conducted to provide an overview of the origin, symptoms, transmission, pathogenesis, diagnosis, and treatment of this virus. 2. source of virus although the source of covid-19 has not yet been fully clarified, epidemiological studies and pathogen analysis suggest that the southern chinese seafood market in wuhan may be an important source of infection. although the evidenced source of this virus is wild animals, it has not been clarified which animal became infected with the virus and transmitted it to humans [4]. other studies have suggested that covid-19 may have originated from a coronavirus with bat origin. evidence suggests a high degree of ace2 receptor homology of diversity in animal species (except mice and rats). thus, these animal species may be considered as intermediate hosts or animal models for covid-19 infection. also, these viruses have an open reading frame (orf) and intact on gene 8, which is another indicator for coronaviruses with bat origin [1, 13]. also, the genomic analysis of viruses isolated from patients has a similarity of 96.2% to coronaviruses isolated from bats [14]. nevertheless, the source of the infection has not been completely recognized and more studies are needed in the future in this regard. 3. ways of virus transmission the main way for transmission of the virus is the transmission of respiratory droplets and close contact with patients suffering from coronary pneumonia. studies suggest that the pathogen can also be isolated from patients’ feces and urine. in infected people, respiratory droplets contain a large number of infectious agents that enter the environment through coughing and sneezing. the spread of respiratory droplets is unlikely without the help of environmental conditions [15]. rather, the transmission of this virus is more likely to occur in crowded and poorly ventilated public places such as conference rooms, restaurants, stations, public transportation, elevators, and temporary shelters. there are usually two ways for its transmission through content, including direct contact and indirect contact. in the indirect contact, pathogens spread by the source of covid-19 virus can easily infect the environment and surrounding devices, thereby infecting susceptible individuals [4, 14]. unlike the sars virus, the covid-19 can be transmitted during the incubation period [16]. analysis of 22 studies suggests that human viruses table 1. epidemiological comparison of covid-19 with mers and sars (15 march 2020) characterization covid-19 sars mers onset of infection wuhan, china guangdong, china saudi arabia year of the outbreak 2019now 2002-2003 2012 primary host not clear, probably bats probably cats and bats probably camel human-to-human transmission yes yes yes hospital transfer yes yes yes number of countries involved 216 29 27 number of cases * 50,586,712 8437 2494 mortality rate ~2.5% 11% 34.4% * retrieved 8 november 2020 mansury et al. 3 such as sars, mers, and human coronavirus (hcov) can remain active on non-living surfaces such as metal, glass, or plastic for up to 9 days [17]. also, a study reported the probability of transmitting the infection through the hospital at 41% from suspected patients [18]. a small number of patients with covid-19 have reported gastrointestinal symptoms. also, ace2 protein is highly expressed in glandular cells in the stomach, duodenum, and rectal epithelium, allowing sars-cov-2 to enter cells [19]. researchers also detected sars-cov-2 rna from patients’ feces in guangdong, shenzhen, and the united states. according to these researchers, the duration of rna detection in feces is 1 to 12 days [20]. most importantly, while some sars-cov-2 infected patients experience gastrointestinal symptoms such as diarrhea, it has also been revealed that virus can be isolated from patients’ urine, testing stool and urine samples. in this way, it is possible to remove a potential alternative route of transmission, especially through health care staff, patients, and so on. thus, it is essential to develop some methods to identify different modes of transmission such as stool and urine samples to achieve solutions to inhibit or minimize transmission and develop methods to control the disease [1]. however, isolation of pathogens from feces is not necessarily transmission through the fecal-oral route, but may also be positive through close contact or respiratory infections. there is also no strong evidence for the survival time of sars-cov-2 in different environments [4]. however, the use of surface disinfectants such as ethanol, 0.5% hydrogen peroxide, or 0.1% sodium hypochlorite for 1 minute can effectively inactivate the virus and play a significant role in controlling the infection [17]. 4. virus receptors in the human body analysis of recent studies shows that covid 19 binds to the same sars receptor as the angiotensinconverting enzyme (ace2) [13, 21-23]. it has been also shown that the surface protein of covid 19 (s protein) has 10 times more affinity than the sars virus for binding to the ace2 receptor. this result might explain the faster transmission of covid 19 in humans than the sars virus [23]. expression and distribution of ace2 in the human body may indicate possible routes of infection of covid-19, such as high expression of ace2 in cells of various tissues like lung, gastrointestinal tract, heart, kidney, and nasal and oral mucosa [24, 25]. in addition to identifying ace2 as a receptor, researchers have found downstream ace 2 as an important target for the type ii transmembrane serine proteases (tmprss2), which could be considered as a drug design site [11]. in covid-19, six mutation sites have been found that can affect the characteristics of the virus transmission and the tendency of the virus to bind to cells of various organs. in addition to ace2, other receptors may be involved in binding the virus to host cells [11]. 5. pathogenicity the mechanism of pathogenicity of covid-19 is generally unclear and may be affected by various organs. covid-19 enters cells primarily by binding to human ace2. unlike other beta-coronaviruses, primary viral replication is not limited to the upper respiratory mucosal epithelium and may occur in other organs such as the gastrointestinal tract. some patients also show non-respiratory symptoms such as acute inflammation of the liver, heart damage, kidney failure, and diarrhea [18, 24, 26]. attacks on various organs result in a complex condition and a variety of clinical manifestations, especially in the early stages of the disease [27]. patients infected with covid-19 show higher leukocyte counts, abnormal respiratory findings, and elevated plasma pro-inflammatory cytokine levels [1]. a male case study of a leukopenia patient showed a leukocyte count of 2.91 × 109 / l, of which 70% were neutrophil. also, 16 mg c reactive protein (crp) was observed in the blood that was higher than normal (0-10 mg/l). increased erythrocyte and d-dimer sedimentation were also observed [28]. the main pathogenesis of covid-19 infection as a targeted virus of the respiratory system is severe pneumonia, viral rna in the blood, glassy background turbidity, and acute heart damage in some cases. increased levels of cytokines and chemokines have been observed in patients infected with covid-19 including il1-β, il1ra, il7, il8, il9, il10, basic fgf2, gcsf, gmcsf, ifnγ, ip10, mcp1, mip1α, mip1β, pdgfb, tnfα, and vegfa. some severe cases admitted to the intensive care unit showed high levels of pro-inflammatory cytokines including il2, il7, il10, gcsf, ip10, mcp1, mip1α, and tnfα [24]. cytokine secretion syndrome is a major cause of disease exacerbation and progression. high levels of il-6 and il-10 have been observed in patients with mansury et al. 4 covid-19 with increasing the severity of the disease [29]. il-6 and il-10 are major cytokines that are consistently elevated in patients with cytokine secretion syndrome. significant reductions in cd4+t cells and cd8+t cells counts have been observed in covid-19 patients [6]. 6. disease symptoms symptoms of covid-19 manifest after the incubation period (approximately 5.2 days) [13, 16]. the onset of symptoms until death is about 6 to 41 days with a mean period of 14 days. this time depends on the immune system conditions and age of patients [30]. various studies have reported the main symptoms of the disease as fever, coughing, shortness of breath, fatigue, and muscle pain while others have also reported sputum, headache, hemoptysis, and diarrhea [22, 24, 30, 31]. it is of note that there are similarities in symptoms between covid-19 and beta-coronaviruses such as fever, dry coughing, and shortness of breath [24]. nevertheless, covid-19 has some unique clinical characteristics such as lower airway involvement and upper respiratory tract symptoms such as the runny nose, sneezing, and sore throat [32, 33]. 7. laboratory findings in a study conducted on 41 patients in wuhan, china, 63% of patients with lymphopenia and 37% of patients with elevated levels of ast enzyme showed an increase in high levels of il2, il7, and il10. moreover, compared to nonintensive care unit (icu) patients, icu patients showed higher levels of il2, il7, il10, gscf, ip10, mcp1, mip1a, and tnfα. data analysis also showed that the likelihood of having lymphopenia, high levels of ast, high levels of ddimer, and elevated cardiac enzymes were directly associated with the severity of patients and the rate of transfer to icu [24]. in another study conducted on 123 coronary patients, low levels of cd4+ t cells and cd8+ t cells were observed in patients with severe pneumonia, but il-6 and il-10 levels were higher in these patients. thus, it can be stated that the t cells and cytokines can be used as a basis for predicting mild to severe disease [29]. 8. diagnosis in a study conducted on 1,014 patients suspected of covid-19, rt-pcr tests and ct imaging of the chest were used for the disease diagnosis and 59% and 88% positive cases were reported, respectively. using rt-pcr as a reference, the sensitivity of ct imaging was calculated at 97%. in this study, serial methods of these two tests showed that in 60 to 90% of patients, the initial ct test was positive before positive rt-pcr. also, in the follow-up of 42% of patients, a ct scan of the normal chest before rtchest ct was negative. thus, chest ct had a higher sensitivity (not specificity) for diagnosing covid-19 compared to rt-pcr from swab samples in the chinese epidemic area [34]. in another research, 49 out of 51 patients with covid 19, confirmed by molecular tests, had a positive ct test (96.1%). for this reason, this study recommended that ct imaging may be useful as a standard method for the rapid diagnosis of covid 19 to optimize patient management (28). in another study, the sensitivity of ct imaging was reported to be 95% based on clinical findings. also, it was found that ct imaging could reduce cases of false-negative nucleic acid testing [22]. however, the final confirmation of patients includes rt-pcr real-time pcr or sequencing of viral genes in respiratory or blood samples [16]. 9. treatment and prevention there is no specific antiviral drug or vaccine against covid 19 infection for possible treatment and prevention in humans. the only available option is to use broad-spectrum antiviral drugs such as nucleoside analogs and hiv-protease inhibitors, which can weaken virus infection until a specific antivirus is available [35, 36]. some of the antiviral drugs used to treat these patients include oseltamivir, lopinavir, ganciclovir, and ritonavir [37]. another study reported that the broad-spectrum antiviral drugs of remdesivir and chloroquine were very effective in controlling covid-19 infection in vitro [26]. moreover, a laboratory study and a clinical study showed that remdesivir (which is a new nucleotide analog) and adenosine analog (which acts as a viral protein inhibitor) improved one patient [38, 39]. the chinese national health commission states that tocilizumab (actemra) can treat covid-19 patients who have severe lung damage and high levels of il-6. this drug applies its effect by inhibiting high levels of protein interleukin-6, which can cause inflammation [40]. although the treatment of some cases with interferon inhalation showed no clinical effect, it worsened the condition as pulmonary turbidity progresses [28]. mansury et al. 5 however, more studies are needed to identify new therapeutic drugs to treat covid-19 infection. many scientists are currently trying to develop a non-human model for studying covid-19 infection for rapid treatment and testing of potential vaccines, as well as providing a better understanding of virus-host interaction [1]. 10. iranian experiences at a glance between 20 february 2020 and 2 april 2020, a hospital based-study study was conducted in one of the first and largest hospital that was specifically designated for the hospitalization of individuals with suspected covid-19 in tehran (capital of iran). their findings showed that of 1,061 suspected patients, 570 patients had rt-pcr positive results and confirmed as a covid-19 infection. among them, 15.6% patients died (with a median age of 64 years), and 4.4% were referred to the icus. the median (iqr) age of these patients was 56.5 (45–66) years and approximately 65% were male [41]. another study in above mentioned hospital 231 suspected cases of covid-19 were studied during two weeks. of which 31.2% were confirmed as a covid-19 cases by rt-pcr and admitted to icu with different level of acute respiratory distress syndrome (ards). in overall, 25% of ards cases were died over two weeks. different therapeutic regimens were employed for these patients including the use of oxygen therapy, supplementation (vitamin d3, thiamine, and selenium), steroid therapy, immunoglobulin therapy, and interferon beta-1a and ribavirin combination [42]. ghanbari on forecasting the spread of the covid-19 in iran, predict that the second wave will be more challenging and is predicated to happen between august and december 2020 [43]. 11. conclusion based on the studies and experiences mentioned in this article, the following recommendations are presented: 1) preventing and 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https://pubmed.ncbi.nlm.nih.gov/33042553/ https://pubmed.ncbi.nlm.nih.gov/32922466/ https://pubmed.ncbi.nlm.nih.gov/32922466/ https://pubmed.ncbi.nlm.nih.gov/32922466/ https://pubmed.ncbi.nlm.nih.gov/32922466/ https://pubmed.ncbi.nlm.nih.gov/32922466/ https://doi.org/10.1016/j.chaos.2020.110176 https://doi.org/10.1016/j.chaos.2020.110176 journal of current biomedical reports jcbior.com volume 2, number 1, 2021 eissn: 2717-1906 1 original research the effect of non-surgical periodontal therapy on glycemic control and c1-reactive protein levels among type 2 diabetic patients with periodontitis: a clinical trial mohammad moshtaghi moghaddam1, arman keymoradzadeh2, rashed ghorbandoust1,* 1department of periodontics, dental school, guilan university of medical sciences, rasht, iran 2student research committee, school of medicine, guilan university of medical sciences, rasht, iran abstract it suggests that non-surgical periodontal therapy without systemic antibiotic therapy may not achieve a significant improvement in glycemic control and systemic inflammation in diabetic patients. however, it is important to highlight that the decision of whether or not to use antibiotics to treat periodontitis. thus this study aimed to evaluate over 3 months the consequence of non-surgical periodontal treatment without systemic antibiotic therapy on serum levels of hba1c and c-reactive protein (crp). forty-two participants with type 2 diabetes mellitus (dm) and moderate periodontal disease were randomized into intervention (ig) and control (cg) groups. the ig received non-surgical periodontal therapy in the form of full-mouth scaling and root planing. participants were followed up for 3 months. the cg received non-surgical periodontal therapy after 3 months. clinical parameters, including plaque index (pi), gingival bleeding index (gbi), probing pocket depth (ppd), clinical attachment level (cal), and hba1c and crp levels, of all patients were recorded at baseline and after 3 months. a p-value less than 0.05 considered significant. at the end of 3 months, ig showed improvement in all the clinical parameters compared to cg. there were no significant differences in hba1c and crp after 3 months when compared to the baseline level in both groups. clinical parameters were significantly improved by the employment of non-surgical periodontal treatment without systemic antibiotic therapy, but hba1c and crp levels were not significantly affected. keywords: antibiotics, diabetes, non-surgical periodontal treatment, periodontal disease 1. introduction diabetes is a chronic disease that affects 8.5% of the world's population. it is estimated that by 2040, the number of people with the disease will increase to 642 million worldwide. based on the epidemiological studies it is estimated that in the year 2030 nearly 9.2 million iranians likely to have diabetes [1]. it has been described that periodontal disease is the sixth complication of diabetic patients after * corresponding author: rashed ghorbandoust, md department of periodontics, dental school, guilan university of medical sciences, rasht, iran tel/fax: +98 911 8069141 email: rashed.ghd@gmail.com https://orcid.org/0000-0003-0540-3768 received: december, 12, 2020 accepted: february, 09, 2021 diabetic nephropathy, neuropathy, retinopathy, vascular disease, and delayed healing [2]. periodontitis is a chronic inflammatory disease that causes progressive damage of periodontium and leading to tooth loss. it is associated with an undesirable impression on the patient's quality of life [3]. the production of pro-inflammatory mediators such as and c-1 reactive protein (crp) in patients with periodontitis is increased. all these mediators can https://jcbior.com/ moshtaghi moghaddam et al. 2 cause insulin resistance and also alter the lipid and glucose metabolism [4]. glycosylated haemoglobin (hba1c) forms by irreversible binding of glucose to haemoglobin. hba1c levels represent glycaemic control over the previous 1-3 months [5]. thus, periodontal disease can affect hba1c and crp levels in diabetic patients [4]. one of the most common therapy that used for the treatment of periodontitis is scaling and root planing. although scaling and root planing improves the periodontal status in the majority of patients, it is usually insufficient to alter the bacterial profile followed by periodontal status [6]. the aim of systemic periodontal antibiotic therapy is to killing subgingival pathogens that remain after conventional mechanical periodontal therapy to support mechanical periodontal therapy and the host defense system [7]. it suggests that in patients with a long history of diabetes, the conventional periodontal therapy alone cannot achieve a significant improvement in glycemic control and systemic inflammation [7-9]. however, it is important to highlight that improper administration of antibiotics can lead to side effects of various intensities, and their indiscriminate use might lead to the tolerance to the antibiotics [10]. therefore, the need to use or not to use these agents by dentists can be a challenge. thus, this study aimed to evaluate the consequence of non-surgical periodontal therapy without systemic antibiotic treatment on serum levels of hba1c and crp over 3 months. 2. materials and methods 2.1 study population this study received ethics approval by guilan university of medical sciences (approval code: ir.gums.rec.1398.307). also, the clinical trial protocol was registered (irct registration number: irct20190721044287n1) and available online. the study population consisted of 42 participants with type 2 dm (hba1c ≥ 7%). inclusion criteria included the following: 1) patients with chronic periodontitis who received no periodontal treatment in the past 6 months; 2) receiving periodontal and antibiotic treatments during the last 6 months; 3) patients with at least 14 teeth in the mouth and a pocket depth (pd) ≥ 5 mm in at least four areas and 4 ≥ clinical attachment level (cal) ≥ 3 mm in at least four areas; 4) diabetes diagnosed by a physician for the past 6 months; 5) patients with unchanged diabetic medication for the past 3 months. exclusion criteria included: 1) patients with advanced diabetic problems (macrovascular and microvascular); 2) chronic pulmonary disease; 3) kidney and liver diseases; 4) malignancies; 5) heart disease (coronary artery disease); 6) transplanted patients; 7) history of infection and trauma; 8) alcoholic and smoker patients; 9) pregnant women; 10) patients with severe periodontitis. 2.2 groups in this study, using a computer program, participants were randomized into two groups of 21 each: the intervention group (ig) and the control group (cg). all the patients received health education using toothbrush and toothpaste with a modified bass method twice a day, and dental floss and chlorhexidine mouthwash (0.12%) twice a day. the ig received nonsurgical periodontal therapy in the form of full-mouth scaling and root planing in two sessions, 1 week apart, with the use of slim instruments, under local anesthesia. participants were followed up for 3 months. the cg received non-surgical periodontal therapy after 3 months. 2.3 follow up during 3 months of follow up, patients with the following criteria were excluded from the study: 1) patients with diabetic diet changes during the study period; 2) patients with problems requiring emergency treatment; 3) patients who changed their diets or medications or used antibiotics during the study. 2.4 data collection clinical parameters, including plaque index (pi), gingival index (gi), gingival bleeding index (gbi), probing pocket depth (ppd), cal were examined at baseline and after 3 months. patients’ blood samples were obtained from the razi laboratory at 8.5-11 a.m. to assess their hba1c and crp levels at baseline and after 3 months. 2.5 statistical analysis data was compiled and analyzed through spss version 24. quantitative variables like hba1c and crp were presented as mean ± sd. the pearson test was used to test the correlation of the periodontal parameters with hba1c and crp levels, and the moshtaghi moghaddam et al. 3 correlation of serum level of hba1c with crp level. differences among all variables from baseline to the 3month visit were analyzed using the wilcoxon mannwhitney u test because it was not normally distributed. the linear regression test was used to evaluate the effect of age and gender on the hba1c and crp levels. a p-value <0.05 considered significant. 3. results hba1c and crp levels in the cg and ig at baseline and after 3 months are shown in table 1 and table 2, respectively. table 3 showed hba1c and crp levels changes between baseline and after 3 months. based on the results of the present study, the two groups did not differ significantly in the hba1c (table 1) and crp (table 2) levels at baseline. also, the results indicated that age and gender did not significantly affect the hba1c and crp levels at baseline. the results indicated that all periodontal parameters measured (pi, gi, gbi, ppd, and cal) showed improvement after 3 months of non-surgical periodontal treatment in the ig compared to the cg (table 4). based on the result of the present study, the relation between hba1c (table 5) and crp (table 6) 1with periodontal parameter changes was not statistically significant. t a b le 1 . h b a 1 c le v e l in th e c o n tr o l g ro u p (c g ) a n d in te rv e n ti o n g ro u p ( ig ) a t b a se li n e a n d a ft e r 3 m o n th s g r o u p s u b g r o u p n u m b e r m e a n s d b a se li n e c g > 9 h b a 1 c 1 5 8 .0 7 0 .4 6 ≤ 9 h b a 1 c 6 9 .7 6 0 .5 4 ig > 9 h b a 1 c 1 5 7 .8 5 0 .6 4 ≤ 9 h b a 1 c 6 1 0 .3 5 1 .1 5 a ft e r 3 m o n th s c g > 9 h b a 1 c 1 5 7 .9 1 0 .7 8 ≤ 9 h b a 1 c 6 9 .6 0 .3 5 ig > 9 h b a 1 c 1 5 7 .9 2 0 .8 0 ≤ 9 h b a 1 c 6 1 0 .0 6 1 1 .9 0 t a b le 3 . h b a 1 c a n d c r p l e v e ls c h a n g e s b e tw e e n b a se li n e a n d a ft e r 3 m o n th s in c o n tr o l g ro u p ( c g ) a n d i n te rv e n ti o n g ro u p ( ig ) p a r a m e te r g r o u p s u b g r o u p n u m b e r m e a n s d h b a 1 c c g > 9 h b a 1 c 1 5 -0 .1 3 0 .5 4 ≤ 9 h b a 1 c 6 -0 .2 3 0 .4 7 ig > 9 h b a 1 c 1 5 0 .1 7 0 .5 7 ≤ 9 h b a 1 c 6 -0 .5 5 0 .5 6 c r p c g 2 1 0 .0 0 4 0 .6 7 ig 2 1 -0 .6 0 0 .7 0 t a b le 2 . c r p l e v e l in t h e c o n tr o l g ro u p ( c g ) a n d in te rv e n ti o n g ro u p ( ig ) a t b a se li n e a n d a ft e r 3 m o n th s g r o u p n u m b e r m e a n s d b a se li n e c g 2 1 3 .6 0 2 .3 6 ig 2 1 3 .0 4 2 .1 0 a ft e r 3 m o n th s c g 2 1 3 .6 1 2 .3 8 ig 2 1 2 .4 3 2 .0 9 moshtaghi moghaddam et al. 4 discussion based on the result of the present study, nonsurgical periodontal therapy without systemic antibiotic therapy could improve the periodontal status of patients with type 2 dm. similar to the result of the present study, vaghani et al. showed scaling, and root planing is associated with significant reductions in the gi, oral health index (ohi-s), ppd, and cal levels of patients with periodontitis [4]. these findings are similar to a study done by obeid et al., which showed the effectiveness of scaling and root planing in improving the periodontal status of patients with periodontitis [11]. based on the results of the present study, nonsurgical periodontal therapy without systemic antibiotic therapy did not affect the serum level of hba1c. similarly, a study done by aldridge et al. reported a significant improvement in periodontal parameters after scaling and root planing without systemic antibiotic therapy but did not found any improvement in hba1c levels in 22 patients with type 1 diabetes and severe periodontitis [9]. grossi et al. table 4. differences of periodontal parameters in intervention group (ig) compared to control group (cg) after 3 months periodontal parameter group statistic p value cg (mean difference ± sd) ig (mean difference ± sd) pi 16.89 ± -4.95 8.16 ± -40.62 4.70 <0.001 gi 0.12 ± 0.08 0.28 ± -0.88 5.61 <0.001 pd 0.15 ± 0.07 0.55 ± 1.10 5.60 <0.001 cal 0.15 ± 0.03 0.31 ± 0.50 5.24 <0.001 gbi 8.41 ± 0.19 19.01 ± -46.24 5.04 <0.001 plaque index (pi), gingival index (gi), pocket depth (pd), clinical attachment level (cal), gingival bleeding index (gbi) table 5. relationships between periodontal parameters and hba1c serum level changes in the control group (cg) and intervention group (ig) after 3 months group subgroup pi gi pd cal gbi cg >9 hba1c r = 0.06 p = 0.820 r = -0.26 p = 0.342 r = 0.04 p = 0.869 r = -0.23 p = 0.39 r = 0.23 p = 0.410 ≤9 hba1c r = 0.47 p = 0.340 r = -0.25 p = 0.633 r = 0.24 p = 0.641 r = -0.61 p = 0.191 r = 0.69 p = 0.125 ig >9hba1c r = 0.36 p = 0.182 r = -0.23 p = 0.409 r = 0.05 p = 0.851 r = -0.27 p = 0.329 r = -0.06 p = 0.831 ≤9 hba1c r = -0.50 p = 0.313 r = 0.51 p = 0.313 r = 0.01 p = 0.984 r = -0.58 p = 0.222 r = -0.19 p = 0.718 plaque index (pi), gingival index (gi), pocket depth (pd), clinical attachment level (cal), gingival bleeding index (gbi) table 6. relationships between periodontal parameters and crp serum level changes in the control group (cg) and intervention group (ig) after 3 months group pi gi pd cal gbi cg r = -0.20 p = 0.383 r = 0.32 p = 0.158 r = 0.10 p = 0.679 r = 0.04 p = 0.876 r = 0.007 p = 0.976 ig r = -0.23 p = 0.318 r = 0.24 p = 0.299 r = -0.20 p = 0.381 r = -0.20 p = 0.393 r = 0.36 p = 0.109 plaque index (pi), gingival index (gi), pocket depth (pd), clinical attachment level (cal), gingival bleeding index (gbi) moshtaghi moghaddam et al. 5 indicated that that topical antimicrobial and systemic doxycycline along with mechanical therapy (100 mg/day for 14 days) resulted in a significant gain in attachment level and a reduction of almost 1% in the levels of hbalc at 3 months after treatment [12]. another study conducted by singh et al. showed that non-surgical periodontal treatment followed by systemic doxycycline is associated with improved glycaemic control in type 2 diabetic patients [10]. this may be due to their antimicrobial effects. based on the results of the present study, nonsurgical periodontal therapy without systemic antibiotic therapy did not affect the serum level of crp. similarly, in a study, non-surgical periodontal therapy without systemic antibiotic therapy did not affect the serum level of high‐sensitivity c‐reactive protein (hscrp) in sixty participants with type 2 dm and moderate to severe periodontal disease [8]. also, katagiri et al. evaluated the effects of periodontal treatment simultaneously on both a1c and crp levels in dm [13]. in that study of 49 participants, scaling and root planing plus topical treatment with minocycline ointment showed no effect on hscrp levels after 6 months. it seems that the clinical beneficial results achieved with non-surgical periodontal treatment in the short-term are frequently not maintained in the long term, particularly in more advanced cases [6] or in those associated with risk factors, such as smoking [14] and diabetic patients [15], because of tolerance to the treatment observed in the case of smokers, or due to the higher prevalence of severe periodontitis observed in patients with diabetes mellitus [5, 15]. for this reason, it has been suggested that the administration of systemic antibiotics can be used together with scaling and root planing. the limitations of the study were the small sample size. also, serum levels of pro-inflammatory mediators such as tnf-α, il-1 β, il-6 were not measured. in conclusion, there is a need for more randomized controlled clinical trials with larger sample sizes, longterm follow-ups, and strict adjustments for confounding factors, such as age, gender, body mass index (bmi), race/ethnicity, and smoking status to confirm the findings of this study despite the improvement of clinical parameters after three months of non-surgical treatment due to no change in important serological markers associated with inflammatory disease and there 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of carbapenem resistance and virulence genes among acinetobacter baumannii isolated from hospital environments in center of iran mohsen nazari1, zohreh youzbashi2, mansoor khaledi3, javad fathi4, hamed afkhami3,* 1department of bacteriology, pasteur institute of iran, tehran, iran 2department of biology, damghan branch, islamic azad university, damghan, iran 3department of medical microbiology, faculty of medicine, shahed university of medical science, tehran, iran 4department of bacteriology and virology, school of medicine, shiraz university of medical sciences, shiraz, iran abstract carbapenem-resistant acinetobacter baumannii are the top urgent antibiotic resistance threat in the world. the aims of this study were the determination of carbapenem-resistant genes and virulence genes among isolates from hospital environments. in this study, a. baumannii isolated from hospital environments and evaluated its antibiotic resistance, virulence factors, and resistance genes. of 258 samples, 58 showed growth of the target organism. antibiotic susceptibility test results considered all the a. baumannii to be multidrug-resistant isolates with the highest resistance being 36.2% to ciprofloxacin; while the most effective antibiotics with 98.3% susceptibility was piperacillintazobactam. of these 58 hospital environment isolates, 18 isolates were positive for metallo betalactamase. overall, 65% of the isolates from hospital environments had many virulence factors. pcr assays demonstrated the highest and lowest positive results in csga and cvac gene among hospital environment isolates. results indicate that the determination of carbapenem-resistant genes and virulence genes among isolates from hospital environments is very important. keywords: carbapenem resistance, virulence gene, non-clinical isolates, acinetobacter baumannii 1. introduction multidrug-resistant acinetobacter baumannii is an opportunistic pathogen that causes nosocomial infections [1-3]. infections caused by this bacterium have a high prevalence in hospitalized and immunocompromised patients who are admitted to intensive care units [4, 5]. these infections are ventilator-associated pneumonia, soft-tissue, urinary tract, and meningitis infections [6-8]. a. baumannii has an ability to survive for long periods the surfaces, sometimes even for several years [9]. due to resistance to a broad range of antimicrobial agents can long-term * corresponding author: hamed afkhami, ph.d department of medical microbiology, faculty of medicine, shahed university of medical science, tehran, iran tel/fax: +98 919 6652678 email: hamedafkhami70@gmail.com http://orcid.org/0000-0002-1110-6447 received: october, 27, 2020 accepted: december, 11, 2020 persistence in the clinical settings, surviving on nutrient sources and transmission by healthcare staff [10, 11]. carbapenems and colistin are the last choices of antibiotic therapies against multi-drug resistant (mdr) a. baumannii strains [12]. the first carbapenem-resistant a. baumannii (crab) originated in the usa and was reported in 1991 [13]. recent studies have reported that crab is a major causative organism in hospital-acquired infections [14]. several mechanisms are responsible for resistance to carbapenems in crab [15]. the https://jcbior.com/ nazari et al. 2 production of carbapenemase enzymes is one of the important mechanisms of carbapenem resistance. these enzymes are class a, b, and d according to molecular ambler classification. the members of class a carbapenemases include sme, imi, nmc, ges, sfc, and kpc families. also, class b enzymes are called metallo-beta-lactamases (mbls), including imp, vim, sim, and ndm. class d β-lactamases referred to oxa-type enzymes or oxacillinases which are the most prevalent carbapenemases in a. baumannii [16, 17]. prevalence of virulence factors (vf) is contributed to pathogenesis in a. baumannii [18]. some of the most significant vf of the a. baumannii strains are curli fibers (csg), colicin v production (cvac), siderophores like aerobactin (iuta), and cytotoxic necrotizing factor (cnf) [18, 19]. the characterization of latent virulence genes, antimicrobial resistance, and molecular detection of carbapenemases of this bacterium in the hospital environments on abiotic and biotic surfaces are the important epidemiological issue. the aims of this study were the determination of antimicrobial resistance and molecular detection of antibiotic resistance and virulence genes among a. baumannii isolated from hospital environments in qom hospitals. 2. materials and methods 2.1 bacterial strain collection and identification the study was conducted at pasteur institute in the period from october 2019 to december 2019. totally, 58 isolates were collected from different hospital environments of qom hospitals (kamkar and beheshti hospitals) (table 1). samples were washed with pbs and transferred to brain heart infusion (bhi) media for further incubation at 37 °c. samples were inoculated into blood agar and macconkey agar for standard aerobic growth and placed at 37 °c overnight. the isolates were identified by the standard biochemical tests. the final confirmation of a. baumannii isolates was performed by pcr blaoxa-51like gene [20]. 2.2 antimicrobial susceptibility testing according to the clsi 2019 guidelines, the disk diffusion test was performed on mueller-hinton agar using a panel of nine antibiotic disks including ciprofloxacin (cip), levofloxacin (lvx), gentamicin (gm), imipenem (imi), piperacillin-tazobactam (ptz), ampicillin-sulbactam (sam), ceftriaxone (cro), and trimethoprim-sulfamethoxazole (sxt) (mast diagnostic, uk). 2.3 phenotypic detection of mbl production combined disk diffusion test (cddt) was performed using an imipenem disk (mast diagnostic, uk) and in combination with edta (sigma, uk) to identify mbls. the inhibition zones of the imipenem, and imipenem + edta disks were compared after 20 h of incubation at 37 °c. in the combined disc test, if the increase in inhibition zone with the imipenem + edta disk was >7 mm than the imipenem disk alone, it was considered as mbl positive [21]. 2.4 detection of carbapenem resistance and virulence genes genomic dna extractions were performed based on the protocol as described elsewhere [22]. pcr reaction mixtures were prepared in total volumes of 25 μl. the presence of the carbapenemase-encoding genes including blaoxa23-like, blaoxa24-like, blaoxa58like, blaimp, blandm, and blavim genes and virulence genes cnf1, csga, cvac, iuta were investigated by pcr assays in all isolates [23-28]. all pcr primers are shown in table 2. table 1. samples source collection source amount percent (number/total) sink 5.1% (3/58) floor 6.8% (4/58) pillow 12.0% (7/58) desk 13.7% (8/58) bed 3.4% (2/58) under-bed wheels 8.6% (5/58) door knob 12.0% (7/58) nurses' fingers 5.1% (3/58) walls 10.2% (6/58) touchpads 22.4% (13/58) nazari et al. 3 3. results antibiotic susceptibility testing showed out of 58 hospital environment isolated strains, the resistance against to cip, lvx, gm, imi, sxt, sam, cro, and ptz were 36.2% (21/58), 31% (18/58), 18.9% (11/58), 15.5% (9/58), 8.6% (5/58), 8.6% (5/58), 5.1% (3/58) and 1.7% (1/45), respectively. of these 58 hospital environment isolates, 21/58 (36.2%) showed resistance to imipenem and were therefore further tested for mbl production. eighteen of these isolates showed positive results for mbl production by cddt method as shown in table 3. pcr analysis in all carbapenem-resistant isolates revealed that prevalence of blavim, blaoxa-23-like, blaoxa-24-like, and blaimp were 10/21 (47.6%), 3/21 (14.3%), 3/21 (14.3%), and 1/21 (4.7 %) of the strains, respectively. none of the hospital environment isolates carried blaoxa-58-like or blandm (table 3). also, pcr assays demonstrated positive results in 43.1% (25/58) of strains for csga, 32.7% (19/58) of strains for cnf1, 12% (7/58) of strains for iuta, and 3.4% (2/58) of strains for cvac genes among hospital environment isolates (table 3). 4. discussion a. baumannii is an opportunistic pathogen and also has the ability to cause nosocomial diseases due to antibiotic resistance and can survive on surfaces, the body of the treatment staff, and patients [29, 30]. this bacterium has the ability to survive on different surfaces and objects, so it has a high potential for spread and colonization in hospitalized patients [31]. through the mechanisms of acquisition of determinants of resistance and upregulation of intrinsic resistance mechanisms, this bacterium is able to resistance against a wide range of available antibiotics [30]. a. baumannii with multidrug resistance causes severe infections and high mortality, especially in patients with impaired immune systems or immunocompromised [2, 32]. although the virulence factors and pathogenicity mechanism of a. baumannii are not fully understood and require further study and research, but this bacterium has the ability to cause a wide range of infections and deaths in hospitals. the virulence factors of this bacterium play an important role in resisting the host's defense mechanism [33, 34]. also these factors are important table 2. primers used for amplification target gene primers sequence (5’ to 3’) size (bp) reference blandm f: cggaatggctcatcacgatc r: cggaatggctcatcacgatc 621 [23] blaimp f: gtttatgttcatacwtcg r: ggtttaayaaaacaaccac 432 [24] blavim f: tttggtcgcatatcgcaacg r: ccattcagccagatcggcat 500 [25] blaoxa-23-like f: tctggttgtacggttcagc r: agtctttccaaaaattttg 606 [26] blaoxa-24-like f: atgaaaaaatttatacttcc r: ttaaatgattccaagattttc 246 [26] blaoxa-58-like f: atgaaattattaaaaatattgagtttag r: ttataaataatgaaaaacacccaac 843 [26] blaoxa-51-like f: taatgctttgatcggccttg r: tggattgcacttcatcttgg 353 [27] cnf1 f: aagatggagtttcctatgcaggag r: cattcagagtcctgccctcattatt 498 [28] csga f: actctgacttgactattacc r: agatgcagtctggtcaac 200 [28] cvac f: cacacacaaacgggagctgtt r: cttcccgcagcatagttccat 680 [28] iuta f: ggctggacatcatgggaactgg r: cgtcgggaacgggtagaatcg 300 [28] f: forward; r: reverse nazari et al. 4 role in binding and invasion bacteria to host cells [28]. according to the contents, it is necessary to study the prevalence of antibiotic resistance and prepare a useful antibiotic treatment model to control and treat diseases caused by a. baumannii. the most important factor in resistance to carbapenem antibiotics is the presence of the 𝑏𝑙𝑎oxa genes, which causes the production of carbapenem hydrolyzing enzymes. in the present study, out of 58 isolates, the rate of antibiotic resistance in a. baumannii based on antibiotic susceptibility tests was the highest and lowest resistance for cip and ptz antibiotics, respectively. according to nourbakhsh et al. (2018) studies, the highest resistance is related to cip (97.2%) [35], which is similar to our study in terms of the highest antibiotic resistance. in the study of shakibaie et al., resistance to cip and ptz was reported to be 66% and 93.3%, respectively [36]. although the table 3. positive and negative genes among hospital environment isolates sample no. mbl blavim blaoxa-23-like blaoxa-24-like blaimp blaoxa-58-like blandm csga cnf1 iuta cvac 1 + 2 + 3 + + 4 + + + 5 + 6 + + + 7 + + + 8 + + + 9 + + + 10 + + + 11 + 12 + + + 13 + 14 + + + + + + 15 + 16 + 17 + + + 18 + 19 + + + + + 20 + 21 + 22 + + 23 + + 24 + + 25 + + + + 26 + + + 27 + 28 + + 29 + 30 + + + + + 31 + + + 32 + + + 33 + 34 + + + 35 + 36 + + + + + 37 + 38 + 39 – 58 nazari et al. 5 bacterial resistance to cip was as high as in our study, the resistance to ptz differed greatly from our study. according to a study by shirmohammadlou et al., a. baumannii is completely resistant to cip [37], and this result is consistent with our study. these results are consistent with the study of kabbaj et al., which showed that mbl production is among the 74% of bacteria resistant to imipenem [38]. among carbapenem-resistant isolates, the frequency of blavim and blaimp genes had the highest and lowest, respectively. also none of the hospital environment isolates carried blaoxa-58-like or blandm. in the study of amudhan et al. [39], the most resistance is related to blavim in 45%, and in the study of shirmohammadlou et al. [37], resistance to blaimp was less common, which met with our study. regard to prevalence of virulence genes, the results of our study were similar to those of al-kadmy et al. (highest frequency was csga 66.7% and lowest frequency was cvac 9.5%) [40], momtaz et al. (highest frequency was csga 55% and lowest frequency was cvac 10%) [41], although our results contradicted the results of darvishi study (highest frequency was cnf1, 35.53% and lowest frequency was csga 12.39%) [28]. the present study had some limitations. mainly, this was a two-center study; therefore, the generalization of the results to other regions requires further investigations. in conclusion, increasing antibiotic resistance due to the acquisition of resistance genes and mutations due to selective pressures is a global problem. the increase in antibiotic resistance in a. baumannii is spreading, especially against effective antibiotics. therefore, studying and determining the pattern of antibiotic resistance for the preparation of a treatment protocol and the use of appropriate antibiotics is effective and reduces the increase in antibiotic resistance. in the current study, we isolated a. baumannii from hospital environments and determinated its antibiotic resistance, virulence factor, and resistance gene. antibiotic susceptibility testing showed the most resistance and sensitivity was against cip and ptz respectively. pcr analysis in all carbapenem-resistant isolates revealed a high prevalence of blavim and blaimp. also, results demonstrated the highest and lowest positive results for csga and cvac gene among hospital environment isolates. acknowledgments we would like to thank the pasteur institute of iran and kamkar and beheshti hospitals of qom for supporting this study. author contributions mn, ha: conceived and design the study. jf, mn, mk: supervised data collectors. mk, jf, zy and ha: drafting the article or revisiting it critically for important intellectual content. all authors read and approved the final manuscript. conflict of interest we declare that we have no conflict of interest. ethical declarations this study was in accordance with the declaration of helsinki and ethical permission was sought from the institutional ethics committee of pasteur institute of iran, tehran, iran (ethical code: 1398057). however, because we only used leftovers from clinical specimens, the local ethics committee waived the need for informed consent. financial support this work was supported by pasteur institute of iran, tehran, iran (grants no. 41443). references 1. hashemi b, afkhami h, khaledi m, kiani m, bialvaei az, fathi j, et al. frequency of metalo beta lactamase genes, bla imp1, int 1 in acinetobacter baumanii isolated from burn patients north of iran. gene rep. 2020; 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https://doi.org/10.5402/2013/154921 https://doi.org/10.5402/2013/154921 https://doi.org/10.5402/2013/154921 https://doi.org/10.5402/2013/154921 https://pubmed.ncbi.nlm.nih.gov/23277500/ https://pubmed.ncbi.nlm.nih.gov/23277500/ https://pubmed.ncbi.nlm.nih.gov/23277500/ https://pubmed.ncbi.nlm.nih.gov/23277500/ https://pubmed.ncbi.nlm.nih.gov/29204285/ https://pubmed.ncbi.nlm.nih.gov/29204285/ https://pubmed.ncbi.nlm.nih.gov/29204285/ journal of current biomedical reports jcbior.com volume 1, number 2, 2020 eissn: 2717-1906 1 original research evaluation of the correlation between serum lipid characteristics of obese subjects and adipoq gene rs266729 polymorphism in chaharmahal and bakhtiari province of iran negar rabiee1, roohollah nakhaei sistani1, ali mohammad ahadi2,*, reza baharloo3 1 department of cellular and molecular biology, faculty of chemistry, kashan university, kashan, iran 2 department of genetics, faculty of science, shahrekord university, shahrekord, iran 3 department of medical laboratory sciences, shahrekord university of medical sciences, shahrekord, iran abstract obesity is a multifactorial disorder that is influenced by various factors such as behavior, diet, environment, metabolic and genetic. this disease is the result of an imbalance between energy absorption and expenditure. mutations in genes that are responsible for appetite control and metabolism are considered as the genetic component of obesity. adiponectin protein is one of the most effective adipokines in regulating the body's energy homeostasis and fat storage, which is expressed by the adipoq gene and secreted from white adipose tissue. the concentration of this protein in the blood decreases in obesity. in this study, the relationship between rs266729 (-11,377 c>g) polymorphism in the adipoq gene with the level of biochemical parameters such as total cholesterol and triglyceride and hdl and ldl in the blood of obese people in borujen (a city in iran) was investigated. this study was performed on 100 people who referred to the tamin ejtemaee clinic due to obesity problems in borujen. in this study, the arms-pcr method was used to determine the genotype of individuals. based on the results of this study, no significant relationship was found between biochemical parameters including total cholesterol, triglyceride, and ldl with rs266729 polymorphism genotypes in adipoq gene in obese subjects. we concluded that rs266729 polymorphism cannot be useful as an index parameter for predispose genotype for imbalance in total cholesterol, triglyceride, and ldl levels in a person. keywords: obesity, adipoq gene, cholesterol, triglyceride 1. introduction obesity is an epidemic disorder in the 21st century. according to epidemiological studies, the prevalence of overweight/obesity has been estimated to have increased by 41% from 1980 to 2013 (1,2). obesity is an important issue for the public health organization because obesity and overweight increase the risk of several serious chronic diseases such as type 2 diabetes, cardiovascular disease, hypertension and * corresponding author: dr. ali mohammad ahadi, ph.d department of genetics, faculty of science, shahrekord university, shahrekord, iran tel/fax: +98 38 32324419 email: ahadi_al@sku.ac.ir https://orcid.org/0000-0001-9580-2740 received: september, 27, 2020 accepted: october, 23, 2020 stroke, hypercholesterolemia, hypertriglyceridemia, osteoarthritis, asthma, and certain types of cancer (3). obesity phenotype occurs when food intake constantly exceeds energy expenditure, resulting in a positive long-term energy imbalance (1). overeating and decreased physical activity are two common causes of obesity, but in addition to them, decreased diversity of intestinal microflora, sleep deprivation, and endocrine disorders are also among the effective factors in https://jcbior.com/ rabiee et al. 2 increasing the prevalence of obesity (4). scientists around the world have intensified their efforts to elucidate the pathophysiological mechanisms which lead to obesity or reinforce its main consequences, to create effective treatments for obesity and related disorders. in this process, the concept of adipocytokines has been defined and dysfunction of adipocytokine pathways has been identified as an important factor in obesity-related disorders. also, the logical manipulation of adipocytokines is becoming a promising way to treat obesity and its metabolic abnormalities (5). since adipokines regulate several important metabolic pathways, genetic variants that affect their function and efficiency may be involved in a variety of pathophysiological conditions. there is evidence that genetic variants in adipokine genes can modulate circulating adipokine levels, which can result in a specific metabolic change (e.g., obesity, insulin resistance, etc.). therefore, a comprehensive analysis of genetic variants, including various and rare single nucleotide polymorphisms, may provide new insights into the specific role of studied adipokines in the pathophysiology of metabolic diseases (6). adiponectin is an adipokine that is mainly secreted from adipocytes (7,8) and its expression is reduced in obesity (9). human adiponectin is encoded by the adipoq gene with the access number of nm_001177800 for mrna in the ncbi genomic database. this gene is located at chromosomal locus 3q27 (chromosomal locus nc_000003.12) (5,10,11). circulating adiponectin may act as a biomarker, and its decreased circulating levels may play a mediating role in the pathophysiology of type 2 diabetes, metabolic syndrome, obesity, and atherosclerosis (7,12). this plasma protein increases fatty acids oxidation and reduces circulating fatty acids concentration (13). regarding the effect of adiponectin on lipid metabolism, adiponectin may increase hdlc levels. also, adiponectin can inhibit the catabolism of triglyceride-rich lipoproteins, and consequently lowering triglyceride levels (14). however, in one meta-analysis, no association was reported between adiponectin gene polymorphisms and lipids (15). in general, adiponectin is negatively correlated with hdlc and positively correlated with ldlc and triglyceride concentrations (16). a positive correlation has also been reported between adiponectin and lipoprotein lipase (17). a similar association has been reported between adiponectin, hdlc, and triglyceride in adolescent and postmenopausal women (18). even in the elderly, adiponectin is positively associated with hdlc and the tc/ldlc ratio (19). however, in patients with type 2 diabetes mellitus (t2dm), conflicting results have been reported due to the association between adiponectin, hdlc, and triglycerides (20,21). hdl dysfunction may be involved in increasing the risk of cardiovascular disease (cvd). adiponectin is involved in increasing hdl particle size and thus improves hdl quality (22). due to the contradictory results observed in the effect of adiponectin gene on the level of serum lipid profiles in obese and other metabolic diseases, in this study, we aimed to investigate the correlation between rs266729 polymorphism of the adiponectin gene and the level of serum lipid characteristics of obese individuals in southwest of iran. 2. materials and methods 2.1 patients and control group participants in this study were selected based on the criteria defined in the world health organization for obesity. therefore, subjects with bmi≥30 were included in our study. participants did not have underlying diseases, like diabetes. the study protocol was approved by the ethics committee of the kashan university of science according to the declaration of helsinki and written informed consent was obtained from each subject. 2.2 sampling and measurement of biochemical characteristics in this study, blood samples were taken from 100 individuals who had referred to the tamin ejtemaee clinic, borujen, charmahal va bakhtiari province, iran, due to the obesity problem. biochemical parameters such as total cholesterol, triglyceride, hdl, and ldl levels were measured by an autoanalyzer (alpha-6-isfahan sanjesh equipment company, iran). 2.3 dna extraction and genotyping dna extraction was performed by the phenolchloroform method from whole blood samples and the arms-pcr method was used to determine the genotype of individuals. this method is a specific allelic pcr for wild type and mutant sequences that rabiee et al. 3 use specific primers. in the arms-pcr method, two complementary reactions are performed for the detection of each allele in polymorphism. one of the reactions involves a specific primer for the mutant allele and the other contains a specific primer for the wild type allele (23). this method is one of the most suitable methods for detecting known mutations and is based on the 3 ́ end nucleotide of the primers. in other words, if this nucleotide is the complementary base of the target base, a pcr product would be obtained, otherwise, the product would not be seen in the gel, and a product for a primer that its 3 ́ end complementary of another allele must be detected. if pcr products are present in both cases, the person would be heterozygous. the absence of the product in both cases of primers indicates a technical problem in pcr and the lack of optimal conditions. many studies can be mentioned regarding the application of the arms technique. also, a comparison of the advantages of this technique has been considered in some researches and its applicability in molecular detection has been proven (24–26). to perform this technique, 4 primers were designed using gene runner software (version 6.5.51 beta) including two specific primers for the detection of wild type and mutated alleles and two other primers for both sides of the amplicon. the names and sequences of the primers are given in table 1. in this study, the final volume of the reaction mixture in each microtube was considered to be 25 μl and a separate pcr reaction was used to identify each of the c and g alleles. the amount of substances used in each of the reactions was as follows: the concentration of substances used to detect the g allele; pcr 1x buffer, magnesium chloride 1.5 mm, dntp 200 µm, forward primer 0.4 µm, reverse primer specific for g allele 0.4 µm, template dna 0.1-1 µg/ml, taq dna polymerase 1 u (sinagen company, iran), the concentration of substances in the pcr reaction for detection of the c allele is the same. the pcr reaction was started with initial denaturation for 5 minutes at 94 °c and followed by 35 cycles of 3 steps consisted of denaturation at 94 ° c for 30 seconds, annealing at 55 for the g allele, and at 58 ° c for the c allele for 40 seconds and elongation step at 72 ° c for 30 seconds. finally, the final extension was performed at 72 ° c for 5 minutes. after the armspcr reaction, the samples were run on 2% agarose gel. arms-pcr product length was 115 bp for g allele and 211 bp for c allele identification. 2.4 statistical analysis in this study, spss ver. 26 software (ibm corp., usa) was used for statistical analysis. kolmogorovsmirnov test was used to calculate the normality of data distribution and the anova parametric test was utilized to calculate the significance of data with normal distribution and the kruskal-wallis nonparametric test was used for data with a normal distribution. tukey and lsd tests were also employed to evaluate the significant relationship between biochemical characteristics and each of the polymorphism genotypes. in all tests, a p value of less than 0.05 showed a significant difference between the two groups. 3. results demographic information and mean serum lipid profile levels of obese individuals are listed in table 2. data normality was assessed using the kolmogorovsmirnov test. hdl and ldl indices had a normal distribution and total cholesterol and triglyceride indices had abnormal distribution. therefore anova test was used to evaluate the significant relationship between hdl and ldl indices and the kruskal-wallis test was utilized to evaluate the total cholesterol and triglyceride indices (table 3). according to table 3, no significant correlation was found between biochemical characteristics consisted of the levels of cholesterol (p = 0.182), triglyceride (p = 0.431), hdl (p = 0.451) and ldl (p = 0.188) and cc, cg and gg genotypes of adipoq rs266729 polymorphism. in this study, the collected data were analyzed by tukey and lsd methods. these two methods focus on comparing means, while anova and kruskal-wallis tests focus on the variance of the whole data. tables 4 and 5 show the results of tukey and lsd tests for rs266729 polymorphism. according to the results of these tests, no significant correlation was found table 1. names and sequences of primers name sequence fnra377 5ʹ-cctttctcacccttctcacc-3ʹ rnrb377 5ʹ-cgcccatgttttgtttttgaagc-3ʹ fnrc377 5ʹgaaccgactcagatcctgcc-3ʹ rnrd377 5ʹgcctggagaactggaagctg -3ʹ rabiee et al. 4 between any of the cc, cg, gg genotypes of adipoq rs266729 polymorphism, and biochemical characteristics including total cholesterol, triglyceride, hdl, and ldl levels in obese subjects. table 2. mean parameters measured in obese subjects parameter obese subjects mean ± sd gender (male/female) 52/48 age (year) 50.19 ± 13.42 bmi (kg/m2) 39.77 ± 5.03 total cholesterol (mg/dl) 236.05 ± 31.55 triglyceride (mg/dl) 238.96 ± 99.68 hdl (mg/dl) 41.57 ± 11.85 ldl (mg/dl) 146.55 ± 31.18 t a b le 4 . e v a lu a ti o n o f th e re la ti o n sh ip b e tw e e n b io c h e m ic a l p a ra m e te rs a n d e a c h o f a d ip o q rs 2 6 6 7 2 9 c /g p o ly m o rp h is m g e n o ty p e s in t h e o b e se i n d iv id u a ls b y t u k e y t e st t u k e y h s d t o ta l c h o le st e r o l t r ig ly c e r id e h d l l d l p v a lu e c c v s c g 0 .3 3 3 0 .8 8 0 0 .4 2 3 0 .2 5 9 c c v s g g 0 .4 5 3 0 .9 9 1 0 .9 7 9 0 .3 7 0 c g v s g g 0 .9 9 9 0 .8 7 9 0 .7 0 4 1 .0 0 0 t a b le 3 . e v a lu a ti o n o f th e re la ti o n sh ip b e tw e e n b io c h e m ic a l p a ra m e te rs a n d a d ip o q rs 2 6 6 7 2 9 c /g p o ly m o rp h is m g e n o ty p e s in th e o b e se in d iv id u a ls u se d to a n o v a a n d k ru sk a lw a ll is t e st s p a r a m e te r c c n = 5 6 c g n = 2 6 g g n = 1 8 p v a lu e m e a n ± s d t o ta l c h o le st e r o l 2 4 0 .6 6 ± 3 2 .8 4 2 3 0 .0 4 ± 3 4 .3 4 2 3 0 .3 9 ± 2 0 .4 0 0 .1 8 2 t r ig ly c e r id e 2 4 1 .3 2 ± 1 0 7 .7 7 2 2 9 .8 5 ± 1 0 0 .8 4 2 4 4 .7 8 ± 7 1 .7 7 0 .4 3 1 h d l 4 0 .5 4 ± 1 1 .0 6 4 4 .0 8 ± 1 4 .7 4 4 1 .1 7 ± 9 .4 3 0 .4 5 1 l d l 1 5 1 .6 1 ± 2 9 .6 3 1 3 9 .9 9 ± 3 5 .8 1 1 4 0 .2 7 ± 2 7 .2 6 0 .1 8 8 t a b le 5 . e v a lu a ti o n o f th e re la ti o n sh ip b e tw e e n b io c h e m ic a l p a ra m e te rs a n d e a c h o f a d ip o q r s2 6 6 7 2 9 c /g p o ly m o rp h is m g e n o ty p e s in t h e o b e se i n d iv id u a ls b y l s d t e st l s d t o ta l c h o le st e r o l t r ig ly c e r id e h d l l d l p v a lu e c c v s c g 0 .1 5 8 0 .6 3 2 0 .2 1 2 0 .1 1 7 c c v s g g 0 .2 3 1 0 .8 9 9 0 .8 4 5 0 .1 7 9 c g v s g g 0 .9 7 1 0 .6 2 9 0 .4 2 6 0 .9 7 7 rabiee et al. 5 4. discussion the role of the adipoq gene in controlling the metabolism of lipids and carbohydrates has been confirmed in many studies (7,27). in the present study, we focused on a polymorphism that could play a role in gene expression. the involvement of this polymorphism in related metabolic disorders has been reported in several studies (28–30). high triglyceride levels and low high-density lipoprotein (hdl) cholesterol levels are of features of the metabolic syndrome which is one of the metabolic syndrome hallmarks of obesity. elevated levels of small, dense, low-density lipoprotein (ldl) cholesterol particles in plasma are key features associated with high triglyceride and low hdl cholesterol levels in people disposed to gain weight. accumulation of dense small ldl particles also increases in subjects with abdominal fat accumulation (large waist circumference). in the mechanism of excessive accumulation of intra-abdominal (visceral) fat, elevated levels of total cholesterol and ldl cholesterol, although common, are mainly associated with the consumption of saturated fats, and are not necessarily associated with weight gain and obesity (31). the importance of parameters including triglycerides, hdl, ldl, and total cholesterol in relation to obesity has been considered in many studies. for example, feingold and granfeld reported that 60 to 70 percent of obese people show blood lipids disturbances, which can indicate ldl, hdl, or triglyceride levels outside the normal range in the blood (32). a study in the chinese population examined the correlation between hdlc levels and obesity and found a negative association between blood hdlc levels and obesity (33). klopp et al. demonstrated that obesity increases the risk of cardiovascular disease due to some risk factors such as elevated triglyceride levels, elevated ldl levels, and decreased hdlc levels (34). dastani et al. showed convincing evidence of an association between several adiponectin-lowering genes and snp with decreased body mass index (bmi), increased waist-to-hip ratio (whr), high triglyceride (tg) levels, and low hdl levels (35). interestingly, in a study of two variants in the adipoq gene promoter, -11391 g>a and -11377 c>g were associated with abdominal obesity, tg levels, and an increased risk of metabolic syndrome in the young population aged 20-33 years (36). for the first time, a study by kaftan and hussein showed an inverse relationship between hdl levels and rs266729 polymorphism in the iraqi population. this was the first study in which such a relationship has been reported (37). sun et al. (2008) examined this relationship and found no correlation between circulating hdl levels and rs266729 polymorphism in the chinese population (38), which confirms the results of our study. this study shows that especially when performing genetic analysis, the origin of the subjects should be carefully considered and because the genetic variants do not necessarily follow the same pattern among the different populations, the origin of study subjects should be considered. in our results and tukey and lsd tests, according to tables 4 and 5 for rs266729 polymorphism, no significant correlation was found between the genotypes of rs266729 polymorphism and biochemical characteristics of obese individuals. the lack of significant differences in the levels of biochemical parameters among the participants indicates accurate sampling, in other words, the lack of specific differences for some reasons such as hormonal diseases or environmental factors in the cause of obesity, for the inclusion of individuals in the study. the variant of rs266729 polymorphism does not show a significant relationship with these characteristics; it can be due to the lack of effect of these variants on changes in adiponectin levels, or the adiponectin level defect may be compensated by other molecules through alternative pathways (39,40). finally, it should be considered that unstudied polymorphisms involved in the imbalance of association with the genetic variants of adiponectin may also play a role in changes in plasma levels of adiponectin (6). one of the limitations of our study was the number of samples. in addition, the relationship between -11,377 c>g adiponectin gene polymorphism and serum lipid characteristics level of obese subjects needs to be investigated in other populations, also other polymorphisms of adiponectin gene need to be considered. our study showed that there is no significant correlation between biochemical parameters including total cholesterol, triglycerides, hdl, and ldl levels with adipoq rs266729 polymorphism. also, according to the results of our study no significant association between different genotypes of this polymorphism in obese individuals with serum rabiee et al. 6 levels of total cholesterol, triglyceride, hdl, and ldl was observed. the lack of relevance of these characteristics can be related to their multifactorial nature, such as diet and nutrition. acknowledgements we thanks kashan university and shahrekord university for their supports. also we thanks tamin ejtemaee clinic, borujen, charmahal va bakhtiari province of iran. author contributions laboratory work has done by nr as her msc thesis, also, collaborated in writing. ama participated as the group leader and the owner of the idea and the main author of the article. rn was collaborated as supervisor of thesis in kashan university. rb was collaborate as advisor of this thesis and sampling and management of participants in this study. all authors read and approved the final version of manuscript. conflict of interests the authors declare that they have no competing interests. ethical declarations the study has been approved by the ethical committee of kashan university of sciences. financial support research director of kashan university financials for msc thesis. references 1. albuquerque d, stice e, rodríguez-lópez r, manco l, nóbrega c. current review of genetics of human obesity: from molecular mechanisms to an evolutionary perspective. mol genet genomics. 2015; 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the validity, quality and efficacy of candidate vaccines in controlling the covid-19 hedyeh maghareh abed1, pardis piri dizaji2, hamed hekmatnezhad3, hoda sabati4, donya zare5,* 1department of biochemistry, faculty of advanced science and technology, tehran medical science, islamic azad university, tehran, iran 2department of biology, faculty of basic sciences, azarbaijan shahid madani university, tabriz, iran 3department of basic sciences, sari agricultural sciences and natural resources university, sari, iran 4biotechnology and biological science research center, faculty of science, shahid chamran university of ahvaz, iran 5department of microbiology, school of medicine, isfahan university of medical sciences, isfahan, iran abstract few would have thought that in this century, a new coronavirus called sars-cov-2 would kill many people around the world, cripple the economy, and leave the medical staff helpless. severe acute respiratory syndrome coronavirus 2 (sars-cov-2), a virus that first appeared in wuhan, china, and spread rapidly around the world, and strict quarantines did not prevent the severe prevalence from spreading worldwide. antiviral drugs do not work well enough for everyone. the mortality rate in the world is still significant. the only thing that gives hope to the people of the world is the hope of being vaccinated, so by producing vaccines in the shortest possible time, science has once again saved humanity. thus, from the very beginning, pharmaceutical companies started to produce safe vaccines. currently, more than 200 types of vaccines around the world are undergoing various stages of production, and about 30 vaccines have entered the clinical trial phase, of which 9 vaccines have entered phase 1 to 3 of clinical trials. dna and rna-based vaccines were first developed and were not licensed before coronavirus disease 2019 (covid-19). other types of vaccines, including nonreplicating viral vectors as well as inactivated vaccines, are undergoing clinical phases. there are currently 9 common vaccines inovio pharmaceuticals, moderna, biontech/pfizer, astrazeneca, cansino biological, gam-covid-vac (sputnik v), wuhan institute of biological products/sinopharm, beijing institute of biological products/sinopharm, and sinovac institutes in the world. vaccination with the pfizer vaccine, which is approved by the world health organization (who), is underway in many countries. the who predicts that by the end of 2021, one billion people worldwide will be vaccinated by the company. keywords: sars-cov-2, covid-19, vaccine, pfizer, moderna, astrazeneca 1. introduction almost one year has passed since the emergence of the deadly coronavirus. the virus was first identified in 2019 in wuhan, china. the virus has spread rapidly * corresponding author: donya zare, msc department of microbiology, school of medicine, isfahan university of medical sciences, isfahan, iran tel/fax: +98 912 756 0121 email: d.zare96@gmail.com https://orcid.org/0000-0001-7827-5600 received: january, 21, 2021 accepted: february, 09, 2021 around the world, killing many people every day [1, 2]. according to official sources, 106 million people have been infected with the virus, of which 59 million have been identified and about 2 million have died. https://jcbior.com/ maghareh abed et al. 2 coronavirus disease 2019 (covid-19) disease which is caused by severe acute respiratory syndrome coronavirus-2 (sars-cov-2), covers a wide range of clinical symptoms, from mild symptoms such as mild flu to severe symptoms such as acute respiratory distress syndrome (ards) followed by pneumonia and death. it should be noted that flock safety is possible by gaining natural immunity through infection, but the losses and consequences can be irreparable [3]. swedish authorities believed that with 60% of the population affected, herd immunity could be effective in controlling the disease, a hypothesis that was ineffective and that the death rate in sweden was five times higher than in germany [4]. thus, the production of an effective vaccine against the disease was prioritized and is considered as the only practical way to establish herd safety. the incubation period of covid-19 is estimated to be 5 days and the onset of symptoms at 11.5 days [5]. studies show that patients with covid-19 excrete the highest levels of viral nucleic acid (rna) at the onset of symptoms [6]. these and other epidemiological data suggest disease transmission in a pre-symptom period [7]. it takes two weeks for the severe symptoms of the disease to appear followed by a cytokine storm created by immunity which causes widespread inflammation and diffuse intravascular coagulation [8, 9]. studies showed the mortality rate of this disease in patients is age-related. a high percentage of people over the age of seventy who have an underlying disease is dying. other factors such as high blood pressure, obesity, diabetes as well as gender (male > female) affect the severity of the disease [10-12]. some anti-cov candidate drugs should be analyzed as preclinical compounds for the potential treatment of covid-19. a small number of covid-19 candidate drugs have reached phase 3 clinical trials such as ritonavir, cobicistat, lopinavir, darunavir, remdesivir, hiv protease inhibitor (asc09f), and oseltamivir [13]. inhibitors of the sars-cov or middle east respiratory syndrome (mers) that were previously available, are being investigated [14]. many institutions around the world have been working from the beginning to develop an effective vaccine against covid-19. in august 2020, more than 200 candidate vaccines were introduced at different stages of development. while about 30 vaccines are currently in clinical trials, the 9 vaccines that have entered phase 1 clinical trials since mid-may 2020 will include astrazeneca/oxford’s azd1222 and moderna’s mrna-1273 vaccines. mrna-1273 in the moderna vaccine encodes the s protein [15]. s protein is released, which stimulates cellular and humoral immune responses. earlier, rna interference technique was used in the treatment of sars and mers, but it did not reach the vaccine production stage [16]. mrna vaccines are a suitable alternative to existing vaccines due to their high efficacy, appropriate immune response, high production rate, harmless administration, and easy processing [17]. in general, vaccines work by training the immune system to identify the part of the virus that causes the disease. in the traditional method, the inactivated virus was used to make the vaccine, but in the case of the mrna vaccine, the situation is different because instead of injecting the viral protein, the person receives the genetic material (mrna) that encodes the viral protein. when these genetic instructions are injected into the arms, the muscle cells translate these codes to make viral proteins directly in the body. the mrna vaccine encodes only an important fragment of a viral protein. mrnas show the immune system what the real virus will cause in the body, and this preview will allow the immune system to design powerful antibodies and elicit an appropriate immune response. mrna is not transmitted to the next generation [18]. given the massive covid-19 pandemic, production capacity soon will certainly not be able to meet global demand for the vaccine in all countries, so vaccination is a priority for medical staff and susceptible individuals. it is estimated that by the end of 2021, the vaccine will be available worldwide to the rest of the population. accuracy and sensitivity in performing preclinical and clinical trials in order to produce a safe and effective vaccine is of great importance in order to prevent severe side effects in individuals [19]. the process of producing a new vaccine is long and usually takes 10 to 15 years. before to covid-19, the mumps vaccine was the fastest vaccine produced and used in 5 years. thus, producing a vaccine against covid-19 over a period of one to two years is a challenge that researchers have finally been able to accomplish. the exploratory phase is the first step in the vaccine production process, which involves preliminary laboratory research that involves identifying the structure and genetics of the virus. the second phase is a pre-clinical studies in which experiments are performed on an animal model such as a mouse to evaluate the immune response of the maghareh abed et al. 3 candidate vaccine and its advantages and disadvantages. clinical trials are performed on humans after the safety and efficacy of the vaccine have been demonstrated in animal models. clinical trials in humans are performed in three phases. in phase 1 (safety), the vaccine is injected into a small number of healthy and immunocompetent individuals. in phase two (expanded safety), the vaccine is tested on hundreds of people in different groups. at this stage, should be evaluated the immune response, appropriate dose, and distance between doses. phase 2 confirms the safety of the vaccine and you can also determine the appropriate dose to test in phase 3. in phase 3 (efficacy) clinical trials in humans, the vaccine is tested on a large scale in thousands of people. in this step, will be evaluated the effect of the vaccine [20]. there are various platforms being looked at for the development of covid-19 vaccines. these include rna, dna, nonreplicating viral vectors and inactivated vaccines. 2. rna-based vaccines the rna-based platform, developed by moderna and pfizer/biontech companies, is a candidate in clinical trials and is in phase 3 clinical trials. the type of candidate vaccine for both moderna and pfizer is lipid nanoparticle [lnp] encapsulated mrna. the target antigen in both of them is spike protein, as well as multiple doses. they will also trigger both humoral and cellular immune responses. their advantage is that they are made using genetic sequencing and do not need to be cell-cultured. one of their disadvantages is that lnp is temperature-sensitive, and no rna-based vaccine has been developed so far. currently, both vaccines are approved by the us government [21, 22]. 3. dna-based vaccines dna-based vaccines encode the target antigen and produce a persistent immune response. transfected cells continuously produce certain transgenic proteins. dna vaccines are temperature stable, but their safety and efficacy require further research. administration of dna vaccines may also cause mutations and integration of the host gene [23]. the dna-based platform, developed by inovio pharmaceuticals (an american biotechnology company), is a candidate in clinical trials and is in phases 1 and 2 clinical trials. the type of candidate vaccine for inovio pharmaceuticals is dna plasmid vaccine with electroporation. the vaccine, called ino4800, is injected intradermally followed by electroporation to ensure that it is absorbed into the cell. the target antigen in these vaccines, such as rnabased vaccines, is spike protein, as well as multiple doses. they will also trigger both humoral and cellular immune responses. the most important advantage of this type of vaccine is that electroporation produces a strong immune response and does not require cell culture. one of the disadvantages of the dna-based vaccine is that electroporation, although safe, can potentially be problematic, and no dna-based vaccine has been developed in the past [24, 25]. 4. non-replicating viral vector vaccines the non-replicating viral vector platform, developed by the astrazeneca/university of oxford and cansino biological inc/beijing institute of biotechnology, and both are in phase 3 and phase 2 clinical trials, respectively. the type of candidate vaccine for astrazeneca is azd1222 and for cansino is adenovirus type 5 vector. both astrazeneca and cansino utilize adenovirus as a vector for their covid-19 vaccine. the target antigen in both vaccines is spike protein, and the astrazeneca vaccine is a single dose. they will also trigger both humoral and cellular immune responses. the advantage of the nonreplicating viral vector vaccine of astrazeneca is that it can be produced on a large scale and provides an effective immune response, as previously demonstrated in the case of ebola. one of the disadvantages of the astrazeneca vaccine from the university of oxford is that the available immunity can disrupt clinical use and reduce the immune response. cansino and astrazeneca intend to offer their vaccines at a low cost, which, given their moderate effectiveness, may be beneficial to some countries [2628]. gam-covid-vac (sputnik v) from russia is a heterologous recombinant adenovirus (rad)-based vaccine. the immunity of the vaccine is satisfactory and produces a strong humoral and cellular immune response and its efficacy is 92% [29]. 5. inactivated vaccines the inactivated platform, developed by wuhan institute of biological products/sinopharm, beijing institute of biological products/sinopharm, and sinovac institutes. they are all in phase 3 clinical maghareh abed et al. 4 trials. the type of candidate vaccine for wuhan institute is inactivated and for sinovac institutes is inactivated aluminum adjuvant. the target antigen in the vaccine produced by these three institutes is the whole virus, and the only wuhan institute is the multiple-dose. the immune response created by the wuhan institute vaccine is more humoral, and also the immune response at the sinovac institute is mostly humoral, which will increase in the presence of aluminum adjuvant. the advantage of using the wuhan institute inactived vaccine is that, due to the use of the inactive virus, the pathogen is killed and there is no risk of recurrence [30, 31]. 6. conclusion in this short review, we discuss ways that can prevent the deadly spread of covid-19 worldwide, including drug therapy and vaccine use. eventually, the efforts of researchers around the world led to the development of effective vaccines against covid-19 to be the sweet end to the most terrible nightmare of the 21st century. of course, vaccination should be done by adopting policies not only in rich countries, but in all countries regardless of nationality, religion, color, and race, and governments should do their best for the health of the people. predictably, the only way to control covid-19 before vaccination by countries is to use new antiviral drugs, social distancing, using facemasks, and regular handwashing with soap and water and if soap and water are not readily available and hands are not visibly dirty, use a hand sanitizer that contains at least 60% alcohol. currently, the bnt162b2 vaccine from pfizer/biontech pharmaceutical companies with 90% efficiency and mrna-1273 vaccine from moderna company has been approved by the us government and is being vaccinated with the pfizer vaccine in the united states, many european countries, and several asian countries [21]. the most important challenge for mrna vaccine production is its inherent instability, as it is more likely to degrade above freezing temperature. pfizer/biontech mrna vaccines require optimal storage at minus 94 fahrenheit degree and are destroyed in about five days at room temperature slightly above freezing. in contrast, moderna pharmaceuticals claims that their vaccine can be stored at most home temperatures or medical freezers for up to six months for long-term transportation and storage. the astrazeneca vaccine, despite being less effective (70%) than its competitors such as pfizer and moderna, can be used in standard refrigerated conditions between 36 and 46 fahrenheit degree for at least six months for storage and transportation, which is a valuable advantage over two the vaccine rivals pfizer and moderna and will be easier to use in developing countries [22]. pharmaceutical companies pfizer and moderna have not joined the covax initiative, but astrazeneca has agreed to provide lower-cost vaccines to middle-income countries. for all covid-19 vaccine candidates, we have only preliminary information on a small number of infections, and none of the covid-19 vaccineproducing groups have released complete information so far, so it is difficult to fully assess the differences between them. however, for evaluating the effectiveness of all covid-19 vaccines to control the pandemic of this disease, we must wait for more follow-up and long-term data. author contributions all authors contributed equally, and read and approved the final version of manuscript. conflict of interest we declare that we have no conflict of interest. ethical declarations not applicable. financial support none. references 1. ahn dg, shin hj, kim mh, lee s, kim hs, myoung j, et al. current status of epidemiology, diagnosis, therapeutics, and vaccines for novel coronavirus disease 2019 (covid-19). j microbiol biotechnol. 2020; 30(3):313-24. 2. merad m, martin jc. pathological inflammation in patients with covid-19: a key role for 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https://pubmed.ncbi.nlm.nih.gov/32778225/ https://pubmed.ncbi.nlm.nih.gov/32778225/ https://pubmed.ncbi.nlm.nih.gov/32778225/ journal of current biomedical reports jcbior.com volume 2, number 2, 2021 eissn: 2717-1906 1 systematic review investigating the effect of ghrelin on improving memory and learning ability in animals: a systematic review mohaya farzin1,2,* parastoo jafarzade1, niloofar faraji2, seyedeh maral mousavi2, mostafa yousefi3 1department of physiology, school of medicine, guilan university of medical sciences, rasht, iran 2razi clinical research development unit, razi hospital, guilan university of medical sciences, rasht, iran 3department of biology, faculty of sciences, university of guilan, rasht, iran abstract ghrelin does an important role in neurological functions such as memory, learning, and cognition as well. there is some evidence that ghrelin can improve memory. this study aimed to evaluate the effects of ghrelin on rat memory. a systematic search was conducted to identify papers published by iranian authors in the web of science, pubmed, scopus, embase, and google scholar electronic databases from january 2010 to december 2019 with the prisma statement. totally, 547 animals were listed in our systematic review. all of the animals were male wistar rats with a mean weight of 250±280 g. the morris water behavior behavioral test and the passive avoidance task were performed to evaluate memory. truly, injection of ghrelin appears to enhance the compactness of the synapse dendritic spines in the hypothalamus. these findings indicate that ghrelin has a prospective role to recover learning and memory ability in animal models, suggesting it as a candidate therapy for memory. keywords: ghrelin, memory, depression, memory disorders 1. introduction ghrelin is a 28-amino acid orexigenic peptide that was identified as an endogenous ligand for the growth hormone secretagogue receptor (ghsr)-1a, performing a significant role in nutrition intake, control of obesity, and plays an important role in homeostasis [1]. based on the findings, it showed that the hippocampus plays an important role in cognitive function and is a vital structure for memory [2]. the presented in different brain tissues for example the hypothalamus and pituitary, ghrelin control brain purposes by entering or highly mainly as ghs-r1a mrna in hippocampal neurons [3]. studies have shown that ghrelin is present in hypothalamic neurons, which are located next to the third ventricle between the abdominal, dorsal, ventricular, and * corresponding author: mohaya farzin, ph.d razi clinical research development unit, razi hospital, guilan university of medical sciences, rasht, iran tel/fax: +98 13 33542460 email: mf99155@yahoo.com https://orcid.org/0000-0003-4739-2454 received: february, 28, 2021 accepted: april, 06, 2021 arched nuclei [4]. notably, ghrelin neurons are located in the hypothalamus and cortex, also the ghrelin neuron fibers in the hypothalamus are straightly thrust to the dorsal vagal complex (dvc) [5]. the above-mentioned ghrelin-induced synaptic growth causes better spatial learning and memory while meddling in ghrelin gene expression diminished the spine synapses density and cholinergic fibers in the hippocampal ca1 which caused reasoning and memory performance to lessen [6]. moreover, intra amygdaloid ghrelin management enhances learning and memory procedures in aversive circumstances in male rats, as studied in the morris water maze (mwm) patterns [6]. intra-hippocampal injection increases long-term memory [7]. it suggests a modulatory performance of © the author(s) 2021 https://jcbior.com/ farzin et al. 2 ghrelin in memory acquisition/consolidation over processes that could contain the promotion of synaptic plasticity and concentration of hippocampal cholinergic fibers [8]. the particular role of ghrelin in depression and anxiety is ambiguous by now, as it has shown, either advanced and lighten depressive and anxiety-behavior in animal studies are with an overweight of indication proposing antidepressant special effects. but a few human investigations demonstrate an interruption of the ghrelin system in a patient with dementia [9]. this study aims to provide a concise yet comprehensive overview of ghrelin's role in memory. ghrelin consistently showed protective and memory-enhancing effects, as well as reducing psychopathology in animal models of dementia. some human investigations demonstrate an interruption of the ghrelin system in a patient with dementia. 2. materials and methods 2.1 search strategies a systematic search from january 2010 to december 2019 on the web of science, google scholar, scopus, pubmed, and embase electronic databases was carried out. the guidelines used for this literature review were from the preferred reporting items for systematic reviews and meta-analyses (prisma). the search was conducted by two reviewers who independently screened the databases through the following terms: “ghrelin” and “alzheimer's disease” or “memory corruption” or “improve memory” or “memory disorder” or “cognition disorders” or “neurodegenerative diseases” and iran. any discrepancies were resolved by consensus with a third reviewer. studies were included without language restrictions. 2.2 inclusion criteria the following articles are included in the study (1) ghrelin was used as a treatment, (2) the tests used in the articles include the maurice water table and the shuttle box, (3) all studies investigating ghrelin in animals. 2.3 exclusion criteria articles were excluded if the following standards were encountered: (1) duplicate publications, (2) figure. 1 prisma flow diagram showing a selection of articles for review farzin et al. 3 articles that compared diverse techniques, (3) studies with a non-control group, (4) intraperitoneal ghrelin injection in mice. 2.4. data extraction the following details were extracted for each of the included studies: the first author’s name, publication date, the study location, sample size, animal details, treatments, and outcome. 3. results ghrelin increases memory and improves map memory performance by becoming longer and richer. ghrelin seems to have a beneficial effect on memory disorders in animals. this will make the memory have better quality. finally, it appears to affect synaptic flexibility in areas involved in memory and has been shown to improve memory capacity in rodents. a total of 15 potential articles were identified. after checking duplicates, reviewing titles, abstracts, and full text, 10 eligible articles were included, which detailed characteristics of them are specified in table 1. the flow diagram outlining the searching procedures is presented in figure 1. over levels of screening, 547 animals were listed in our systematic review. all of the animals were male wistar rats. the weight of all the animals was over 250±280. the morris water behavior behavioral test and the passive avoidance task were performed to evaluate memory. truly, injection of ghrelin appears to enhance the compactness of the synapse dendritic spines in the hypothalamus. according to both types of research by babri, et al. ghrelin can increase memory level [10]. also, other studies by the simultaneous use of narcotics and ghrelin indicated that ghrelin can improve memory level [11, 12]. using the shuttle box and ghrelin block, zahiri et al. and beheshti, et al. showed that the level of memory increased by ghrelin [2]. moreover, eslami, et al. by use of ghrelin blocking demonstrated that it can enhance memory level [6]. 4. discussion this study highlights the conclusion of ghrelin on ameliorating learning and memory aptitude. to our information, this study is a systematic review that discovers the efficacy of ghrelin on enhancing memory and learning in animal trials by the results of the morris water maze test and shuttle box as the result measures in iran. it recommends that ghrelin has a possible role in enhancing learning and memory impairment in animal models. ghrelin appears to use a neuroprotective effect in various models over similar signaling pathways. subsequently, ghrelin could be used to improve existing symptoms in multiple neurodegenerative and also neurological disorders as well as to make disease development slow. in the ca1 region of the hippocampus, ghrelin developed spatial memory and about the ghrelin secretion from the stomach through fasting, it can be theorized that ghrelin may have an ameliorating impact on learning and memory which exposed the enhancing the effect of higher doses of ghrelin on spatial memory in the ptz-treated rats [10]. these findings indicate that the ghs-r1a blocking in the rat’s brain using a selective antagonist impairs memory encoding and emphasis the idea of the signaling of endogenous ghrelin in the rat’s brain is vital for memory attainment and merging [2]. a longterm schedule of ghrelin therapy can have a possible therapeutic value for impaired cognitive function and ghrelin treatment might improve spatial memory and reduce the apoptotic cell number in the hippocampus of methamphetamine-treated animals [6, 13]. advantageous impacts of ghrelin are mediated fairly by the n-methyl-d-aspartic acid )nmda (receptor signaling and ghrelin in the amygdale dependent learning [14, 15]. ghrelin in the dorsal hippocampal network avoids disrupting the influence of morphine in avoidance memory. furthermore, ghrelin empowers the nicotine improving impact on morphine-induced impairment and also the hippocampal nicotinic receptors which are involved in this procedure [12]. infusion of ghrelin into the lateral ventricle was identified to behave this potency to preventing morphine-induced memory impairment loss. the median septal area appears to mediate the ghrelin effects on morphine-induced amnesia [16]. based on the co-administration of ghrelin and compound c highlighted the reductive effect of ghrelin on memory, and it could be considered that this presentation is at least in part, mediated via the ampactivated protein kinase (ampk )pathway [17]. as a limitation, these data must be interpreted with caution because our population was not large enough and we only evaluated studies conducted in a limited area. further studies with larger numbers of articles are needed to confirm our results. farzin et al. 4 in an animal model, ghrelin has a significant role to improve memory ability and learning. but the most effective stimulating (intraventricular injection of ghrelin or systemic) is still imprecise. the results of this review have shown that the effect of ghrelin improves acquisition, consolidation, and retrieval of passive avoidance memory and could reduce the time of escape latency, decrease the frequency of crossplatform. author contributions all authors contributed equally to this manuscript and approved the final version of the manuscripts. conflict of interests the authors declare that they have no conflicts of interest. ethical declarations not applicable. financial support table 1. characteristics of studies included in the meta-analysis study publication year city/ region weight (g) sample size outcome index p-value drug control ref babri 2013 tabriz 230–250 90 1. escape latency 2. total swimming distance 1. p < 0.05 2. p< 0.05 ptz + gh saline [10] beheshti 2015 isfahan 230-280 72 1. stl 2. tdc 3. nst up to 600 s 1. p< 0.05 2. p<0.05 3. p<0.05 bolok ghrelin(dlys-3-ghrp-6) saline [2] eslami 2018 tehran 250 ±20 20 1. stl 2. pal(stl, tlc, and tdc) 1. p<0.01 2. p<0.01 treatment (aβ+ghr) sal+ghr (ghrelincontrol) [6] fallahi 2019 tabriz 220–250 60 1. escape latency 2. traveled distance parameters 3. stl up to 600 s 1. p< 0.05 2. p< 0.05 3. p < 0.001 methamphetamine /simultaneous ghrelin (mesg) saline/saline group (ses) [13] goshadrou 2013 tehran 200 + 20 32 1. stl 2. tdc 1. p <0.05 2. p <0001 different doses of ghrelin (200 ng/rat and 400 ng/rat) ghrelin 200 ng/rat and mk 801 mk-801 [14] goshadrou 2012 tehran 200–250 40 1. stl 1. p < 0.01 sal + ghr saline+ saline [15] mohaddes 2011 tabriz 230–250 50 1. escape latency 2. total swimming distance 1. p< 0.05 2. p< 0.05 ghrelin saline [11] nazariserenjeh 2019 tehran 220–250 35 1. stl 1. p< 0.001 saline/ghrelin/nic otine/morphine saline/saline/morphine [12] shafieifar 2019 arak 220–250 84 1. stl 2. tdc 1. p< 0.001 2. p<0001 ghrelin plus saline morphine/lidocaine [16] zahiri 2019 guilan 200–250 64 1. stl 2. tdc 1. p< 0.001 2. p< 0.001 ptz + ghrelin saline [17] abbreviations: mvm: morris water maze; gh: ghrelin; ptz: pentylenetetrazol; ampk: adenosine monophosphate kinase; pal: passive avoidance learning; stl: step-through latency; tdc: time spent in the dark compartment; nst: step-through into the dark compartment; ghrp: growth hormone-releasing peptide farzin et al. 5 not applicable. references 1. fabel k, kempermann g. physical activity and the regulation of neurogenesis in the adult and aging brain. 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https://pubmed.ncbi.nlm.nih.gov/31260652/ https://pubmed.ncbi.nlm.nih.gov/31152850/ https://pubmed.ncbi.nlm.nih.gov/31152850/ https://pubmed.ncbi.nlm.nih.gov/31152850/ https://doi.org/10.1016/j.peptides.2013.03.022 https://doi.org/10.1016/j.peptides.2013.03.022 https://doi.org/10.1016/j.peptides.2013.03.022 https://pubmed.ncbi.nlm.nih.gov/22487248/ https://pubmed.ncbi.nlm.nih.gov/22487248/ https://pubmed.ncbi.nlm.nih.gov/22487248/ https://pubmed.ncbi.nlm.nih.gov/30553544/ https://pubmed.ncbi.nlm.nih.gov/30553544/ https://pubmed.ncbi.nlm.nih.gov/30553544/ https://pubmed.ncbi.nlm.nih.gov/30553544/ journal of current biomedical reports jcbior.com volume 1, number 2, 2020 eissn: 2717-1906 1 original research the role of metalloproteinase and hypoxia conditions in endometrial cells and embryo implantation maryam mahdavi1, diba bagheri2, elmira vanaki1, mehdi shamsara1, ehsan hashemi1, saeed ansari1, mojtaba dashtizad1, morteza daliri joupari1,* 1department of animal biotechnology, national institute of genetic engineering and biotechnology (nigeb), tehran, iran 2department of genetics, faculty of biological sciences, tarbiat modares university, tehran, iran abstract in the process of implantation, metalloproteinase enzymes play a key role in basement membrane degradation and endometrial extracellular matrix. the activity of these enzymes is impeded by binding tissue inhibitors of metalloproteinase (timp). the oxygen concentration in the mammalian uterus at the time of implantation is about 2-5%. it is seen that the imposition of hypoxia on cancer cells increases the expression of metalloproteinase enzymes and reduces the expression of metalloproteinase inhibitors, resulting in increased cell invasion. to know the effect of hypoxia-inducible factor (hif) and other related factors, we decided to evaluate hypoxic conditions on endometrial epithelial cells of the uterus and the role of matrix metalloproteinases (mmps) on angiogenesis and invasion of the embryo during implantation. in this study, human and mouse endometrial epithelial cells were incubated for 24-48 hours in hypoxic conditions. subsequently, the expression level of timp-1 was measured in mouse and human epithelial cells by real-time pcr technique. the cell viability in hypoxic conditions was evaluated by mtt assay. our results demonstrated that hypoxia reduced the quantitative gene expression of timp-1 in the human and mouse endometrial epithelial cells compared to the control group. it can be concluded that applying hypoxic conditions by reducing the timp-1 expression and consequently increasing mmp expression, may improve the embryo implantation rate. keywords: implantation, embryo, hypoxia, timp-1, mmps 1. introduction implantation is an essential process to develop the embryo, in which the embryo attaches to the outer layer of the uterus and then invades the uterine endometrium ready to accept it in several steps [1], including blastocyst opposition from zona pellucida, blastocyst adhesion to the uterine epithelium, embryo invasion to stromal cells and angiogenesis. the implantation success depends on the ability of the embryo to degrade the basement membrane of the uterine epithelium and invade the uterine stroma. several major factors are involved in this process, * corresponding author: dr. morteza daliri joupari, ph.d department of animal biotechnology, nigeb, tehran, iran tel/fax: +98 21 44580395 email: daliri@nigeb.ac.ir http://orcid.org/0000-0001-9465-7983 received: october, 07, 2020 accepted: november, 11, 2020 including extracellular matrix degradation enzymes. degradation of endometrial extracellular matrix facilitates the trophectoderm invasion to the uterine epithelium; various proteinases play a key role in this degradation, with the emphasis on the importance of metalloproteinase [2]. these enzymes are divided according to substrate specificity, including collagenases, gelatinases, and stromelysins. gelatinase b (gel b; also known as mmp-9) plays a significant role in implantation. the binding of tissue inhibitors impedes the activity of metalloproteinase enzymes [3,4]. according to in vivo studies, the regulation of the https://jcbior.com/ mahdavi et al. 2 balance between metalloproteinase and inhibitors presented in the fetus and the uterus leads to trophoblast invasion into the decidual cells of the endometrium. the tissue inhibitors of metalloproteinase enzymes inhibit processes such as angiogenesis, metastasis, tumorigenesis, and cellular invasion, and include four subsets. in 1970, tissue inhibitors of metalloproteinase-1 (timp-1) secreted glycoprotein (25-31 kda) was purified as the first member of this family from human skin fibroblasts. in 1999 and 2000, timp-2 and timp-3 with a molecular weight of 21 kda, and timp-4 with a molecular weight of 22 kda were identified [5]. timp-1 and timp-2 are both soluble proteins and timp-3 is an insoluble protein that is attached to the extracellular matrix components [6]. the inhibitors have almost the same structure with two domains in mammals, n-terminal (125 amino acids) and c-terminal (65 amino acids) [7]. their inhibitory activity occurs by binding the nterminal domain to the catalytic site of metalloproteinase enzymes [8]. the affinity of timp-1 to mmp-9 and timp-2 to mmp-2 is higher than other enzymes. timp-1, timp-2, and timp-3 are expressed throughout the menstrual cycle in luminar and glandular epithelial cells, veins, and endometrial stromal cells [9]. the timp-1 mrna level is higher during the secretive and menstrual phase compared to the proliferative phase. the timp-2 mrna level is almost constant throughout the cycle. the timp-3 mrna level is higher in the late secretive and menstrual phase compared to the early secretive and proliferative phase. the expression of timp-3 in comparison with timp-1 and -2 has a variety of variations throughout the cycle [10]. vascular endothelial growth factor (vegf) is one of the most important angiogenesis regulators that plays a significant role in the implantation process [11], followed by the formation of the placenta. the highest expression of this factor is affected by the estrogen and progesterone as well as hypoxic conditions in the secretive phase [12], in other words, in the implantation window. evidence suggests that vegf increases the expression of metalloproteinase secreted by blastocyst [13] and reduces the expression of metalloproteinase inhibitors presented in the uterine endometrium [14]. in hypoxic conditions, hypoxiainducible factor (hif) is activated which contains two subunits of hif1α and hif1β [15]. hif1α enters the nucleus and attaches to hif1β and finally by binding to hypoxia responsive elements (hre) located in the promoter region of the target gene, leads to transcript from the desired gene. the hre sequence is located in the promoter region of the vegf and matrix metalloproteinases (mmps) genes. in other words, the hif factor is activated in hypoxic conditions and plays a role in processes such as angiogenesis and invasion, expressed by vegf and mmps, respectively. in studies on cancer, it has been seen that the expression of timp has decreased in hypoxic conditions [16]. given that vegf has an inhibitory effect on timps expression, it can be said that hypoxic conditions can indirectly reduce the timps expression. to know the effect of hif and other related factors, we decided to evaluate hypoxic condition on endometrial epithelial cells of uterus and the role of mmps on angiogenesis and invasion of the embryo during implantation. 2. materials and methods 2.1 preparation and culture of endometrial (uterine) epithelial cells all chemicals were obtained from sigma-aldrich (usa); otherwise, they are specified within the text. human endometrial epithelial cells (c10128) were obtained from the iranian biological resource center (ibrc), tehran, iran. cells were maintained in dmem/f12 medium supplemented with 10% fbs. when cultured cells reached 80-90% confluency, the cultured flask was sub-cultured with 0.25% trypsin/edta solution. to obtain mouse endometrial epithelial cells, pseudopregnant mice were used. to this end, a number of female mice were mated with vasectomized male mice and positive vaginal plaque were detected on the next morning. after 5-6 days, when the uterus had a thicker wall, the mice were euthanized by cervical dislocation and then dissected. animal experiments was reviewed and approved by the local ethical committee (ir.nigeb.ec.1394.8.10.a). mice were group housed 4–5 to a cage on a 12 h light/12 h dark cycle with food and water available ad libitum in a temperaturecontrolled room (22 ± 1 °c). the cells were isolated from the internal surface of the fallopian tubes, using 0.25% trypsin/edta solution and thus a uniform solution of cells was obtained. then, the overall cell volume was evenly distributed between two flasks, containing dmem/f12 medium and 10% fbs. the cells were examined for viability and uniformity mahdavi et al. 3 dispersion using an inverted microscope (nikon, eclipse-ts-100) and finally incubated. after the bottom of the flask is covered by about 70 to 80%, the cells were passaged in three similar flasks to create enough space for growth on a culture dish. 2.2 flow cytometry since verified human endometrial epithelial cells were purchased from ibrc, the evaluation of cell surface markers in contrast to mouse cells did not carry out. mouse endometrial epithelial cells were identified and characterized based on cell surface markers. the flow cytometry performed at the national blood bank, tehran, iran, briefly, 106 cells at passage 3 were cast in 1.5 ml falcon. cells were incubated with primary antibodies of surface markers cd44 (dako, denmark) and cd54 (dako, denmark), and the process was continued by exposure to the fitc-conjugated secondary antibody at 4 °c in a dark place for 30 min. readings were performed with flow cytometry (partec cy-flow), and the results were analyzed using flomax software. 2.3 mtt cell proliferation assay mtt assay was performed to determine the effect of hypoxia on growth and proliferation of cells using 3(4, 5-dimethylthiazol)-2, 5-diphenyltetrazolium bromide (mtt). in each well of the 96-well plate, 10,000 cells/ml were cultured in triplicate. plates were exposed to hypoxic conditions (5% o2) at 37 ˚c with 85% humidity in an incubator for 24 and 48 hours. after the incubation time, the supernatant of each well was removed and replaced with 10 μl of mtt at a concentration of 5 mg/ml in a phosphate buffer solution. further incubation for 4 hours, the supernatant was removed and formazan crystals were dissolved with 50 μl of dmso. after the complete dissolution of crystals, the od of each well was read at 580 nm versus the dmso control solvent by the enzyme-linked immunosorbent assay (elisa) reader. 2.4 hypoxic condition and rna extraction human and mouse endometrial cell suspension was made separately; 10,000 cells/ml were cultured in triplicate in different 96-well plates. plates were incubated at 37 ˚c in an incubator having the hypoxic condition (5% o2) with 85% humidity and control plates were incubated at 37 ˚c having 5% co2 with 85% humidity for 24 and 48 hours for gene analysis evaluation. total rna was extracted from cultured cells in hypoxic and control group in two different incubation times by total rna extraction kit (roche co., germany), in accordance with the manufacturer’s protocol, extracted rna was then synthesized to cdna using cdna kits (k-2101, bioneer, s. korea). to verify the accuracy of synthesized cdna, pcr with a final volume of 15 μl was performed using a pcr master mix (ampliqon 190301). finally, the gene expression was analyzed by real-time rt-pcr technique. the reaction was prepared in a volume of 20 µl using maxima sybr green qpcr master mix kit (fermentas, usa) as the following thermal profile: 95 ºc for 10 min, 40 cycles of 95 ºc for 20 seconds, 57.8 ºc for 30 seconds, and 72 ºc for 20 seconds. data were standardized to the corresponding ovine gapdh (glyceraldehyde 3-phosphate dehydrogenase) level. specific primers (timp-1 for human and mouse) were designed using the beacon designer 7 software, as listed in table 1. t a b le 1 . p ri m e r s e q u e n c e s p r im e r t m s e q u e n c e a m p li c o n si z e ( b p ) g a p d h 6 1 f : g g t g a a g g t c g g t g t g a a c g 2 3 3 6 0 r :c t c g c t c c t g g a a g a t g g t g t im p -1 h u m a n 5 9 f : g g t g a a g g t c g g t g t g a a c g 1 6 2 6 0 r :c t c g c t c c t g g a a g a t g g t g t im p -1 m o u se 6 1 f : g g t g a a g g t c g g t g t g a a c g 1 5 2 6 1 r :c t c g c t c c t g g a a g a t g g t g mahdavi et al. 4 2.5 statistical analysis all experiments were repeated in triplicate and results were expressed as means ± standard deviation. after obtaining the ct of genes from different treatments, the data was calculated to obtain the expression level by the livac method and then analyzed by spss 16.0 software using one-way anova and duncan’s test for data comparison. p<0.05 was considered significant. 3. results 3.1 surface markers results obtained from flow cytometry showed that cd44 and cd54 levels on the surface of mouse endometrial epithelial cells were 75.46% and 51.94%, respectively (figure 1). 3.2 effect of hypoxic conditions on cell viability the growth and proliferation of human and mouse endometrial epithelial cells were assessed by mtt assay after 24 and 48 hours in hypoxic conditions. the viability of mouse endometrial epithelial cell proliferation in hypoxic conditions (5% oxygen) compared to 20% oxygen was reduced by 18.61%, and the viability of human endometrial epithelial cells in hypoxic conditions was also reduced by 16.83%. the results obtained (the number of cells) are shown in figures 2. the data is derived from three replications. 3.3 relative expression of timp-1 pcr product on gel electrophoresis showed a 152bp band of timp-1 in mouse sample and a 156-bp band of timp-1 in the human sample (figure 3). these figures confirm the accuracy of the initial stages of the test, showing the cdna synthesis and proper performance of primers at 61°c. 3.4 decrease in expression of timp-1 since the sybr green dye used in the reaction is a non-specific detector and binds to any doublestranded dna, a melting curve is required to ensure the proper and specific proliferation. as shown in figure 4, the expression of timp-1 was significantly reduced after 24 hours of incubation in hypoxia (6.5fold decrease in expression) as well as in 48 hours of hypoxia (0.412-fold decrease in expression) as compared to the control group. the result also show timp-1 gene expression in the human endometrial epithelial cells was significantly decreased in 24 hours of hypoxia (0.70-fold decrease in expression) as well as in 48 hours of hypoxia (0.31-fold decrease in expression) as compared to the control group. figure 1. surface markers of endometrial epithelial cells in the mouse uterus figure 2. comparison of the number of mouse and human uterine endometrium cells in hypoxic conditions after 24 and 48 hours compared to normal conditions 18.6116.83 100100 0 20 40 60 80 100 120 mousehuman c e ll p e rc e n ta g e hypoxia control figure 3. gel electrophoresis mahdavi et al. 5 4. discussion previous studies on surface markers showed that cd44 and cd54 are presented in the mouse endometrial epithelial cell surface throughout the cycle [17]. according to our flow cytometry results, cd44 and cd54 were found on 75.46% and 51.94% of cells surface, respectively; so it can be concluded that isolated cells were the mouse endometrial epithelial cells. results on the viability of pc12 cells (derived from rat kidney) indicated a reduction by 14% under hypoxic conditions than normal conditions [18]. our cell viability in hypoxic conditions (5% oxygen) also showed a decrease of 18.16% in mouse and 16.83% in human endometrial cells in comparison with 20% oxygen. as a result, it can be said that hypoxia has a slightly destructive effect on cell viability. in fact, oxygen depletion can be considered as the environmental stress that changes intracellular functions and pathways. embryonic development depends on the immediate access of the embryo to the maternal blood flow. implantation is the first step in achieving this capability. the ability of embryonic invasion into endometrial tissue is the most important means to achieve this goal, and invasion needs to secrete extracellular matrix (ecm) degrading enzymes. studies on the implantation process showed that the most important proteases involved in this process were mmps and serine proteases, in which mmp9 and urokinase plasminogen activator (upa) had the highest expression at the time of implantation [19]. the advancement of laboratory techniques such as rt-pcr, immunocytochemistry, plasminogen-casein zymography demonstrated that the upa expression was increased in medium from 4-8 days after fertilization [20]. the zymography test also showed an increase in the protein in the medium on the 6-9th day, this can be attributed to the increased expression of this protein when leaving the zona, which predisposes the blastocyst for implantation [20]. the highest mmp9 expression in the mouse trophoblastic cells was observed on the day of 4.5-7.5 as parallel with the time of implantation, indicating the importance of this enzyme in trophoblast invasion. inhibition of this enzyme expression reduces trophoblast invasion [21]. also, this enzyme expression level at in vitro embryos is less than at in vivo ones. given its importance in implantation, it can be considered as one of the causes of implantation reduction in laboratory conditions [22]. observations suggest that inhibiting this enzyme by timp-1 could prevent adhesion of the mouse embryo to the uterus. the expression of timp-1 and timp-2 is increased in the stroma and basement membrane within 15-24 figure 4. hypoxic effect on timp-1 gene expression in epithelial cells of the human and mouse uterine endometrium after 24 and 48 hours 48h hypoxia24h hypoxiacontrol human 0.310.71 mouse 0.4120.1541 0 0.2 0.4 0.6 0.8 1 1.2 r e la ti v e e xp re ss io n o f t im p -1 /g a p d h treatments human mouse mahdavi et al. 6 days of the menstrual cycle, the time of maximal progesterone levels. this plays a key role in protecting the uterine endometrial tissue against proteins secreted from the embryo (mmps) but decreased the activity of timps during implantation due to the presence of inhibitory factors such as cytokines, especially leukemia inhibitory factor (lif), and decreased progesterone receptors, resulting in an embryo invasion into the uterine endometrial epithelium [23]. on in vivo, none of the human tissues, except for the alveoli, is exposed to high concentrations of oxygen. measuring the oxygen content of the female reproductive system also shows that gametes and embryos have never been exposed to oxygen concentrations of more than 10% [24]. for example, the oxygen content is between 2% to 6% in the rabbit oviduct and about 8% in the hamster and rhesus macaque oviducts [25]. the embryo encounters an oxygen gradient when passing from the fallopian to the uterus so that the oxygen pressure in the uterus is lower than other parts of the reproductive system and the amount of oxygen is lower (3.5% to 5%) during implantation [26]. the previous report has shown that no studies have ever been done on the expression of timp-1 gene in uterine endometrial epithelial cells under hypoxic conditions, and the evaluations only focus on the cultivation of other cells. changes in the oxygen level cause new reactions in the cell to create the ability to cope with new conditions. adaptation with hypoxia involves a wide range of responses, such as changing the expression of genes, altering metabolic functions and changing the activity of ion channels; all of which are essential for cell viability. the ability of cells to respond to oxygen depends on the activation of the family of transcription factors called hif that binds to hre in the promoter region of the target gene to initiate transcription. the significance of this factor is where the main transcription factor is in response to hypoxia and transcribes over 40 genes, including glucose transporters, glycolytic enzymes, factors involved in angiogenesis, and other genes enhancing oxygenation [27]. the purification of dna-binding factors revealed that hif consists of a heterodimer protein complex comprising hif1_α and hif1_β. hif stays in the cell only when the cell is cultured in hypoxia. the level of this protein increases with oxygen reduction slowly from 20% to 7% and reaches its maximum at 5%. the rapid degradation process does not occur in the beta subunit and this subunit has a permanent expression [28]. the promoter of this factor is rich in gc and it has a very low expression in most cells of the body, but its expression is greatly increased in hypoxia [29]. two subunits of hif1-α and hif2-α accumulate rapidly in hypoxia. the reason for this is the stability of these two subunits. the hif is attached to dna within the cgtg sequence (a/g) in the hre region, which in turn increases the expression of the target gene. although the most important factor in regulating hif1-α is the amount of oxygen, research has shown that this transcription factor is affected by other factors, such as nitric oxide, cytokines, and growth factors. the study highlighted the role of this factor in embryonic development because the female reproductive system has an environment rich in growth factors, cytokines, reducing molecules, and oxygen. this strengthens the theory that the activation of this factor on in vitro fertilization (ivf) causes a simulation of the conditions in which the embryo is in its normal state [30]. misregulation or overexpression of this factor by hypoxia or genetic changes is effective in the development of cancer and tumor invasion [31]. as a result of the observations, hif1α is considered to be the most important factor in regulating gene expression in hypoxia conditions. some genes containing this particular region in their promoter are those involved in angiogenesis, energy metabolism, erythropoietin, and cell proliferation and invasion [30]. the hre sequence is located in the promoter region of the vegf and mmps genes. in other words, the hif factor is activated under hypoxia and it is involved in some vital processes of the body, such as angiogenesis and invasion, by expressing vegf and mmps respectively. vegf is one of the most exclusive regulators of angiogenesis that is expressed under the influence of estrogen, progesterone and hypoxia in implantation. jung et al. showed that the lif cytokine (maximum expression of lif in the uterine epithelium cells in implantation as well as in the embryo before implantation) under hypoxia, activates the stat3 transcription factor that in the interaction with the hif factor, is associated with the increased expression of the vegf factor [32]. on the other hand, pufe et al. reported that vegf, as an autocrine growth factor, under cartilage hypoxia, mahdavi et al. 7 increases the mmp expression and decreases the timp-1,2 expression. in fact, the balance between the enzyme and the inhibitor is eliminated [33]. another study in 2001 demonstrated that the cultivation of mcf-7 and htr8-s/vneo cells (which is a human trophoblast cell line) under hypoxia for 24 hours, decreased timp-1 expression and increased mmp-9 expression, thereby increased cell invasion [16]. the results of this study showed that timp-1 gene expression is decreased in 24 and 48-hour hypoxia samples, compared to control endometrial epithelial cells of human and mice. therefore, it can be concluded that hypoxia reduces the expression of genes involved in implantation. in short, it can be said that applying hypoxic conditions reduces the expression of genes involved in invasion. given the importance of invasion in the implantation process as the most important stage in pregnancy, this treatment will improve this phase. author contributions all authors contributed equally to this manuscript, and approved the final version of manuscripts. conflict of interests the authors declare that they have no conflicts of interest. ethical declarations animal experiments was reviewed and approved by the local ethical committee of national institute of genetic engineering and biotechnology (nigeb) (ir.nigeb.ec.1394.8.10.a). financial support the authors would like to acknowledge the national institute of genetic engineering and biotechnology (nigeb, tehran, iran) for technical and financial support under contract grant number 941113-i-557. references 1. koot ye, macklon ns. embryo implantation: biology, evaluation, and enhancement. curr opin obstet gynecol. 2013; 25(4):274-9. 2. bischof p, campana a. a putative 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decoction of some vitamin c enriched plants for the management of covid-19 in jos, nigeria: a case report uchejeso m. obeta1,*, goodluck a. k. ohanube2, maureen o. ekpere-ezeugwu3, eno c. mantu4, nkereuwem s. etukudoh5, muhammad akram6, imtiaz mahmood tahir7 1department of medical laboratory management, federal school of medical laboratory science, jos, nigeria 2department of biomedical sciences, university of applied sciences, bonn-rhein-sieg, germany 3department of pharmaceutical microbiology and biotechnology, university of nigeria, nsukka, nigeria 4department of special duties, federal school of medical laboratory science, jos, nigeria 5department of haematology, federal school of medical laboratory science, jos, nigeria 6department of eastern medicine, government college university faisalabad, faisalabad, pakistan 7college of allied health professionals, faculty of medical sciences, government college university faisalabad, faisalabad, pakistan abstract the worldwide influence of coronavirus disease 2019 (covid-19) pandemic compelled the global health organizations to prioritize the situation over all other related community health issues. the lack of specific treatment modalities against covid-19 is a major concern nowadays. here is a case of a 44year-old man of 176 cm height and 87 kg body weight, who took some powdered mixture equally composed of bitter kola, cinnamon, garlic, ginger, and turmeric sweetened with three teaspoons full of honey. all ingredients were dissolved in 100 ml hot water and taken three times a day during isolation as a positive covid-19 patient and reversed all associated signs and symptoms after 7 days as confirmed with rapid diagnostic techniques (rdts). this case revealed that homogenized herbal tea in jos nigeria possesses a potential therapeutic that could be a treatment protocol against covid-19. keywords: covid-19, herbal tea, vitamin c enriched plants, epigenetics, homeostasis, coronavirus introduction coronavirus disease 2019 (covid-19) pandemic as declared by world health organization (who) has not gotten a confirmed cure worldwide [1], giving rise to many postulations with regards to treatment and management of the disease. while nonpharmaceutical ways of controlling the disease are duly encouraged to be practiced, many have tried various drugs for the treatment [2], and others have equally adopted many natural formulae to manage the situation depending on what is available in various domains [3, 4]. * corresponding author: uchejeso m. obeta, msc.,mpa department of medical laboratory management, federal school of medical laboratory science, jos, nigeria tel/fax: +2348039664892 email: uchejesoobeta@gmail.com http://orcid.org/0000-0002-1382-6034 received: february, 25, 2021 accepted: april, 04, 2021 such natural/herbal remedies included the use of natural plants [3, 5], nutrients, and vegetables. the use of herbs can be in various prepared forms like powder, cold or boiled water extracts, or alcohol-based extract depending on the choice of the herbalist or the consumer. such use of natural or herbal means to treat diseases has been practiced more in africa in the past [3] and has been supported by who provided it follows a scientific and standardized approach [6]. the use of vitamin c [7, 8], vitamin c enriched plants [9] in other forms such as water and alcoholbased extracts [10] have been encouraged for trials but © the author(s) 2021 https://jcbior.com/ http://orcid.org/0000-0002-1382-6034 obeta et al. 2 this case was interested in the homogenized powder of such natural resources. medical laboratory scientists, public health physicians, and academic researchers are seriously in search of prevention and cure of covid-19 especially from natural products around africa and across the world of which bitter kola, ginger, garlic, citrus, turmeric, and giloy are mostly mentioned not excluding vitamin c and vitamin c enriched plants and products [10-15]. this study aimed to educate healthcare professionals and caregivers in covid-19 that the using decoction of some vitamin c enriched plants may be useful for the management of covid-19 in jos and possibly other parts of the world. this case study, therefore, was well monitored to ascertain if the dry products can as well be effective having tried liquid extracted ones within the pandemic with encouraging reports [7-10] as the dry products could easily be transported across the globe for trials and management of covid-19 cases. case presentation a certain business man 44-years-old in jos developed covid-19 like symptoms such as cough and catarrh, incessant sneezing, high temperature, loss of appetite, and general body pains. he is well built with 176 cm height and 87 kg body weight. he isolated himself at home and managed himself with home remedies as we recommended. the homemade remedy adopted was a powdered mixture of vitamin c enriched plants, namely; bitter kola, cinnamon, garlic, ginger, and turmeric. the equally measured homogenized powdered mixture of the five plants was prepared by dissolving three tablespoons full into a 100 ml teacup with hot water and sweetened with honey. a cup of tea was taken in the mornings, afternoons, and evenings for 10 days. the patient was tested for covid-19 after two days of commencing the herbal tea using the rapid diagnostic technique (rdt) called sd biosensor ag that was made in south korea. after seven days, the patient was retested using the same covid-19 rapid testing kit and the result was negative. whereas all the signs and symptoms were completely ceased. the subject was impressed with such wonderful tea and gave his consent for his data to be published as of 20th september 2020. the subject could not disclose any past medical history but the authors present the details of the number of days it lasted, symptoms, tests performed, and recommended decoctions and medicines as presented in table 1. discussion covid-19 pandemic ravages across the globe. there is an urgent need to discover a product that is effective and can be produced and packaged very well for effective logistics and delivery to every nook and cranny of the world. a very dry material or powder can be one of the ways to achieve this height. in this case, the product in the powder is bitter kola, cinnamon, garlic, ginger, and turmeric with honey and hot water, and at various points have been analyzed to contain alkaloids, saponins, tannins, flavonoids, glycosides, sterols, phenols, carbohydrates, reducing sugars, proteins, oils, steroids fiber, enzymes, vitamin c with other vitamins, and vast micronutrients [16-19]. just as honey contains water, fructose, glucose, sucrose, and other sugars, total protein, minerals, acids (such as gluconic acid), vitamins, enzymes, aromas, phenolic compounds, and some minor constituents [20-22]. these components including vitamin c are enriched with antioxidants, anti-inflammatory properties that seem to be good and effective remedies against the virus [10, 23-26] and in some cases against covid-19 [27] just as vitamin c could be used for the treatment of other diseases [28]. there is no doubt that the herbal tea must have contributed to the fast recovery of the covid-19 patient in this case. the effect and action may not be far from the epigenetics and homeostatic mechanisms advocated by our previous works [7-9]. close observation in this case study showed that the patient’s response to treatment was progressive. this result is in line with girija and sivan [29] as seen in ayurvedic treatment of covid-19. it sounds encouraging that yang et al. [30] and ren et al. [31] gave the perspectives of traditional chinese medicine in relation to covid-19 just as presented in this nigerian case study. this case can be said to be a nigerian herbal tea for covid-19 and calls for more research and better ways to do trials on the products used in the tea to enable, validations, registrations, and licensing by responsible government agencies. confirmation of this case through further studies would encourage packaging and exportation to areas where covid-19 is seriously affecting people to save lives across the world. though obeta et al. 3 the case might be limited by the fact that it is just one case at a time, further studies can tell more. also, the dietary component, in this case, might have affected or contributed to the quick recovery of the patient. another study could be carried out on the covid-19 patients treated with the decoction herbal tea with diets of different climes. covid-19 has killed many especially in countries with good healthcare systems like the usa and uk. there is no confirmed conventional medical protocol and strategy for the cure. this case gives a hint towards herbal and natural remedies to the virus. edible plants, vegetables, and nutrients could contribute immensely to the management of the covid19 pandemic and should not be neglected no matter the area or country of origin. such plants like bitter kola, cinnamon, garlic, ginger, and turmeric have consistently shown effectiveness against covid-19 as liquid extracts and now as a powder in consideration of this case report. notably, this case has further given credit to the nigerian herbal remedies using vitamin c enriched plants including vegetables as they may have antitable 1. case presentation and management s/n day – date noticed signs and symptoms test/ result diet/food treatment 1 day 1-10/09/20 dry cough, fever, body pains, stomach pains, loss of smell and taste temperature 37 oc pounded yam and okro soup, rice and stew, cold water paracetamol (2 tablets, 3 times daily); emzor cough syrup (20 ml 3 times daily) 2 day 2-11/09/20 no difference temperature 38 oc garri and vegetable soup, yam and beans, warm water same as above; vitamin c tablet (500 mg, 3 times daily); decoction herbal tea (3 times daily) with advice to sleep very well and drink warm water 3 day 3-12/09/20 reduction in stomach pains, others remained temperature 38.5 oc; home rdt test covid-19 positive pounded yam with vegetable soup, locally prepared fruit juice, warm water vitamin c as above; decoction herbal tea as above 4 day 4-13/09/20 reduction in body pains, stomach pains and cough temperature 37 oc same as above same as above 5 day 5-14/09/20 same as above temperature 36.7 oc same as above same as above 6 day 6-15/09/20 stable and normal same as above same as above same as above 7 day 7-16/09/20 same as above; partial restoration of sense of smell and taste same as above same as above same as above 8 day 8-17/09/20 all signs and symptoms gone same as above same as above same as above 9 day 9-18/09/20 same as above same as above same as above 10 day 1019/09/20 temperature 36 oc home rdt testcovid19 negative same as above same as above 11 day 1120/09/20 same as above obeta et al. 4 covid-19 properties that require consideration for curative medicine. it is suggestive to posit that homogenized herbal hot tea has potential therapeutics and could be a curative modality against covid-19. acknowledgments the authors appreciate the subject who gave consent to use the information for publication. authors contribution all authors contributed equally and approved the final version of manuscript. conflict of interests the authors declare no competing interests. ethical declarations this study was performed in accordance with the declaration of helsinki and a signed informed consent was obtained from the patients for using the recommended remedy. consent for publication written informed consent was obtained from the patients for publication of this case report. a copy of the written consent is available for review by the editor-in-chief of this journal. financial support self-funded. references 1. cucinotta d, vanelli m. who declares covid-19 a pandemic. acta biomed. 2020; 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[4]. in addition, they are important and potent pathogens of tooth decay that occur in dental plaque after adhesion to the tooth surface due to the accumulation of lactic acid, © the author(s) 2021 https://jcbior.com/ jafarzade et al. 2 therefore, acid producers are considered to be likely to cause tooth decay [5]. although streptococci such as streptococcus sanguinis and streptococcus salivarius have been reported to produce organic acid, their acid tolerance is weaker than that of mutant streptococci, limiting the contribution of these oral streptococci to caries [6]. the presence of s. sanguinis in the oral cavity may also be the result of moderate caries in children [7]. the use of probiotics as a new method to eliminate pathogenic bacteria in the oral cavity can be considered. the use of probiotics is a promising way to fight infections with the help of beneficial bacteria and replace them with pathogenic microorganisms [8]. mutans streptococci are one of the most important cariogenic bacteria and various in vitro studies have shown that lactobacillus rhamnosus and lactobacillus paracasei can reduce the number of mutans streptococci significantly [9]. different species of bacteria have been introduced as probiotics, the most common of which are lactobacillus and bifidobacterium. today, the benefits of using probiotics in systemic diseases, especially gastrointestinal diseases, have been identified [10]. however, there are limited studies in this field and their role in maintaining oral health needs further investigation. probiotics can work through several mechanisms; probiotic bacteria inhibit the colonization of pathogenic bacteria by colonizing the intestinal tract, binding to each other, using nutrients in the body before being consumed by pathogenic microorganisms, and producing toxins [11]. other potential benefits of probiotics in host health include reduced susceptibility to infections, prevention and treatment of allergic manifestations, treatment of lactose intolerance, regulation of blood pressure and serum cholesterol, treatment of cardiovascular disease and cancer, it also eliminates the side effects of overuse of antibiotics (such as diarrhea) and especially the emergence of bacterial resistance [12]. lactobacilli have no role in initiating the caries process and are mostly associated with dentin caries and are present at the site of carious lesion progression [13]. it has been shown that salivary streptococci are significantly reduced immediately after the end of daily use of the probiotic lactobacillus [14]. however, in people with advanced dentin caries, consumption of probiotic products may lead to an increase in lactobacilli in the carious lesion and further progression of caries [15]. in this study, we aimed to evaluate the antibacterial and antibiofilm activity of probiotic bacteria against streptococci that cause tooth decay. 2. materials and methods 2.1 bacterial strains in a study conducted at guilan university of medical sciences (ethics code ir.gums.rec.1399.343), 42 isolates of lactobacillus which was previously isolated from human fecal samples were studied. of these, the results of 5 strains which belonged to lactobacillus plantarum, l. rhamnosus, lactobacillus reuteri, lactobacillus fermentum, and lactobacillus brevis species were more promising and can be considered as potential probiotics sources for functional products. two strains of bacteria including s. mutans atcc 35668, s. sanguinis atcc10556, were used. the strains were recovered from stocks by cultured overnight at 37°c. 2.2 determination of antimicrobial activity to investigate the antibacterial effect of probiotic producing lactobacillus strains, microbial susceptibility testing by the well-diffusion method was used. mcfarland 0.5 standard concentration was obtained from the bacteria and cultured on mueller hinton agar enriched with 5% sheep blood using a swap. in the next step, wells with a diameter of 6 mm were created on this medium, and then supernatant without cells of lactobacillus strains was added to each well (62.5 – 500 µg/ml). finally, the plates were incubated for 24 hours at 37°c and then the diameter of the growth inhibition zone was measured [16]. 2.3 determining the minimal inhibitory concentration (mic) probiotic bacteria mics were determined by standard broth microdilution method based on the clinical and laboratory standards institute (clsi) guidelines [17]. muller hinton was used to determine the mic for the studied strains. to evaluate the inhibitory effects of probiotics on bacterial growth, each 96-well microplate was filled with a concentration range (15.6 to 500 µg/ml). after 24 hours of incubation at 37°c, the wells were examined for microbial growth and the lowest concentration at jafarzade et al. 3 which no growth was observed was considered as mic. 2.4 biofilm formation assay probiotic bacteria were added to the microtiter plate wells and incubated at 37°c for 48 h. after staining with crystal violet and adding ethanol, the absorbance was measured at 570 nm [18]. the tests were done at least three times. 2.5 statistical analyses in this study, the obtained results were entered into spss software ver.25. to describe quantitative variables from the mean and standard deviation and for qualitative variables numbers and percentages were used. 3. results the diameter of the inhibition zone was measured by the well-diffusion method at different concentrations (supplementary figure 1) that was estimated between 10 and 16 mm (table 1). most isolates have an inhibition zone at concentrations above 125 µg/ml. the mics values of the five lactobacillus isolates against s. sanguinis and s. mutans bacteria were estimated at 62.5 to 250 µg/ml and 125 to 500 µg/ml, respectively. the detailed results of mic of were shown in table 2. probiotic bacteria could not significantly inhibit the formation of biofilms in the suspension tested at concentrations of 62.5 to 500 µg/ml (data not shown). 4. discussion in the present study, the antibacterial and antibiofilm effects of native probiotic bacteria on cariescausing bacteria and dental plaque were measured. the minimum inhibitory concentration for tested bacteria was 125 μg/ml. at this concentration, the diameter of the inhibition zone was estimated to be approximately 10 mm. consistent with our results in a study conducted by rayani et al. to compare the effect of probiotic and regular buttermilk on two common oral microorganisms, s. mutans and enterococcus faecalis, thier results indicate a significant difference in the mic of the two types of probiotic dough used against tested bacteria [19]. our current results in a laboratory study indicate that probiotic lactobacilli affect the growth and inhibition of oral streptococci. this finding is in agreement with the results of a study that showed that consumption of yogurt or probiotic drinks can significantly reduce the level of s. mutans [20, 21]. the results of a study in denmark showed that l. rhamnosus significantly reduced the risk of caries. therefore, milk containing probiotic bacteria may have beneficial effects on children's dental health [22]. several studies have shown that the amount of s. mutans in saliva decreases significantly immediately after the end of daily consumption of probiotic lactobacilli [23, 24]. previously, a study conducted in ahvaz (southwestern iran) to investigate the effect of consumption of probiotic yogurt containing bifidobacterium lactis b12 on s. mutans and lactobacilli in students with early stages of tooth decay, contrary to the results of our study, in which the studied probiotics had little effect on the removal of bacterial biofilm, a significant decrease in biofilm was observed in the intervention group compared to the control group [25]. another study conducted in china examined the effect of probiotic lactobacilli on s. mutans and the development of biofilms in children with severe caries. their results showed lactobacillus table 1. the diameter of the inhibition zone in different concentration of probiotic isolates bacteria s. sanguinis s. mutans 500 µg/ml 250 µg/ml 125 µg/ml 62.5 µg/ml 500 µg/ml 250 µg/ml 125 µg/ml 62.5 µg/ml l. plantarum 14 mm 12 mm 0 0 12 mm 11 mm 0 0 l. rhamnosus 14 12 11 0 15 12 10 0 l. reuteri 15 12 10 0 14 12 10 0 l. fermentum 12 12 10 0 11 0 0 0 l. brevis 16 13 10 10 14 11 10 0 jafarzade et al. 4 casei significantly reduced the number of s. mutans, s. sanguinis, and all bacteria in mixed biofilms compared to the control group [26]. as the main limitations of the present study, a narrow range of tested bacteria and the lack of toxicity evaluation should be acknowledged. the results of the study showed that probiotic bacteria with potential antibacterial properties against bacteria that cause tooth decay can be used to prevent dental caries. probiotics will be used as a new method in the future due to their cost-effectiveness and fewer side effects compared to other antibacterial compounds. however, our local probiotics did not show a significant effect on preventing biofilms formation. supplementary files supplementary file 1. author contribution hs, ma, mh: conceived the study. hs, shr, ma, gr, kd: participated in the design of the study and performed the statistical analysis. pj, hs, shr, em, gr, ty, mh: interpreted the data. hs, mh, ma, shr: obtained ethical clearance and permission for study. hs, ma, hs: supervised data collectors. pj, hs, shr, em, gr, kd, ty, mh: drafting the article or revisiting it critically for important intellectual content. hs, ma, mh were project leaders and primary investigators of the study. all authors read and approved the final manuscript. conflict of interests there is no conflict of interest. ethical declarations the study protocol was approved by the guilan university of medical sciences. the ethical approval code is ir.gums.rec.1399.343. financial support self-funding. 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https://pubmed.ncbi.nlm.nih.gov/28851857/ https://pubmed.ncbi.nlm.nih.gov/28851857/ journal of current biomedical reports jcbior.com volume 2, number 1, 2021 eissn: 2717-1906 1 original research the role of proteases in the invasion of sars-cov-2 virus into human host cells maryam saeidi1, fatemeh rouhollah1,* 1department of cellular and molecular, faculty of advanced science and technology, tehran medical sciences, islamic azad university, tehran, iran abstract the new coronavirus, which originally broke out in wuhan, china, in december 2019, increase quickly around the world, causing a pandemic all over the world. the virus uses its spike protein to enter the human host cells. protein s binds to the angiotensin-converting enzyme ii (ace2) receptor and enters the host cell. for the more successful binding, several proteases facilitate and optimize this binding, the most important of which are transmembrane protease serine 2 (tmprss2), furin, and cathepsin l proteases. after binding of protein s to the ace2 receptor, cleavage of protein s is required for membrane fusion by protein s, which causes viral entry into host cells. this proteolytic activity may be cathepsin l-dependent and occurs with changes in ph in cell endosomes, or it may occur through serine proteases activity at the surface of the host cell membrane or within vesicles. finally, the cell becomes infected with the virus. several studies have tried to reduce the rate of viral infection by using inhibitors of these proteases. keywords: sars-cov-2, tmprss2, furin, cathepsin l, covid-19 1. introduction the new coronavirus, which primary started in december 2019 in wuhan, china, caused cases of pneumonia [1], so that by february 15, 2021, more than 108 million 400 thousand people worldwide infected with severe acute respiratory syndrome coronavirus 2 (sars-cov-2), and about 2 million people died due to coronavirus disease (covid-19) [2]. there are many diverse kinds of coronaviruses. some of them can cause colds or other mild respiratory (nose, throat, and lung) sicknesses. further coronaviruses can cause more severe diseases, as well as severe acute respiratory syndrome (sars) and the middle east respiratory syndrome (mers). the name of corona derives from the latin name * corresponding author: fatemeh rouhollah, ph.d department of cellular and molecular, faculty of advanced science and technology, tehran medical sciences, islamic azad university, tehran, iran tel/fax: +98 21 22006660 email: frouhollah@iautmu.ac.ir https://orcid.org/0000-0001-5243-129x received: january, 18, 2021 accepted: february, 22, 2021 crown meaning circular crown [3]. this virus belongs to the beta-coronavirus family based on phylogenetic studies [1]. investigation of the sars-cov-2 rna sequence showed that it is most similar to the coronavirus isolated from bat species [4, 5]. bats are natural reservoirs of coronaviruses, and human-tohuman transmission of the disease has been confirmed [6, 7]. this positive single-stranded virus structurally requires four vital proteins to form a whole viral particle: spike (s) protein, nucleocapsid (n) protein, membrane (m) protein, and envelope (e) protein [8]. this virus binds to its surface cell receptor through its spike protein, causing the virus to enter the host cell. this s protein has two domains, s1 and s2. proteases cut the link between n terminal s1 and c https://jcbior.com/ saeid et al. 2 terminal s2 [9]. s1 subunit is the receptor-binding motif (rbm) located in the receptor-binding domain (rbd), which interacts straight with host cell receptor angiotensin-converting enzyme ii (ace2) and mediates virus binding to host cells. sub-unit s2 exists membrane-terminal c-terminal and amphipathic heptad repeats that predict to be involved in coiled-coil formation in virus-cell binding [9]. sars-cov-2 enters the cell by binding to the ace2 cell receptors [10, 11]. the virus detects human ace2 with high efficiency and therefore increases the ability of sarscov-2 to be transmitted from person to person [12]. ace2 is a carboxypeptidase that converts angiotensin ii to angiotensin-(1-7), anti-fibrosis, anti-hypertrophy, and vasodilation [11, 13, 14]. the ace2 gene locates in the xp22 chromosomal area with a length of 39.98 kb of genomic dna. this gene produces two transcripts that finally form a protein with 805 amino acids. high levels of polymorphisms observed in this gene, several single nucleotide polymorphisms (snps) are associated with vulnerability to diseases such as type 2 diabetes and hypertension [15, 16]. figure 1 (https://www.proteinatlas.org), shows ace2 expression in different tissues, suggesting that a lot of organs may be the host for viral attack due to their ace2 receptor. numerous host proteases can degrade s protein in sars-cov-2, including transmembrane protease serine 2 (tmprss2), cathepsins, and furin. among these proteases, tmprss2 is necessary for viral entry and occurrence in the sars-cov2 infected host [1]. in this review, we will deal with the most important protease involved in the cleavage of protein s. 2. tmprss2 sars-cov-2 uses ace2 to enter the cell, and tmprrs2 is a serine protease involved in the cleavage of protein s [17]. early preparation of s protein by tmprss2 is necessary for the entry and spread of the sars-cov-2 virus by its mediator with ace2 [18]. the tmprss2 gene is located on human chromosome 21q22.3, encodes a 492-amino acid polypeptide with five different domains: serine proteinase, scavenger receptor, low-density lipoprotein, transmembrane, and cytoplasmic [18]. the tmprss2 gene essentially expresses in the adult prostate [19]. regulation of tmprss2 gene expression is affected by androgen signaling [20]. androgen receptor activity required for transcription of the tmprss2 gene, no other regulatory element found for the tmprss2 promoter up till now [20, 21]. the human tmprss2 promoter has an androgen response. also, tmprss2 mrna expression is affected by androgen regulation in prostate cells [21]. ace2 is involved in anchoring the sars-cov-2 virus to the cell surface, which is also affected by androgens and is more active in men [22]. one research reported that men were at higher risk for developing the disease with severe symptoms [23]. the tmprss2 gene is mostly expressed in the adult prostate but also expressed in other tissues, including the adult colon, small intestine, pancreas, kidney, lung, and liver [19]. figure 1. the expression of ace2 in different tissues in terms of consensus normalized expression (nx) levels, obtained from three transcriptomics datasets (hpa, gtex, and fantom5) saeid et al. 3 as well, tmprss2 is express in the lungs, liver, and kidneys, which are target organs for covid-19 [24]. expression of tmprss2 in the small intestine facilitates virus binding and entry into host cells, which may contribute to the increase of the disease because it provides a potential site for virus replication in the small intestine enterocytes [25]. a study showed that genetic polymorphisms ace2 and tmprss2 could be associated with genetic susceptibility to covid-19. it is better to treatment directed to personal medicine [26]. tmprss2 can help sars-cov-2 to enter host cells [18]. tmprss2 is useful at neutral ph and loses its activity in acidic situations [27]. therefore, tmprss2 can play a critical function in the proteolysis of the s1 subunit of the virus at the surface of the host cell, while membrane-bound or released cathepsin l (catl) should also target the same substrate [28]. sars-cov viruses are ph-sensitive viruses, and their intracellular trafficking requires an acidic environment [29]. tmprss2 acts locally on the plasma membrane of the host cell and probably acts along with the endocytotic vesicle trafficking [30]. also, mechanisms suggest for virus entry, including the breakdown of ace2 by membrane-bound serine proteases, which leads to increased virus entry [30]. regarding the role of serine proteinases, success in inhibiting serine proteinase may be efficient in virus entry to the cell in vitro [18]. sars-cov-2 requires tmprss2 and catl to enter. camostatmesylate, an inhibitor of tmprss2 performs a critical role in reducing virus entry into the calu-3 lung cell line [18]. hoffman et al. have recently shown that the tmprss2 inhibitor blocks the entry of the virus and may be a treatment option [18]. they reported that camostatmesylate a serine proteinase inhibitor accepted in japan for the treatment of distinct diseases blocks the activity of tmprss2 [31, 32]. 3. cathepsin l sars-cov-2 also enters the cells through endocytosis. phosphatidylinositol 3-phosphate 5kinase and catl are significant for endocytosis [33]. after binding, protein s cleavage, required for fusion membrane fusion with protein s, which causes the virus to enter host cells. this proteolytic activity may be catl-dependent and occurs with ph changes in cell endosomes, or it may bind via serine proteases to the surface of the host cell membrane or within vesicles [33, 34]. after sars-cov-2 endocytosis, the s protein is cleaved by catl, which permits the virus membrane to combine with the endosomal membrane. following that, the viral genome is released into the host cell [35]. endosomal proteases for instance cathepsins can cause viruses to enter the host cell through an endosomal pathway that occurs at low ph. as well, the virus can enter the cells through cell surface proteases, particularly tmprss2 [36-38]. both endosomal and non-endosomal pathways are involved in the entry of the sars-cov-2 virus, and low ph is essential for virus activity within the cell [33]. catl, with 220 amino acids, is an enzyme involved in other pathologies, including osteoporosis and periodontal disease. as a result, a host of catl inhibitors are available [39]. this protein is a lysosomal cysteine peptidase and has a double chain (l and r) [40]. catl is involved in protein turnover and cell apoptosis. excessive expression of catl in cancer cells has made it suitable as a target for anti-cancer strategies [41]. it has been found that in some food proteins and peptides naturally have the inhibitory activity of catl, some peptides in food proteins may be able to inhibit catl and therefore help prevent covid-19 [42]. inhibition of catl may be practical in reducing infection by sars-cov-2. many inhibitors of catl, such as the epoxydipeptide ketones used to suppress sars-cov, are fundamentally peptidomimetic [35]. by inhibiting the activation of protein s, teicoplanin prevents catl in pseudoviruses [43]. sid26681509 is also a catl inhibitor that reduces the entry of sars-cov-2-like viruses, demonstrating the importance of catl in priming the sars-cov-2 protein [33]. chloroquine can prevent the activity of proteases and s protein, neutralize endolysosomal ph. then reduce viral entry into the host. it can also trap ace2 in nuclear vacuoles [44]. the anti-malarial drug chloroquine can efficiently block sars-cov-2 infection in cultured cells [45]. but to date, there is no recorded clinical trial to support this inference. chloroquine affects ace2 terminal glycosylation [46]. while chloroquine may have short-term benefits for covid-19 patients by inhibiting catl activity, it can influence to cardiac arrhythmia [47]. the mechanism of chloroquine is related to the function of catl, due to which its activity increases the endosomal ph [48]. chloroquine prevents proteolysis of the s1 subunit in endosomes by raising endosomal ph and decrease the saeid et al. 4 release of viral genetic material [49]. treatment with catl inhibitors or protease inhibitor cocktails can have compensation over chloroquine. patients or cells treated with specific catl inhibitors without camostatmesylate do not show a decrease in the optimal activity of further endosomal proteases, unlike those treated with chloroquine [50]. 4. furin in the sars-cov-2 genome, although it’s many similarities to the sars-cov genome, the furin protein cleavage site was not found in other sars-like covs sequence, which may play a role in virus entry into the cell and pathogenicity [51]. the study of proteolytic activation of glycoproteins in enveloped viruses shows the relationship between viral infection and furin protein, which indicates the role of this protein in the activation of spike protein [52]. this protein can consider as a mediator in the processing and maturation of sars-cov-2 virus s protein [53]. regarding the role of furin in the processing of essential surface proteins and its role in viral infectivity has been identified [52]. coronaviruses attach to the host cell receptor to enter the cell through their s protein, and then fuse their envelope to the cell membrane to release its genome into the host cell cytoplasm and then replicate its genome inside the cell [54]. observations indicate that the furin-cleavage site reduces the constancy of protein s in sars-cov-2 and provides the essential conformational for binding to the ace2 receptor [55]. furin is present in all vertebrates as well as many invertebrates and requires calcium for its enzymatic activity [56]. this enzyme is a type of serine protease that cleaves the amino acid sequence -arg-x-x-argconsensus cleavage site r ‐ x ‐ k / r ‐ r ↓, where x represents any amino acid except cysteine and rarely proline [56]. seven distinct families from the family of proprotein convertases find out in mammalian species, furin is one of them. this family processes and activates essential biological functions. furin is an endoprotease that expresses in all tissues. it is located in the trans-golgi network, and some proprotein located between other parts of the cell and at the cell surface. furin can cleave protein precursors labeled with specific sequences, including proteases of the blood-clotting, complement systems, matrix metalloproteinase, receptors, viral-envelope glycoproteins, and bacterial exotoxins [57]. this protein requires the activity of pathogens such as bacterial toxins, enveloped viruses such as the human immunodeficiency virus (hiv), ebola, and sarscov-2. certainly, it is also involved in increasing the pathogenicity of the virus because cutting the target site, activates the virus functionally [58]. in vitro studies have shown that suppression of furin can decrease viral infections [59]. the use of furin suppressors has been shown to protect host cells against furin-dependent viral infections, providing a new basis for host cell-based treatment for acute diseases [60]. one study showed that the use of the mi-1851 inhibitor in calu-3 epithelial cells strictly concealed sars-cov-2 replication, and the mixture of tmprss2 and furin inhibitors had a better result against the activity of this virus [61]. consequently, furin inhibitors can use as a preventative and therapeutic agent against this virus [62]. 5. conclusion various proteolytic enzymes either of the host or the virus act in a 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screening methods amin mohsenzadeh1, alireza fazel1, shirin bavari2, sanaz borji3, sahar pourasghar3, taher azimi4, hoda sabati5,* 1department of microbiology, ardabil branch, islamic azad university, ardabil, iran 2department of microbiology, faculty of medicine, iran university of medical sciences, tehran, iran 3department of biology, faculty of science, rasht branch, islamic azad university, rasht, iran 4department of pathobiology, faculty of public health, tehran university of medical sciences, tehran, iran 5biotechnology and biological science research center, shahid chamran university of ahvaz, ahvaz, iran abstract biofilm producer bacteria cause nosocomial, chronic, and recurrent human infections. it is hard to treat biofilm-embedded bacteria because they are more resistant to antimicrobials than planktonic bacteria. the present study aimed to investigate different methods for detecting biofilms in the clinical isolates of escherichia coli and pseudomonas aeruginosa. the study was carried out at the department of microbiology, ardabil branch, islamic azad university, ardabil, iran, from january 2019 to june 2019. a total of 320 clinical samples were collected from educational and medical centers in tehran, iran which from them 100 isolates of e. coli and p. aeruginosa were identified by standard microbiological procedures and subjected to biofilm detection methods. biofilm detection was tested by congo red agar (cra), tube method (tm), microtiter plate assay (mtpa). the mtpa was considered to be superior to cra and tube tm. from the total of 100 clinical isolates, mtpa detected 38 (38%) isolates as biofilmpositive phenotype, of which 30 as strong, and 8 as moderate biofilm-forming isolates. it can be concluded from the current study that the mtpa is a more quantitative and dependable assay for the detection of biofilm-forming microorganisms as compared to other methods, and it can be recommended as a general screening method for the detection of biofilm-producing bacteria in laboratories. keywords: biofilm detection, microtiter plate assay, congo red agar, tube method 1. introduction microorganisms can live in one of two phenotypes: sessile or plankton. planktonic are freefloating microorganisms. the sessile phenotype results from the binding of microorganisms to solid surfaces, their irreversible binding, and the development of exopolysaccharides with a gradual * corresponding author: hoda sabati, msc biotechnology and biological science research center, shahid chamran university of ahvaz, ahvaz, iran tel/fax: +98 922 6092051 email: h.sabati@yahoo.com https://orcid.org/0000-0002-4473-7245 received: march, 27, 2021 accepted: april, 08, 2021 increase in cell secretions' thickness [1]. a biofilm is a collection of microbial cells that are irreversibly enclosed in a polysaccharide matrix and bonded together on biotic surfaces such as host cells or abiotic surfaces such as medical devices [2]. microbial cells that grow in a biofilm are physiologically different from planktonic cells of the same organism, which, in © the author(s) 2021 https://jcbior.com/ mohsenzadeh et al. 2 contrast, are single-cell and may float or swim in a liquid environment [3]. biofilms are considered a serious public health problem due to the increased resistance of the constituent organisms to antimicrobial drugs and the ability to cause infection in patients who have been implanted with medical devices [4, 5].the biofilm matrix blocks the penetration of antibiotics and prevents them from reaching the cells inside the biofilm [6]. studies have shown that bacterial biofilms cause 80% of microbial infections that are very difficult to eradicate and treat with antibiotics [7]. one of the most important pathogens that produce strong biofilms is escherichia coli. previous studies on the survival of biofilm-related pathogens also indicate that biofilms may play an essential role in the persistence and release of stress-sensitive organisms into the environment. the results show that e. coli resists food deficiency conditions, and the interaction of this bacterium with pseudomonas aeruginosa can increase e. coli growth in biofilm [8]. biofilm formation contributes to the pathogenesis of p. aeruginosa in acute as well as chronic infections in clinical cases [9]. p. aeruginosa is currently considered the leading cause of ventilator-associated pneumonia (vap) in the icu. infections caused by this bacterium occur mainly in patients with critical condition and immunodeficiency and increase the morbidity and mortality in these patients as well as increase the incidence of resistance of p. aeruginosa to antibiotics [10]. to control chronic and recurrent infections, it is essential to identify microorganisms' biofilms, maturity, and dispersion [11]. there are various laboratory methods to evaluate the production of biofilm in bacteria. biofilm growth and observation with microscopes such as confocal scanning laser microscope (clsm), quantitative method of closed systems under static conditions such as tissue culture plate (tcp) method and observation with non-specific color, floating biofilms (air liquid interface (ali)), bioluminescent assay, observation of bacteria colonies on the surface of a solid medium such as congo red agar (cra) and the simple tube method (tm) are among the biofilm measurement methods [12]. microtiter plate assay (mtpa) is a screening method for comparing adhesion patterns. it is the most widely used quantitative method for detecting biofilm production. in the cra method, a positive result is shown by the black colony and the dry crystal consistency. a pink colony appears in the absence of biofilm [13]. laboratory study of biofilm production by bacteria can be affected by the choice of a specific culture medium. the conditions that the bacterium encounters during growth can encourage or suppress biofilms production or cause biofilms with unusual structures [14]. this study aimed to investigate the methods (microtiter plate methods, tube method, and congo red agar) for the detection of biofilm formation in the clinical isolates of e. coli and p. aeruginosa. the sensitivity and specificity of the methods have been determined through statistical analysis. the mtpa method was considered the gold standard for this study and compared with data from tm and cra assays. true positives were biofilm producers by mtpa, tm, and cra assays. false-positive were biofilm producers by tm and cra assay and not by mtpa method. false-negative were the isolates that were non-biofilm producers by tm and cra but were producing biofilm by mtpa method. true negatives are those which were non-biofilm producers by all the methods. 2. materials and methods 2.1 sample collection out of 320 clinical samples collected from educational and medical centers in tehran, iran from january 2019 to june 2019, 50 isolates of e. coli and 50 isolates of p. aeruginosa were identified and subjected to biofilm formation detection. these isolates were identified by, colonial morphological analysis on eosin methylene blue (emb) agar (for e. coli isolates) and cetrimide agar (for p. aeruginosa isolates) and standard microbiological procedures such as gram staining, catalase test, cytochrome oxidase reaction, motility, and biochemical tests. these isolates were selected for biofilm formation evaluation. reference strain of positive biofilm producer p. aeruginosa atcc 9027, e. coli atcc 25922 were used as controls. 2.2 microtiter plate assay (mtpa) each of the collected and reference strains was cultured on tryptic soy agar (tsa) (merck, germany) medium and incubated overnight at 37°c. fresh colonies were cultured in 10 ml tryptic soy broth (tsb) (merck, germany) medium in a sterile tube and mohsenzadeh et al. 3 incubated for 15-18 h at 37°c with a shake at 200 rpm. the optical absorption of each liquid culture was adjusted using a fresh medium with an od of 0.1 at 620 nm. bacterial cultures were diluted, and a suspension equivalent to half mcfarland was prepared in tsb medium. 200 μl of the suspension prepared from each bacterial strain was transferred into 96 polystyrene plate wells. the plates were incubated at 37 °c for 96 h. the control organism was also incubated, diluted, and added to the culture plate. negative control wells contained inoculated sterile broth. after this time, each well's contents were removed and washed twice with phosphate buffer solution (pbs) (ph 7). this removed free-floating bacteria. after drying, the wells were fixed with 95% methanol for 10 minutes and then stained with crystal violet (1%) for five minutes. the plates were emptied, and an excess stain was washed by sterile distilled water. the wells were filled with 100 μl of glacial acetic acid solution (33%) and biofilm formation has been evaluated at 570 nm with an elisa auto reader (biotek elx800, usa) [9]. 2.3 congo red agar (cra) the congo red indicator (8 g/l) was prepared separately from the other medium constituents as a concentrated aqueous solution and autoclaved (121°c for 10 minutes). then it was added to the autoclaved muller hinton agar medium (merck, germany) (10 g/ml) with sucrose (50 g/l) at 55°c. test organisms were cultured on the cra medium and incubated aerobically for 24 48 h at 37 °c. black colonies with a dry crystalline consistency indicated biofilm production [1]. 2.4 tube method (tm) a loopful of the bacterial suspension was transferred to a tube containing 10 ml of lb broth (merck, germany) medium with glucose (2%). the tubes were incubated for 24 h at 37°c. the contents of the tubes were then removed and washed with pbs (ph 7.3). then the dried tubes were stained with crystal violet (1%). excess dye was washed with sterile distilled water. tubes were dried in the inverted position. isolates forming slime or biofilm-based on crystal violet color's thickness on the tube's bottom and the wall was observed and recorded. the observation of a visible thin layer on the wall and bottom of the tube was considered a moderate biofilm. the observation of a thick layer visible on the wall and bottom of the tube was considered a strong biofilm. the lack of a visible layer on the wall and bottom of the tube was considered non-biofilm formation and negative. the experiment was performed in three replications [3]. 2.5 statustical analysis the results were entered into microsoft excel 2016 and were analyzed by using spss version 25 (spss inc., chicago, il, usa). spearman's correlation test was used to define the direction of association. pvalue <0.05 was considered statistically significant. 3. results of the 100 e. coli and p. aeruginosa isolates, 38 (38%) isolates showed a biofilm-positive phenotype under the optimized conditions in mtpa (figure 1) and isolates were further classified as strong, moderate, weak, and no biofilm (table 1). cra assay led to different results, only 26.3% of isolates showed black colonies with dry crystalline morphology after 24-48 hours (table 2). though the cra assay can well detect biofilm-producing strains, weak producers were difficult to discriminate from biofilm negative isolates. no direct correlation between colony morphology on cra and mtpa results was observed. in tm, strong biofilm producers could be easily detected, whereas it was difficult to differentiate between moderate and weak biofilm-forming isolates which affected its performance in terms of sensitivity and specificity. statistical analysis of tm and cra methods for detection of biofilm formation in isolates using mtpa as a gold standard (n=100) showed that tm assay has more sensitivity and specificity than the cra method (table 3). biofilm formation in the isolates by either of the methods was 16% and 60% for e. coli and p. aeruginosa, respectively. discussion biofilm-producing bacteria cause many chronic and recurrent infections that are difficult to treat. treatment and control of biofilm-associated infections is an essential issue in hospitals and medical care. these bacteria exhibit resistance by different assays such as restricted penetration of antibiotics into biofilms and expression of resistance genes by facilitating the exchange of plasmids responsible for drug resistance due to proximity of cells within mohsenzadeh et al. 4 biofilms. there are a lot of different assays for detecting biofilm [15, 16]. in this study, we evaluated 100 isolates by three screening methods for their ability in biofilm formation. in the mtpa, the number of isolates showing biofilm formation was 38 (38%) (30 strong and 8 moderate) and no weak biofilm producers were detected. a previous study showed from the 50 clinical figure 1. biofilm formation detection in pseudomonas aeruginosa by microtiter plate assay 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 o p ti c a l d e n si ty ( o d ) a t 5 7 0 n m no. of isolate control+ s54 s66 s57 s62 s40 s59 s43 s35 s53 s63 s45 s46 s52 s19 s61 s15 s14 s48 s49 s4 s21 s18 s8 s64 s22 s47 s34 s16 s26 control table 1. interpretation of biofilm formation by average od value in microtiter plate assay biofilm formation od isolate (at 570 nm) odc isolates strong od isolate>0.929 od isolate>4×odc e41, e42, e43, e45, e48, e58, e60, s66, s57, s62, s40, s59, s35, s53, s63, s46, s34, s16, s52, s19, s61, s15, s48, s4, s21, s18, s8, s26, positive controls moderate 0.464<od isolate≤0.929 2×odc <od isolate≤4×odc s54, s43, s45, s47, s14, s49, s64, s22 weak 0.232<od isolate≤0.464 odc <od isolate≤2×odc no biofilm detected od isolate≤0.232 od isolate≤ odc optical density cut-off value (odc) = average od of negative control + 4x standard deviation (sd) of negative control = 0.232 standard deviation (sd) = 0.03214 e: e. coli; s: p. aeruginosa table 2. biofilm formation (38 isolates) by each of the methods biofilm formation mtpa no. (%) tm no. (%) cra no. (%) strong 30 (78.95) 13 (34.21) 10 (26.32) moderate 8 (21.05) 13 (34.21) 14 (36.84) weak/none 0 12 (31.58) 14 (36.84) mtpa: microtiter plate assay, tm: tube method, cra: congo red agar table 3. statistical analysis of tm and cra methods for detection of biofilm formation in isolates using mtpa as a standard (n=100) detection methods sensitivity specificity tm 70% 83% cra 50% 71% mtpa: microtiter plate assay, tm: tube method, cra: congo red agar mohsenzadeh et al. 5 isolates, 32 were biofilm producers (3 strong and 29 moderate) and 18 were weak/non-biofilm [17]. in another study, of the 50 isolates, 25 each were environmental and clinical. the number of biofilm producers reported in cra and mtpa was 7 (14%) and 30 (60%), respectively which is consistent with the results of the current study [12]. in the current study with the cra assay, 29 (76.32%) were found to be biofilm-producing bacteria and 9 (23.68%) as nonbiofilm producers. the cra method showed little correlation with the other methods. siddhiqui et al. reported that the cra method is not appropriate for biofilm detection. out of 112 clinical isolates, cra detected only 14.25% as biofilm producers as compared to tm which detected 29.46% as biofilmforming bacteria [1]. the tube method also detected 38% isolates as biofilm producers. this method correlated with mtpa for identifying biofilm producers, but it was difficult to differentiate between high, moderate, and weak producers due to the variability in the results detected by different observations. several studies recommend the analysis of biofilm formation via tm by different observers [18, 19]. tm and cra are considered to be less sensitive than the mtpa, as some clinical isolates that were non-adherent, weak, or moderate biofilm producers in the tm and cra assays showed strong biofilm patterns in the mtpa. so mtpa was selected as a gold standard for statistical analysis. in the current study sensitivity and specificity of tm were 70% and 83%, respectively. for the cra assay, sensitivity and specificity were lower at 50% and 71%, respectively. so following the previous studies, tm and cra cannot be recommended as general screening tests to identify biofilm-producing isolates since they can yield false positive or false negative results [12, 16, 20]. no significant relationship can be reported from the correlation of biofilm formation with organisms isolated because other factors such as the isolation source or the type of infection, antibiotic resistance, and genetic, physiological, and biochemical differences of the isolates should be considered to investigate the relationship between the genus of the isolates and the techniques used to identify biofilm formation. conclusively, mtpa is a more reliable quantitative assay for detecting biofilm formation among clinical isolates. the tube method may also be considered in a fast screening of biofilm producers. hence, measures for the early detection of biofilm producers in hospital environments and patients should be considered. this will decrease the rate of mortality of infections that are seemingly untreatable due to the presence of biofilms. limitations encountered in this study were sampling, analytical and equipment limitation to investigate other screening and confirmatory assays of biofilm formation and not considering the effect of gene factor in biofilm formation. prevention and control against potential biofilm forming clinical pathogenic isolates is an essential step towards the management of chronic and recurrent infections. there are different biofilm detection methods like mtpa, tm, and cra. a suitable method that is cost-effective easy to do and requires less technical expertise is the need of the hour. results of the current study showed that mtpa is a suitable and reliable assay that is more efficient than the other detecting biofilm formation methods. considering the ease of doing the test, rapidity, and cost-effectiveness, the cra method and tm can be considered for biofilm detection but not as general and reliable detection methods. author contributions all authors contributed equally to this manuscript and approved the final version of the manuscripts. conflict of interests the authors declare that they have no conflicts of interest. ethical declarations this study received ethics approval by ardabil university of medical sciences (approval code: ir.arums.rec.1398.022). financial support none to be declared. references 1. siddhiqui s, afreen u, kotgire s. evaluation of biofilm formation by three different methods and its antibiogram with special reference to indwelling medical devices from a tertiary care hospital. ann pathol lab med. 2018; 5(2):171-76. 2. giaouris e, heir e, hébraud m, chorianopoulos n, langsrud s, møretrø t, et al. attachment and biofilm formation by foodborne bacteria in meat processing environments: causes, implications, role of bacterial interactions and control by alternative novel 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https://pubmed.ncbi.nlm.nih.gov/26557518/ https://pubmed.ncbi.nlm.nih.gov/23326076/ https://pubmed.ncbi.nlm.nih.gov/23326076/ https://pubmed.ncbi.nlm.nih.gov/23326076/ https://pubmed.ncbi.nlm.nih.gov/23326076/ journal of current biomedical reports jcbior.com volume 2, number 4, 2021 eissn: 2717-1906 1 original research studying the suppressing effect of mesenchymal stem cells derived from amniotic membrane on colorectal cancer niloofar mesbah1, nooshin barikrow2,*, masoumeh heshmati2 1pharmaceutical sciences research center, tehran medical sciences branch, islamic azad university, tehran, iran 2department of molecular and cellular sciences, faculty of advanced sciences and technology, tehran medical sciences branch, islamic azad university, tehran, iran abstract mesenchymal stem cell therapy is considered as a proper tool for biological activities and treatment of diseases and cancer. here, the suppressing effect of mesenchymal stem cells derived from the amniotic membrane (am-mscs) on colorectal cancer (ht29 cell line) was studied. mscs were isolated from the human amniotic membrane and identity tests were performed. am-msc and ht29 cells were cocultured and mtt assay was used to determine proper inhibitory concentration. the apoptotic effect of am-msc on ht29 cells was studied by acridine orange staining. expression of cdk2 protein and the antioxidant effect of am-mscs on ht29 was performed by immunocyto chemistry test. the effect of msc on the ht29 cell line migration was determined by scratch test. the result showed that am-msc had proliferation-inhibitory effects and caused apoptosis of the ht29 cell line. real time-pcr assay showed the increase of gene expression of p53, caspase3 in apoptosis, and p21 in tumor cell line treated with am-msc. am-msc arrested the cell cycle of tumor cells by reduction of cdk2 protein. the migration of ht29 and ros expression levels was decreased. altogether, am-msc may play a role in suppressing colorectal cancer ht29 cell line by inhibiting angiogenesis, cell cycle, and induction of apoptosis and has antioxidant properties. keywords: amniotic membrane, colorectal cancer, co-culture, mesenchymal stem cell 1. introduction colorectal cancer (crc) is known to be the third deadliest cancer in the world, accounting for 9.7% of all cancers [1, 2]. crc progresses slowly, at first as a mass in the internal rectum, and then continues to grow as unusual growth in the cells, which can then extend to the blood vessels, increasing the chance of distant metastasis to liver and lung sites [1, 3, 4]. although great progress made in clinical treatment and diagnostic developments, effective treatment for this cancer is now a major challenge because metastasis and recurrence of tumors are two important factors in *corresponding author: nooshin barikrow, phd department of molecular and cellular sciences, faculty of advanced sciences and technology, tehran medical sciences branch, islamic azad university, tehran, iran tel/fax: +98 21 88991117 email: nooshinbarikrow@gmail.com http://orcid.org/0000-0001-9367-3885 received: february, 18, 2021 accepted: november, 08, 2021 colorectal cancer [5, 6]. inhibition of apoptosis and uncontrolled proliferation of tumor cells are considered to be factored in the development of cancer [5, 7, 8]. there are many ways to treat cancer, including chemotherapy, radiation therapy, and surgery. due to the side effects of each of these methods, they should be replaced with new therapies [9]. for this reason, cell therapy in various ways over the last few years has been very much considered. in the cell therapy process, a wide array of cells are used, one of the most important is mscs, mesenchymal stem cell therapy is © the author(s) 2021 https://jcbior.com/ mesbah et al. 2 considered as a suitable tool for biological activities, treatment of diseases, and also cancer [7, 10, 11]. mscs are multipotent cells, which have selfrenewal ability and differentiate into different cells; they have unique immunological properties that make them a powerful source of clinical treatment [10, 12]. among different types of stem cells, mesenchymal stem cells (mscs) have numerous benefits and can differentiate into different cells or generally to mesodermal and nonmesodermal lineages. there are many types of non-tumor cells in the tumor microenvironment (tme) that affect the function of the tumor, mscs are an important part of the tme. they can be used to transmit inhibitory and pro-apoptosis agents to the tme, they communicate with the host cell through the desmosomes, tight junctions, adherens junctions, gap junctions and release antitumor paracrine factors which can cause cancer cell apoptosis and resensitization [12-14]. they play an important role in suppressing cancer tumors by inhibiting angiogenesis, suppressing wnt signaling, and increasing inflammatory infiltration [12]. mscs can be isolated from a variety of sources such as bone marrow, adipose tissue, cord blood, and amniotic membrane (am). one of them is the amniotic membrane, access to which is noninvasive, reduced donor damage, less dna damage, and doesn't decrease in the capacity and the amount of mscs with the increase in age despite other sources. also, because of their greater self-renewal ability which differentiates mscs from other sources, and for a paucity of study on these types of cells, we studied them from this source [15-18]. in recent years, according to the biological roles and specialized capacities of stem cells and amniotic membrane, special attention has been paid to their use in both the medical and clinical settings [11, 19]. am membrane consists of a thick base membrane layer and an avascular stroma layer; there are two types of stem cells in this membrane, aescs, and am-msc. ammscs are located in an avascular stroma. am membrane is considered to be a postpartum tissue, which can be used for cell therapy due to its antiinflammatory properties, low immunity, antimicrobial, anti-apoptotic, and anti-angiogenesis properties [19-22]. in this research, the effect of mscs on inhibition of cell cycle was studied by assessing the gene expression of p21, cdk2 protein, and induction of apoptosis by studying the gene expression of p53 and caspase3, the mechanism of p21protein: it is regulated and activated by p53, this protein binds and inhibits the activity of cdk2 at the end of the cell cycle in g1 phase, and also caspase-3 is activated in the apoptotic cell both by intrinsic and extrinsic pathways. in general, the kinase (pi3k)/akt and wnt/β-catenin signaling pathways control cell survival, proliferation, growth, migration, and cell metabolism. researches indicated that in some cancers, mscs exert their apoptotic effects and inhibitory activity by activating the jnk apoptosis signaling pathway and inhibiting the akt/p13k pathway [23, 24]. due to the importance of colorectal cancer and the clinical application of am-mscs, we study the suppressing effects of am-msc on colorectal cancer. 2. materials and methods 2.1 cell line the human colon adenocarcinoma ht29 cell line was purchased from the cell bank of pasteur institute of iran. the cells were cultured in a culture medium containing dulbecco's modified eagle's medium (dmem-lg), 10% bovine serum albumin (bsa), and 5% penicillin/streptomycin (all from gibco, usa). 2.2 mscs isolation and identification the human amniotic membrane was obtained from the cesarean section of the milad hospital after obtaining the consent of the pregnant women. all ethical principles were performed in accordance with the declaration of helsinki and study design approved by ethical committee of tehran medical sciences branch, islamic azad university. isolation of mesenchymal stem cells from the amniotic membrane was performed using alviano’s isolation protocol [25]. after isolating, the cells were cultured in dmem-lg, 10% fbs, and 5% penicillin/streptomycin. thereafter, cells were trypsinized with 0.25% trypsin/edta, centrifuged, washed twice with bsa/pbs, and incubated for 30 min with antibodies against cd105, cd90, cd73, cd29 and cd45, cd34. they were conjugated with primary antibodies with pe, fitc, and percp. the negative control staining was used a pe-conjugated mouse igg1 isotype, fitc-conjugated mesbah et al. 3 mouse igg1 isotype antibody and percp-conjugated mouse igg1 isotype antibody. flow cytometry was performed with bd facs calibur instrument (bd facslyric, germany). the cells were analyzed by flowjo 7.6 [26]. all material is from sigma aldrich, usa. 2.3 differentiation into osteoblast and adipocyte amniotic mesenchymal cells are adherent cells and are propagated and maintained in tissue culture between 5-10 passages. cells in passage 6 were prepared for differentiation into osteoblast and adipocytes. in the osteoblast differentiation plate, collagen was first added, after 24 h, collagen was removed. then am-msc cells were added with the differentiation medium in osteoblast and adipocytes plates, after 21 days of differentiation, the alizarin red staining was used to check the differentiation of osteoblast, and the oil red o staining was used to check the adipocytes (all from sigma aldrich, usa) [27]. 2.4 mtt assay msc was cultured to study the inhibitory effect of am-msc on the proliferation of ht29 cell line. ammscs were treated with 50 mg/ml mitomycin c (mmc; sigma aldrich, usa) at 37°c for one hour to inhibit the proliferation. the cells were trypsinized and transferred to a 96-well plate. after 24h, ht29 was added to wells with am-msc and without am-msc. the ratios of am-mscs to ht29 were co-cultured as 1:2, 1:5, 1:10 and 1:20; all groups had at least three replicate wells. after 24, 48, 72, and 96 h, 0.5 mg/ml mtt reagent was added to wells and incubated for 4 h until a visible purple precipitate was formed, then 150 μl of dmso was added and shaken for 5 min at room temperature and absorbance was read at 570 nm (sigma aldrich, usa) [28]. the formula for calculating the inhibitory effect of am-mscs on the ht29 cell line is presented below [29]. formula = (od ht29 – od co-culture) + od ammsc/od ht29 × 100 2.5 acridine orange staining (ao/eb) acridine orange will stain both live and dead cells and is a vital dye. after preparing co-culture am-msc with ht29, the cell culture was removed and washed with pbs, then the cells were fixed with 4% formaldehyde, 250 µl of cell suspension was mixed with 10 µl of ao/eb solution. the sample should be mixed before quantification and study. then 10 µl mixture was placed on a microscopic slide and examined with the olympus fluorescence microscope, 400 x objectives [30]. 2.6 real time-pcr assay total rna was extracted from the control ht29 cell, am-msc, and co-cultured using the total rna isolation system (qiagen, usa), and the ratio of absorbance values at 260 and 280 nm indicated an estimate of rna purity. in the next step, cdna was synthesized and samples were compared based on the expression of the gapdh gene (qiagen, usa). the primer is determined according to the most appropriate concentration found in the initial tests. also, deionized distilled water was added until the final volume reached 20 µl (supplementary table 1). the real time-pcr temperature program is shown in supplementary table 2. pcr products were subjected to electrophoresis in 2% agarose gel. then real timepcr was performed to measure the expression level of p21, caspase3, and p53 genes (supplementary table 3). 2.7 immunocytochemistry as a result of the mtt test, co-culture cells were prepared and fixed with 4% p-formaldehyde for 20 min at room temperature and incubated for one hour with blocking buffer which consists of pbs containing 10% goat serum and 0.3% triton x-100. in the next step, the primary antibodies diluted with pbs (1:100, ab32147) were added and incubated at 4°c for 24 h in the dark. after washing with pbs, the secondary antibodies diluted (1:150, ab7007) with conjugated fitc were added and incubated for 2 h. finally, the sample was washed and dapi was added. the images were captured with a microscope (olympus bx51, japan), 400x objective [26]. 2.8 measurement of reactive oxygen species (ros) a 1 µl of dcfh-da staining solution was added to1 ml of the co-culture and ht29 control cells suspension. cells were mixed and incubated for 15 min at 37°c in the dark, in the next step, cells were washed and resuspended in 300 µl of cold pbs, and then ros generation was monitored using flow cytometry. mesbah et al. 4 2.9 scratch assay in this test, the first 6 well-plate of am-msc was added, after 24 h of incubation, ht29 cancer cells were added to am-mscs and without am-mscs wells. after 72 h, the cells were washed once with pbs buffer, and the dmem medium containing 10% fbs was added to the cells. then with the tip of a pipette, scratches were created on the bottom of the wells. in the next stage, the images were taken by microscope (olympus bx51, japan) at the start and after 3 days of the test, the migration of cells from the gap in the coculture and control samples was compared. 2.10 statistical analysis all experimental data were analyzed with spss software version 23 (spss inc., usa). all data are presented as mean ± standard deviation. for comparison and study of more than two groups, oneway anova was performed, and also values of p< 0.05 were considered statistically significant. 3. results 3.1 characterization of cultured am-msc expressed surface markers of am-msc were studied by flow cytometry. the expressed am-msc markers are as follows: cd105: 99.8%; cd90: 81.1%; cd73: 98.2%; cd29: 98.5% and the negative markers are: cd45: 1.22% and cd34: 2.88% (figure 1). 3.2 differentiation of am-msc into osteoblast and adipocyte the results showed that am-msc cells are affected by differentiation and the culture can differentiate am-msc into osteoblast and adipocyte tissue (figure 2). the cells in passage 5 were exposed to the adipocyte differentiation medium and then stained using oil red o, figures 2 are inverted under a microscope. the red dots are fat vesicles that indicate the differentiation of amniotic membrane stem cells into adipocytes. also, the cells in passage 5 were exposed to the differentiating medium of the bone and then stained using alizarin red dye. the red dots indicate calcium accumulation, indicating differentiation of amniotic membrane stem cells into osteoblast (figure 2). 3.3 mtt result mtt assay was used to determine the proper inhibitory concentration in vitro inhibition of ht29 cell growth by am-msc. co-culture am-msc with ht29 has performed the ratios 1:2, 1:5, 1:10, and 1:20, at times 24, 48, 72, and 96 h, using the mtt method and read absorbance at 570 nm. also, the inhibitory effect of mscs on the viability of tumor cells was expressed as follows: inhibitory rate (%) = (od570 of tumor cells od570 of the tumor with mscs + od570 of mscs control)/od570 of tumor cells x 100%. the result indicated am-msc significantly inhibited of proliferation ht29 cells, a proper concentration of (am-msc: ht29) 1:5 for 72h was determined (30%, p<0.05). based on the results am-msc showed antiproliferative effects against ht29 cell line (figure 3). 3.4 acridine orange staining am-msc cells were co-cultured with ht29, acridine orange staining test was performed to determine the apoptotic effect of am-msc on ht29 cells. the green area represents living cells and the orange area represents apoptotic cells, the figure indicated apoptosis of cell line ht29 was increased after co-culture with am-msc. however, don't occur apoptosis in ht29 and am-msc control cells. the results showed that am-msc cells induce apoptosis in cell line ht29 (figure 4). figure 1. characterization of cultured am-msc, studied expressed surface marker by flowcytometry. mesbah et al. 5 3.5 real time-pcr after am-msc cells were co-cultured with ht29, to evaluate gene expression of p21, p53, and caspase3 for apoptosis and cell cycle, tumor cells were used. in the real time-pcr assay, the gapdh was control of internal gene expression on all cells group. the results showed that in the co-culture sample, the genes expression of p21: 27.83 fold, p53: 1.64 fold, and also caspase3: 9.25 fold was significantly increased compared with the ht29 control cell line (p <0.001) (supplementary figure 1). 3.6 immunocytochemistry an immunocytochemistry test was performed to determine the level of cdk2 protein in ht29 cells after co-culture with am-msc, the results indicated that the protein expression of cdk2 in tumor cells in the co-culture sample was significantly reduced by 63% compared with the ht29 control cell line (p <0.001). the results showed that am-msc cells inhibited the cell cycle at the end of the g1 phase by reducing cdk2 on tumor cells (figure 5). 3.7 measuring reactive oxygen species after co-culturing ht29 with am-msc, to study the antioxidant properties of am-msc on ht29 cell line, the expression of ros was measured by flow cytometry. the result showed significant reduction of ros expression in co-cultured cells (fluorescence amount = 223) compared with ht29 control cells (fluorescence amount = 233) (p <0.05) (figure 6). the results indicated that am-msc can induce antioxidant properties by reducing ros in ht29 tumor cells. 3.8 scratch assay after co-culturing am-msc with ht29, the scratch assay was used to examine the effect of ammsc on the ht29 cells migration. the scratch test showed that ht29 co-cultured with am-msc did not migrate on the wounding space between cells but the ht29 control cell line migrated and filled the wound, figure 2. differential am-msc to osteoblast and adipocyte. after staining alizarin red, the pointers indicated calcium accumulation. (a): am-msc control cells. (b): am-msc differentiated into osteoblast cells. after staining oil red o, the pointers indicated adipose vesicles. (c): am-msc control cells. (d): am-msc differentiated into adipose cells (x 25). the results demonstrated that am-msc cells have the ability differentiate to osteoblast and adiposity tissue. figure 3. mtt assay was performed to determine the proper inhibitory concentration in vitro inhibition of ht29cell growth by am-msc. (am-msc: ht29) 1: (1:2), 2: (1:5), 3: (1:10), 4: (1:20). am-msc significantly inhibited of ht29 cells, a proper concentration of (am-msc: ht29) 1:5 for 72h was determined (30%, p <0.05). mesbah et al. 6 the result indicated that am-mscs inhibited ht29 cell migration (figure 7). 4. discussion colorectal cancer is known as one of the deadliest cancer, despite the great medical progress, there is no effective treatment for this cancer. in this research, we examined the suppressing effect of am-msc on ht29 cell line, studied the effective induction of apoptosis, cell cycle arrest, migration, and antioxidant properties of am-msc cells on ht29 cells. the results showed that am-msc cells induced apoptosis by increasing the expression of the caspase3 and p53, inhibiting the cell cycle by increasing the expression of the p21 gene and decreasing the cdk2 protein. furthermore, these results showed that am-msc cells inhibited the migration of cancer cells and the induction of antioxidant activity to ht29 cancer cells by reducing the amount of ros. mscs are multipotent cells, which have selfrenewal ability and can differentiate into other tissues. am-mscs have unique immunological properties and figure 4. acridin orange staining. am-msc cells were co-cultured with ht29, acridin orange staining test was used to determine apoptotic effect. a, b, c: ht29 cell lines are co-cultured with am-msc cells. orange regions represent apoptosis of cell line ht29 and green area represent of living cells, don't apoptosis in d, e, f. the control cells ht29 and g, h, i cells control the am-msc cells (x400). the results indicated that am-msc cells induce apoptosis in cell line ht29. figure 5. immunocytochemistry test was used to determine the level expression of cdk2 protein in cell line ht29 after co-culture with am-msc. a: co-culture 1 (am-msc with ht29) 20% expression of cdk2, b: pi staining co-culture 1, c: co-culture 2 (ammsc with ht29) 40% expression of cdk2, d: pi staining co-culture 2, e: control cell line ht29 90% expression of cdk2, f: pi staining cell line ht29, g: control am-msc with 40% expression of cdk2, h: pi staining am-msc (x400). the results showed am-msc cells inhibition cell cycle by reduced cdk2 on tumor cells ht29. mesbah et al. 7 don’t reduce in the capacity and the amount of mscs with the increase in age despite other sources and also because of the greater self-renewal ability and differentiation of mscs from this source compared to others, this cell is suitable for the study of cell therapy [10, 15, 16]. however, the role of msc hasn't been determined about the support or suppressing of the tumor cell, but it may be attributed to tumor models, the animal host, the dose or timing of the mscs injected, and might be due to their activation status [31, 32]. according to previous studies, msc causes resensitization and cancer cell apoptosis by releasing antitumor paracrine factors that seek to release the chemoattractants from the tumor. also, mscs with the secretion of interleukin and interferons inhibit figure 6. measuring ros. after co-cultured ht29 with am-msc, to examined the antioxidant properties of am-msc measuring expression ros by flowcytometry. a: flowcytometry in ht29 control cells, and coculture cells. b: represents a reduction in the amount of ros in co-culture cells compared with ht29 control cells. ros mean of 233 indicate the fluorescence amount in control cells ht29, and 223 fluorescence amount in coculture cells. the results indicate am-msc induce antioxidant properties by reduce ros in tumor cells ht29. * = p <0.05 figure 7. scratch assay. after co-cultured ammsc with ht29 the scratch test was used to evaluate the effect of am-msc on the cell line ht29 migration. a: am-msc; b: cell line ht29. a: co-culture 0 day, b: co-culture after 3 day, c: cell line ht29 0 day, d: cell line ht29 after 3 day (x10). the result indicated cell line ht29 treatment with am-am-msc wasn't migrate on the wounding space between cells but control cell line migrated and filled wound, am-mscs inhibited the ht29 cell migration. mesbah et al. 8 cancer cells, il-18, which inhibits proliferation and cell division in tumor cells, il-15, which also inhibits the growth of tumor cell cells also leads to nk cell, cd8+, and lymphocyte t, and causes the immune system to react in tumor cells [14, 33, 34]. in addition, msc helps in the inhibition of tumors by inhibiting tumor proteins, activating various signaling pathways, and inducing apoptosis in tumor cells. mscs, by activating the jnk pathway and inhibiting the akt/pk13 pathway, induced apoptosis in cancer cells [35, 36]. abd-allah et al. found that msc can arrest cell cycle hcc cell line. the expression levels of p21 and p53 were down-regulated in the hcc cell line treatment with msc [36]. lu et al. examined the effect of mesenchymal cells isolated from bone marrow on the cell cycle of mouse h22 cancer cells. p21 gene expression was increased. this is similar to our result in this study. the results of fold increase in p21 gene expression in ht29 cancer cells compared to controls [29]. also, ahn et al. studied the effect of mscs derived adipose tissue on melanoma cancer cells; they found that at-msc-cm inhibited melanoma cells by arresting cell-cycle and inducing apoptosis [37]. hou et al. examined the suppressing effect of bm-msc cells on hepg2 hepatic cancer cells, they found that bmmsc inhibits the proliferation and promotes the apoptosis of hepg2 cells by down-regulating the gene expression of bcl-2, cmyc, β-catenin, and survivin. also, bm-msc inhibits the wnt signaling in tumor cells by secreting dkk-1 [28]. bm-msc-cm promotes colorectal cancer (cell line hct116) progression through ampk/mtor-mediated nf-κb activation and induces proliferation and survival by downregulating p21, p16, p53, bax and increasing protein expression of bcl2 [38]. further, these results of am-msc cells inhibited the migration of cancer cells and the induction of antioxidant activity to ht29 cancer cells by reducing the amount of ros. the role of mscs has not been determined regarding the support or suppressing of the tumor cell but because the mscs are an important part of the tme, they release antitumor factors, and also in this research, the dose and timing of the mscs were determined. the result indicated that am-msc can play a role in suppressing colorectal cancer (ht29 cell line) by inhibiting angiogenesis, suppressing the cell cycle, induction of apoptosis, and antioxidant properties. also, these cells, have an antitumor effect in acceptable medical ethical issue and non-invasive access are an appropriate option for the treatment of some cancers and proper cell for cell therapy, so further studies in this field seem to be necessary. supplementary files supplementary file 1. acknowledgements this research was supported by pharmaceutical sciences research center, tehran medical sciences branch, islamic azad university, and tehran, iran. we wish to thank nasim iran doost and parisa nikkhoo for their supports. author contributions the authors were equally involved in data collection and writing the article. also, all authors approved the final version of the manuscript. conflict of interests there is no conflict of interest. ethical declarations all ethical principles were performed in accordance with the declaration of helsinki and study design approved by ethical committee of tehran medical sciences branch, islamic azad university (ir.iau.ps.rec.1396.131). also, a signed informed consent was obtained from the patients for taking blood samples. financial support this research was supported by faculty of advanced science and technology, tehran medical sciences branch, islamic azad university, and tehran, iran (registered number: 1087). references 1. marley ar, nan h. epidemiology of colorectal cancer. int j mol epidemiol genet. 2016; 7(3):105-14. 2. pan t, xu j, zhu y. self-renewal molecular mechanisms of colorectal cancer stem 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https://pubmed.ncbi.nlm.nih.gov/17170725/ https://pubmed.ncbi.nlm.nih.gov/17170725/ https://pubmed.ncbi.nlm.nih.gov/17170725/ https://pubmed.ncbi.nlm.nih.gov/17170725/ journal of current biomedical reports jcbior.com volume 2, number 2, 2021 eissn: 2717-1906 1 original research study of phenytoin effect on the genes involved in glucose and lipid metabolism expression in liver: a mouse model study fatemeh samimi1, ali mohammad ahadi2,*, nafiseh boroomand2, farzaneh mohamadzadeh rostami3 1biochemistry laboratory, al zahra hospital, isfahan university of medical sciences, isfahan, iran 2department of genetics, faculty of science, shahrekord university, shahrekord, iran 3department of bacteriology and virology, faculty of medicine, isfahan university of medical sciences, isfahan, iran abstract phenytoin as an anti-seizure medication, is useful for the prevention of tonic-clonic seizures and focal seizures. in this study we focused on the probable effects of phenytoin drug on gene expression profile of liver related to lipid metabolism balance in mouse as a model. in this study, a group including 7 male mice of balb/c were treated with phenytoin 3–5 mg/kg/day orally and a group including 7 male mice of balb/c were took standard food. liver tissue samples were isolated. total rna was extracted and cdna was synthesized. expression of akt1, leptin, adipoq and glut4 genes was measured using realtime rt-pcr method. results showed an increase about 15 and 3 fold changes in akt1 (p <0.001) and adipoq (p <0.001) gene expression respectively in treatment group compare to control mice. also, we detected decreasing in leptin and glut4 genes expression in the mice treated with phenytoin drug. several studies indicated that phenytoin can promote hyperglycemia in human and animal. we proposed here that this effects may resulted from an interference between the phenytoin drug and gene expression profile in liver. decreasing of leptin level here may be a result of glucose level elevation in blood that can induce a satiety situation result in decrease of leptin production. it may that akt1 gene expression is increased to compensate the low level of glut4 protein. we concluded that phenytoin is a relatively high-risk antiepileptic drug for obesity and metabolic syndrome, but more studies are needed. keyword: phenytoin, akt1, leptin, adipoq, glut4 . 1. introduction side effects of drugs is a prevalent problem between the challenges of clinical medicine. many of medications are vital to treat diseases, but sometimes they have serious side effects [1]. phenytoin is one of the most effective drugs in treatment and prevention of acute repetitive seizures [2]. phenytoin is believed to control the seizures by its effects on the voltage-gated sodium channels. this reduces the activity of brain seizure centers by stabilizing the nerve cell membranes *corresponding author: ali mohammad ahadi, ph.d department of genetics, faculty of science, shahrekord university, shahrekord, iran tel/fax: +98 38 32324419 email: ahadi_al@sku.ac.ir https://orcid.org/0000-0001-9580-2740 received: april, 07, 2021 accepted: may, 15, 2021 [3]. this drug blocks the inflow of sodium ions through the membranes of the neurons, thereby limiting neurological impulses [4]. the most common side effects of phenytoin include headache, nausea, fever and enlargement of the gums. phenytoin consumption may be lead to serious side effects include sleepiness, liver problems and low blood pressure [5]. there are several investigations regarding the effects of phenytoin on the metabolism. studies have shown that phenytoin induce hyperglycemia and © the author(s) 2021 https://jcbior.com/ samimi et al. 2 insulin resistance in experimental animal models and human fetus [6, 7]. high levels of blood sugar can lead to complications such as diabetes and metabolic syndrome. diabetes mellitus is a group of metabolic disorder characterized by chronic hyperglycemia and has a significant impact on the health and quality of life in patients [8]. in the present study, we investigated the effect of phenytoin on mrna levels of glut4 (glucose transporter 4), akt1 (protein kinase b), leptin and adiponectin (adipoq) genes in the liver tissue of mouse treated with phenytoin as a models. insulin is a key factor for managing blood glucose levels by facilitating cellular glucose uptake in several important tissues such as skeletal muscle, liver, and adipose tissue, by activating the pi3k-akt signaling pathway [9]. glut-4 is a protein that plays a crucial role in metabolism of carbohydrates [10]. glucose uptake in peripheral tissues is mediated by insulin-dependent glut4. insulin stimulate the pi3k-akt signaling pathway and the activated akt kinase subsequently catalyzes the phosphorylation of akt1 substrate 160 kda (as160), responsible for glut4 translocation to the plasma membrane and allows glucose uptake to the cells [11]. akt1 was involved in this study because of its common role in signaling pathways that mediate the metabolic effects of insulin in numerous physiologically important target tissues [12]. also, one of the most important of hormones in regulation of lipid and glucose level of body is leptin that encoded by lep gene. leptin that previously known as obesity factor, is an adipocyte-derived hormone that plays an important role in the regulation of energy stores and food intake [13]. adiponectin/leptin ratio has been considered as a biomarker for detection of adipose tissue disorders. leptin is an index of fat mass, and adiponectin is an index for triglyceride metabolism and insulin sensitivity. the people with high adiponectin/leptin ratios have better triglyceride profile [14]. leptin may have some effects on specific peripheral targets including liver, and pancreas, adipocytes and skeletal muscle cells. leptin improves hepatic insulin resistance and hyperglycemia through decreasing gluconeogenesis, increases fatty acid oxidation. on the other word, leptin deficiency can lead to insulin resistance diabetes [15]. leptin can suppresses lipogenesis and glucose output in liver [16]. adiponectin is another hormone produced by the adipocytes and encoded by adipoq gene. this gene is down-regulated in obese diabetic patients. adiponectin enhances insulin secretion to inhibit hepatic glucose production by reducing the gluconeogenic gene expression, so it induces glucose uptake and promotes fatty acid beta oxidation [17]. an optimal level between leptin and adiponectin is critical for maintenance of normal metabolism. in leptin-deficient (ob/ob) mice, maintaining adiponectin at lean levels, as opposed to the widely observed drop of adiponectin in the obese state, increases fat mass, yet improves insulin sensitivity [18, 19]. given to the role of akt1, adipoq, lep and glut4 genes in regulation of glucose and lipid level of the blood, we think, phenytoin may influence the expression of these genes in the liver. main goal of this study, was quantitation of the change in expression of these genes after a median term treatment with phenytoin in mouse as an animal model. 2. material and methods 2.1. animals and drug treatment experiments were performed on adult male balb/c mice weighing 24±3 g. the animals were maintained under controlled room temperature (25 ± 2°c) and light and dark (12:12 hr) conditions and were given standard food pellets and water. before conducting the experiment, ethical clearance was obtained from the local ethical committee on animal research and ethical guidelines for investigations were followed in accordance with shahrekord university guidelines. experiments consisted of two groups of animals, seven in each group. group i (standard control), group ii (test drug, intraperitoneal injection, phenytoin 15-20 mg/kg). before injection, phenytoin was dissolved in distilled water and optimum concentration was calculated. the dose of the drug was calculated according to the manufacturer's recommendation per kg of body weight for a 25 g mouse. however, a conversion between mouse and human was carried out [20]. 2.2. liver tissue biopsy, rna extraction and cdna synthesis mice were anesthetized by intraperitoneal injection (ip) injection of pentobarbital (0.7%, w/v) and culled by exsanguination. livers were immediately excised under sterile conditions and directly subjected for total rna extraction procedures using rnxplus buffer (cinnagen co., iran) as samimi et al. 3 recommended by company manual. in the next step, cdna synthesis was performed on about 50ng rna by using the first strand cdna synthesis kit (add bio co., south korea) using oligo (dt) and random hexamer primers according to the manufacturer’s instructions. 2.3. primer design and real-time rt-pcr assay specific primers were designed using gene runner version 6.5.52, 64-bit beta software against akt1, adipoq, lep and glut4 genes that listed in table 1. also a pair primers was designed for actb gene as internal control or reference gene in relative gene expression analysis in real-time rt-pcr assay. the real-time rt-pcr assay was performed by using the one-step sybr green real-time rt-pcr (takara bio inc., japan). each 20μl reaction mixture contained 10 μl of 2x master mix, 0.5 μl of the 20 u/μl rnase inhibitor, 0.8 μl each of 10 μm forward and reverse primers and 1 μl of template cdna. amplification was carried out in 48-well plates on a real-time one-step rt-pcr machine (applied biosystems co., usa). thermal cycling conditions consisted of 10 min at 95°c and 45 cycles of 15 s at 95°c and 1 min at 60°c. melting curve was calculated between 65°c and 95°c with 0.3°c ramping rate. 2.4. gene expression and statistical analyses expression changes of studied genes was calculated based on 2-δδct method [21]. all results are given as the mean ± sem. kolmograph-smirnov method was used for normality assay of given data by spss version 26. probabilities of chance differences between two groups were calculated using graphpad prism version 9.0.0 with an indipendent t-test method. 3. results as described in material and method, total rna was extracted by using rnxplus solution and qualified in gel electrophoresis (figure 1). the extracted rna of 28s and 18s rrna shows an optimal quality. supplementary file 1 shows the melt curves for target genes amplicons. the single peak is typically interpreted as representing a pure, single amplicon. also, amplification plot for every genes is showed. as mentioned in this study, the expression changes of adipoq, glut4, leptin and akt1 genes as candidate genes involved in the metabolism of lipids and carbohydrates under the administration of phenytoin were measured in liver by the 2-∆δct method [18]. the statistical independent t-test method was carried out and the research hypothesis was tested and the results were presented. the distribution of data for table 1. primer sequences for used genes primer name sequence target length target gene accession no. mactf 5’ggactcctatgtgggtgacg-3’ 119bp actb nm_007393.5 mactr 5’aggtgtggtgccagatcttc-3’ faq1 5’-acttgtgcaggttggatgg-3’ 139bp adipoq nm_009605.5 raq1 5’-ctgtctcacccttaggacc-3’ flep 5’-cacacacgcagtcggtatcc-3’ 133bp leptin fj374142.1 rlep 5’-caggtcctcaccagcctgcc-3’ fgl4 5`aatgtcttggccgtgttggg -3` 118bp glut4 nm_009204.2 rgl4 5`gccctgatgttagccctgag -3` fakt1 5’-gcctgaggagcgggaagaatg-3’ 122bp akt1 nm_001331107 rakt1 5’-cttcagcccctgagttgtcac-3’ https://www.ncbi.nlm.nih.gov/nucleotide/nm_009204.2?report=genbank&log$=nuclalign&blast_rank=7&rid=n74wzpbm014 samimi et al. 4 the results of gene expression changes between control group and the animals treated with phenytoin was performed by kolmograph smirnov test. kolmograph smirnov test showed a significant equal with 0.2 that present normality of data distribution. based on the result of kolmograph smirnov test, independent t-test was considered as significant test for analysis of gene expression change. figure 2 (a-d) show the result of glut4, leptin, adipoq and akt1 genes expression analysis. 4. discussion in this study, we investigated the effect of phenytoin drug on the expression of four key genes in metabolism balance of lipids and carbohydrates. several studies indicated that phenytoin can promote hyperglycemia in human and animal [6, 22, 23]. in a study in the rats, phenytoin significantly increased fasting plasma glucose, insulin resistance index, total cholesterol and triglycerides compared with the control group [6]. this phenomena is very important in about diabetic patients and the peoples that affected by obesity. we proposed here that this effects may resulted from an interference between the phenytoin drug and gene expression profile in liver. as a simple view, adiponectin has been shown to enhance insulin sensitivity in several reports that result in entrance of glucose into the adipocytes and liver cells. in this situation, leptin hormone acts in reverse direction in releasing of glucose into the blood stream [18]. in our study, adipoq gene expression showed 15 times increase (p <0.001) compared to the control group. adiponectin level elevates with hyperglycemia independent of body size. optimization of blood glucose in the hyperglycemic patients that tolerate decrease in adiponectin levels is independent of diabetes type and/or body weight [24]. adiponectin played a critical role in regulating plasma glucose and reducing pancreatic islet apoptosis after gastric bypass surgery [25]. so, increasing of adiponectin that we reported in this study, may be a secondary or compensation result of hyperglycemia. in this situation, the body may changes its metabolic source from glucose consumption to lipid catabolism. other result of our study, decreasing of glut4 gene expression to 0.4 times (p <0.001) compare to control group, is compatible with this hypothesis. also, in this study, we investigated the expression change of adiponectin and leptin genes under the phenytoin diet. adiponectin/leptin balance is one of the most important indexes for an optimal metabolic condition in the body [26]. results of this study showed decreasing of leptin to 0.76 (p <0.05) compare to control group. this decreasing of leptin level after the phenytoin consumption, may be a result of glucose level elevation in blood that can induce a satiety situation result in decrease of leptin production. similar to our findings, some studies indicated that carbamazepine cause diminish in level of leptin in the patients’ blood [27]. carbamazepine and phenytoin are two common anti-epileptic drug. decreasing of glut4 gene expression that we reported here, must be considered beside of leptin diminish. low level of leptin in blood can lead to decreasing of insulin secretion and decreased glut4 gene expression. previous researches showed that changes in glut4 gene expression under the high figure 1. separation and analysis of rna. green viewer-stained 1% tbe agarose gel showing rna extracted from liver tissue of treatment (a: 1-7) and control group (b: 1-7) of mice. m: 100bp dna size marker. samimi et al. 5 level of camp is accompanied by similar alterations in glut4 protein expression and glucose uptake, suggesting a role of glut4 gene expression in regulation of cellular insulin role in glucose transport [28]. based on this findings, we proposed that decreased leptin that resulted from phenytoin drug may be act through a camp manner. as supportive reports, correlation analysis indicated that the decrease in glut4 gene levels was only observed in hyperglycemic mice. on the other hand, the glucose catabolic related genes do not exhibit any clear coordinate expression. agitated expression of glucose catabolic genes may contribute to hyperglycemia and muscle insulin resistance [29]. interestingly, a study showed that hyperglycemia correlate with increasing of glut1 protein, while, glut4 gene level is decreased under hyperglycemic condition [30]. it seems, because of a wide role of leptin hormone compared with restricted action of adiponectin, the expression changes in leptin gene shows lower variance. finally we analyzed the expression of akt1 gene after treatment of animals with phenytoin drug. our result showed a more than 3 times increasing in expression for akt1 gene compare to control group of mice (p <0.001). akt1 is expressed in many mammalian tissues. specially, during adipocyte differentiation in vitro, akt1 is expressed at a relatively high level in the early stages of adipogenesis, however its expression is gradually decreased at later stages. thus, it is possible that akt1 is necessary for the early stages of adipogenesis [31]. also. akt1 has a well figure 3. gene expression changes between control and phenytoin treated groups. a: glut4 gene showed 0.456 decrease in expression compared to the control group (p ≤0.001, sig.= 0.00). b. leptin gene expression, which decreased by about 0.76 (p ≤0.05, sig. = 0.01, fold change = 0.76152) compared to the control group. c: changes in the expression of adipoq gene, which is an increase of about 15 times (p ≤0.001, sig. = 0.00, fc = 14.78263) compared to the control group. d: akt1 gene expression with increase of more than 3 times (p ≤0.001, sig. = 0.00, fc = 3.25971) compared to the control group. samimi et al. 6 defined role in phosphorylation and translocation of glut4 protein [32, 33]. this role can explains increasing of akt1 gene expression under phenytoin consumption while glut4 gene level has decreased. on the other word, we think that, akt1 gene expression is increased to compensate the low level of glut4 protein. considering the finding of this study, it can be concluded that phenytoin is a relatively high-risk antiepileptic drug for obesity and metabolic syndrome, but more studies are needed. supplementary files supplementary file 1. acknowledgements we thanks shahrekord university and isfahan medical university for their collaborations. author contributions laboratory work 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https://pubmed.ncbi.nlm.nih.gov/31572181/ https://pubmed.ncbi.nlm.nih.gov/31572181/ https://pubmed.ncbi.nlm.nih.gov/31572181/ https://pubmed.ncbi.nlm.nih.gov/21401839/ https://pubmed.ncbi.nlm.nih.gov/21401839/ journal of current biomedical reports jcbior.com volume 2, number 2, 2021 eissn: 2717-1906 1 review use of nanotechnology in the diagnosis and treatment of coronavirus elham maghareh abed1, seyedeh mahsan hoseini-alfatemi2, hoda sabati3, mohammad amin khajavi gaskarei4, kourosh delpasand4, marzie ghasemi5,* 1department of microbiology, faculty of biological sciences, islamic azad university, north tehran branch, tehran, iran 2pediatric infections research center, research institute for children’s health, shahid beheshti university of medical sciences, tehran, iran 3biotechnology and biological science research center, shahid chamran university of ahvaz, ahvaz, iran 4razi clinical research development unit, guilan university of medical sciences, rasht, iran 5department of biochemistry, school of medicine, ahvaz jundishapour university of medical sciences, ahvaz, iran abstract coronavirus is a beta virus that has caused a worldwide pandemic since december 2019. many treatments such as antiviral drugs, immunosuppressive drugs, neutralizing antibodies, and monoclonal antibodies have been tested on coronavirus disease 2019 (covid-19) that most of them were effective. given that nanotechnology-based approaches have been successful in detection and treatment of viral systems such as human immunodeficiency virus (hiv), influenza a virus subtype h1n1 and middle east respiratory syndrome coronavirus (mers-cov), they also seem to be effective in detecting and treating covid-19. nanotechnology is used in various methods for early and rapid diagnosis of the disease. nanoparticles can be used in products for the diagnosis, treatment and prevention of covid19. these substances are very effective in the controlled delivery of antiviral drugs and biomolecules and they are also used in the manufacture of personal safety equipment, widely, and the production of anti-virus coatings for surfaces, air filters and the production of vaccines. in general, nanomaterial can play an important role in controlling the disease, based on strategies to prevent the virus from entering the host cell, inhibiting virus replication, virus delivery systems, and nano-based vaccines. nanotechnology is a multidisciplinary tool that can offer a variety of solutions based on disease prevention, diagnosis and treatment strategies. keywords: covid-19, nanotechnology, coronavirus, diagnosis, treatment 1. introduction the corona pandemic that has occurred in the world since the last days of 2019 is related to a virus called coronavirus-2019 (covid-19) and belongs to the beta virus family along with severe acute respiratory syndrome coronavirus-2 (sars-cov-2) and middle east respiratory syndrome coronavirus * corresponding author: marzie ghasemi, msc department of biochemistry, school of medicine, ahvaz jundishapour university of medical sciences, ahvaz, iran tel/fax: +98 933 6357377 email: marzieghasemi1985@gmail.com https://orcid.org/0000-0001-6753-8356 received: february, 21, 2021 accepted:, march, 13, 2021 (mers-cov) [1]. this virus has a single-stranded positive rna genome [2]. the virus attaches to the human angiotensin converting enzyme-2 (ace-2) and binds to the host cell. this enzyme is a receptor for the sars-cov-2 virus in the host cell [3]. they cause several diseases in humans and animals, including © the author(s) 2021 https://jcbior.com/ maghareh abed et al. 2 respiratory, intestinal, hepatic, and nervous systems [4]. the rate of human-to-human transmission of coronavirus is significantly high and causes a wide range of clinical manifestations in patients infected with the virus [5]. however, the reproductive number and transmission way are not yet completely understood, the routes of transmission with and without symptoms and the role of environmental factors should be better known. widespread geographical epidemics and prevalence in large populations of the world, rapid transmission, recurrent infections and genomic alteration [6, 7], create difficult barriers to the development of ways to prevent, control, and treat this disease [8, 9]. a wide range of possible treatments is being tested. based on the sars-cov-2 life cycle and its structure, the treatments proposed for covid-19 have been antiviral drugs, immunosuppressive drugs, neutralizing and monoclonal antibodies [10], and vaccines. antiviral drugs effect by inhibiting ribonucleic acid (rna) polymerase, viral protease, membrane occlusion, and widespread of antiviral effects [10]. currently, oxygen therapy and the use of the respiratory tract play a major role in the treatment of covid-19 [11]. recent clinical results for antiviral drugs have not been very satisfactory [12]. some drugs have also been suggested to reduce the damage caused by sars-cov2 to the lungs [8]. in some cases, plasma therapy is also suggested [13]. this method of treatment is used in patients who are not able to produce enough antibodies and is a safe and effective method and seems to reduce mortality [14]. scientists believe nanotechnology-based approaches could help against covid-19. based on previous studies, nanotechnology can be effective in identifying viral systems and overcome the limitations of conventional methods of viral disease strategies [15, 16]. in this study, in addition to reviewing the effects of nanotechnology in the fight against other viral diseases, the proposed nanotechnology-based solutions in the diagnosis, treatment, and prevention of environmental pollution caused by covid-19 are examined. 2. application of nanotechnology in the detection and treatment of viral infections according to previous research, nanotechnology has been considered in the process of diagnosis and treatment of many viral diseases. the use of luminescence electrochemical biosensors for the selective detection of avian influenza virus (h9n2aiv) [17] and the use of magnetic nanoparticles (nps) for the detection of influenza a virus subtype h1n1 virus [18] are examples of nanoparticles applications in virus detection. the use of poly vinyl pyrrolidone (pvp) coated agnps (30-50 nm) on hiv strains has also been shown to have antiviral activity [19]. curcumin nanoparticles also have high antiviral properties against the respiratory syncytial virus (rsv) [20]. dendrimers have also been extensively studied for human immunodeficiency virus (hiv) disinfection applications. these nanoparticles suppress virus replication [21]. lipid-based nanoparticles have previously been tested for the treatment of hiv, herpes, hepatitis b (hbv), and hepatitis c (hcv) viruses. these nanoparticles can encapsulate different classes of antiviral drugs and are a mediator of drug delivery to the target tissue or cell, as well as modulating specific biological responses [22]. aluminum nanoparticles are another class of metal structures that can be used in vaccines for respiratory viruses such as mers-cov and sarscov-2. these compounds elicit cellular and humoral immunity responses [23]. unsaturated liposomes also have an inhibitory effect against hbv, hcv, and hiv [22]. 3. sars-cov-2 and nanotechnology insights nanotechnology-based treatment strategies are a promising way to overcome the current limitations in the prevention, diagnosis, and treatment of covid-19 [24]. nanomaterials can be used for disease diagnosis to develop simpler, cheaper, and faster methods. in therapy, nanosystems can be used in the controlled delivery of antiviral drugs and biomolecules to the lungs and target tissues that are used to prevent virus replication or inactivation of viral particles [25]. there are successful prospects in building safe personal protective equipment [25] and producing vaccines or immune modulators using nanotechnology [16]. maghareh abed et al. 3 4. detection of sars-cov-2 with nanotechnology currently, the only way to diagnose covid-19 infection is to test for nucleic acid by reverse transcription polymerase chain reaction (rt-pcr). but this method also has limitations; it is not able to diagnose asymptomatic patients, not all health care centers, especially in non-urban environments, are equipped with sufficient pcr infrastructure. while accurate and early diagnosis of the disease is a vital requirement for essential care. nanotechnology can meet these demands through nucleic acid testing methods, point-of-care testing (poct), electrochemical sensors, and biosensors [15]. in the nucleic acid test method, the nucleic acid is amplified in nucleic acid isothermal conditions. this method is very sensitive, rapid, and specific [26]. nanomaterials exhibit electrical and optical properties that have been extensively applied in the development of diagnostic methods such as point-of-care testing (poct) biosensors. these substances can improve the sensitivity of diagnosis, rapid isolation of patients, and facilitate the treatment process [24]. different types of nanomaterials include; nanoparticles, nanoclusters, quantum dots, carbon nanotubes, nanocomposites, etc that have been used to increase the performance of biosensors. recently, deoxyribonucleic acid (dna) sensor nanomaterials have been developed for simple, rapid, selective, low-cost, and sensitive detection of infectious diseases [27]. biosensors are color-coded test points that can detect contaminated specimens through simple color changes that are visible to the naked eye [15]. a sample of these pocts has been generated using gold nanoparticles to detect the mers-cov virus [28]. in another method, reverse transcription loop-mediated isothermal amplification (rt-lamp) with nano-based biosensors has been used to detect covid-19 [29]. electrochemical sensors are effective in detecting viruses due to their high sensitivity and the possibility of shrinkage. gold nanoparticles (aunps) are one of the most attractive nanomaterials because they have electronic optical properties that can be detected by a variety of biosensors, especially those based on colorimetric, electrochemical [30]. the use of aunps in these sensors is much more effective in detecting viruses by immobilizing biomolecules while maintaining their function [31]. 5. treatment of sars-cov-2 with nanotechnology the specific antiviral drug for sars-cov-2 is not currently available to the public [32]. production of these drugs is a time-consuming process that must be monitored and tested for safety before use [33]. on the other hand, increasing virus resistance is another limitation of the production and use of antiviral drugs [15]. in some studies, nanotechnology has been widely applied in the treatment of this disease. nanotechnology through methods such as; preventing viruses from entering host cells, inhibiting virus replication, virus transmission systems, and nanobased vaccines is probably successful in treating the disease [15]. also, many of the proposed treatments for covid19 infection that are spreading, use nano-based methods such as; sars-cov-2 virus-like particles by using ibio's fast pharming technology[15]. nanoparticles (np) (organic and inorganic) have been considered for some of their properties. inorganic nps (inps) have properties such as luminescence, adjustable size, shape, composition, high surface-to-volume ratio, and the ability to expose multiple reciprocal locations on the surface. the most common types of inps are mesoporous silica nps, iron oxide nps and metal nps (gold, silver). organic nps include; carbon nanotubes and graphene nanoparticles are polymeric and lipid nps, dendrimers, extracellular vesicles (or exosomes), liposomes, and nanomolecules. organic nanoparticles are being noticed because of their potential sitespecific targeting, drug release control, biodegradation, biocompatibility, and non-toxicity [34]. 6. pharmacological treatment the first step in starting a viral infection is the virus's attachment to the host. nanoparticles have the ability to prevent the virus from adherent to the host cell and entering the cell [35]. one way to use nanoparticles to prevent the virus from entering the host cell is to use carbon quantum dots that if combined with boronic acid show stronger antiviral activity [36]. the use of gold nano rods in antiviral therapy for mers has also been successful [37]. maghareh abed et al. 4 second, if the virus enters the host cell, the spread of infection can be prevented with nanoparticles, mainly as carriers for the delivery of antiviral molecules. higher specificity, improvement of drug solubility, the combination of different drug molecules in one particle, and reduction of toxicity for the host are the advantages of nanoparticles in this field [15]. some nanoparticles, such as ag2s nanoclusters, can prevent the virus from replicating in the host body. these nanoparticles also play a role in enhancing the expression of pro-inflammatory cytokines, which are effective against viral infections [38]. zinc oxide (zno) nanoparticles have similar activity and effective against h1n1 influenza virus infection [39] and it also seems to be involved in inhibiting the proliferation of sars-cov-2 [40]. traditional methods of prescribing antiviral drugs have limitations such as poor bioavailability, systemic toxicity, susceptibility to in vivo destruction, and short half-life. in contrast, nanotechnology drugs have advantages such as higher bioavailability, lower toxicity, protection against degradation, improved circulating half-life, reduced effective dose of the drug, the ability to cross biological barriers [41], and tissue or cellular targeting [42]. also, nanocarriers provide the ability to control drug release in the target tissue, improve biocompatibility and reduce drug toxicity in healthy tissues by confining drugs [24]. nano compounds such as micelles, microspheres, liposomes, and dendrimers have been used for better and targeted delivery of antiviral drugs such as efavirenz, zidovudine, and acyclovir [42]. 7. prevention of sars-cov-2 with nanotechnology 7.1 improving personal protective equipment (ppe) using nanotechnology in cases where a definitive cure for covid-19 has not yet been discovered, prevention is especially important in vulnerable people such as the elderly, those with immunodeficiency, and at-risk individuals include healthcare professionals [43]. although observing physical distance is known as the first step but, it is difficult to implement in the healthcare staff. therefore, equipping these people with highly efficient safety and ppe is a very necessary step [15]. 7.2 environmental, surface disinfectants, and healthcare studies have shown that the sars-cov-2 virus remains on the surface for a long time [44, 45], so even the ppe kits used may be infected with the virus [46]. therefore, the production of anti-virus coatings on surfaces and air filtration devices can help reduce infection. nanomaterials have the ability to prevent viral contamination by air and contact with contaminated surfaces and have the ability to sterilize protective equipment, especially in hospital settings [24]. creating self-disinfecting surfaces is another strategy to prevent the spread of covid-19. coating surfaces using nanomaterials such as silver, zinc oxide, and copper nps is one of these solutions. these compounds oxidize and release ions with antimicrobial properties [25, 47]. according to studies, controlled and long-term diffusion of ions in some metals, such as copper, modulates the antiviral properties of surfaces [48]. these nanoparticles are loaded into a polymer matrix and provide an effective antiviral coating on surfaces [15]. this method can also be applied to polypropylene masks. this method has already been used in the preparation of protective masks against influenza viruses. this was done by depositing a coating of silver or silica nanocoating composite on face masks [49]. graphene in face masks can also sterilize these and allow them to be reused [50]. this coating is also suitable for use on surfaces in public places [51]. there are several products made of nanocomposites that have antimicrobial activity and are used in the disinfection of surfaces. cac-717 is a new disinfectant consisting of calcium hydrogen carbonate mesoscopic crystals that are a compound containing mesostructured nps and are involved in inactivating enveloping [52] and non-enveloping [53] viruses. tungsten carbide nanoparticles also have extensive antiviral activity [54]. agnps also have various medical, environmental, and industrial applications as disinfectants [55]. these compounds have antiviral activity against several types of viruses, including rsv, hbv, and hiv [56]. these nanoparticles can also be used in air filters [55] and clothing fabrics [57]. maghareh abed et al. 5 8. nano-based vaccines many efforts are currently underway to develop the covid-19 vaccine, including clinical trials. nanotechnology approaches can help develop nanobased vaccines. the apparent similarity of sars-cov2 with other viruses (mainly sars-cov-2 and merscov) and the therapeutic progress and challenges of sars-cov-2 infection with other infectious diseases as well as oncological research are not significantly different [58], and it can help a lot in making a successful covid-19 vaccine [59]. vaccine production has significant similarities in the fight against sars, mers coronaviruses [16]. on the other hand, conventional vaccines have problems such as the risk of reversal of viral virulence (live attenuated virus vaccines), weak immune response (inactivated viruses), and limited immunogenicity (subunit vaccines) [15]. another advantage of vaccine nanocarriers is their size. many biological systems, such as viruses, including the sars-cov-2 virus and some proteins, are nano-scale [16]. in this way, nanoparticles can be loaded with a wide range of antigenic components which is very important in the production of vaccines [60]. nanoparticles can also target both humoral immune systems and cell-mediated immunity [61]. modulation of antigen-presenting cells (apcs) using nanoparticles is of particular importance in vaccine fabrication [62]. organic nanoparticles such as liposomes, lipid-based nps, and polymeric nps are significant in vaccine formulations [24]. also, biopolymer-based nps, like protein-based nps, have low toxicity and biodegradability [63]. aunps, as another nanosystem for vaccine production, have been studied on more than 45 pathogens in various sizes, shapes, and functions [64]. 9. conclusion the outbreak of the covid-19 virus over the past year has led to the infection of a large population of the world and high mortality. as a result, rapid progress in discovering ways to diagnose, treat, and control the disease is a necessity. early and rapid diagnosis of the disease, rapid and effective treatment measures, the use of appropriate and safe equipment are the most important strategies to control the disease. currently, the diagnosing method of this disease is timeconsuming and it is not applicable in public populations and far from urban health centers. on the other hand, many therapeutic approaches to this disease are based on symptomatic treatment and are performed using antiviral drugs previously used in other viral diseases. these drugs prevent the virus from being absorbed into the tissue and block the activity of proteases in infected cells. however, this strategy only reduces the number of viruses and the symptoms. according to reports, the most important barrier to the use of these drugs is their delivery to target cells and tissues in the respiratory tract, which reduces their effectiveness. use methods based on nanotechnology is one of the best ways to overcome these barriers. due to the aerodynamic size of the nanoparticles, they can be effective in predicting the treatment process. also, the lack of an approved vaccine with high efficacy and low side effects is another problem and obstacle in controlling the disease. the unpredictable and unknown nature of the disease and the similarity of the specific properties of covid-19 and the physicochemical properties of nanosystems have led to the discussion of solutions based on new technologies. this study attempts to review possible strategies for the prevention, diagnosis, and treatment of covid-19 by focusing on current experiences and knowledge about nanotechnology and its successful measures in curing other viral diseases. the use of proprietary antiviral drugs, animal models, in vitro and in vivo studies to help screen for antiviral drugs, prevent disease transmission, and produce vaccines with minimal side effects and high efficacy as soon as possible will determine the prospects of disease control. in summary, nanotechnology is a powerful 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https://pubmed.ncbi.nlm.nih.gov/23142821/ https://pubmed.ncbi.nlm.nih.gov/23142821/ https://pubmed.ncbi.nlm.nih.gov/23142821/ https://doi.org/10.1038/nature06175 https://doi.org/10.1038/nature06175 https://doi.org/10.1155/2014/180549 https://doi.org/10.1155/2014/180549 https://doi.org/10.1155/2014/180549 https://pubmed.ncbi.nlm.nih.gov/32306785/ https://pubmed.ncbi.nlm.nih.gov/32306785/ https://pubmed.ncbi.nlm.nih.gov/32306785/ journal of current biomedical reports jcbior.com volume 3, number 3, 2022 eissn: 2717-1906 1 original research differentiation of mesenchymal stem cells isolated from the amniotic membrane and umbilical cord to osteocytes and the expression of runx2, osteonectin, alp genes amin tabasi1, nooshin barikrow1,* 1department of molecular and cellular sciences, faculty of advanced sciences & technology, tehran medical sciences branch, islamic azad university, tehran, iran abstract mesenchymal stem cells (mscs) are multipotent cells and able to differentiate into connective tissues such as bone, fat, cartilage, tendon, and muscle. they show to be very potent tools for tissue engineering and regenerative medicine. several researches have shown that amniotic membrane mesenchymal stem cells (am-mscs) and umbilical cord mesenchymal stem cells (ucb-mscs) are both multipotent in nature differentiating into several cell types such as adipocytes and osteoblasts. in this study, mesenchymal stem cells were derived from the human amniotic membrane (ham-dmscs) and umbilical cord then characterized with their surface antigens using flow cytometry. these cells differentiated to osteocyte and adipocyte in induction medium then the expression of runx2, osteonectin, and alp genes were calculated by real-time pcr. we showed that am-mscs and ucbmscs can discriminate to osteogenic and adipogenic cells in the specific induction medium. the capability of am-mscs and ucb-mscs differentiation to osteogenic cells was confirmed by enhanced expression of runx2, alp and osteonectin gene and deposition calcium shown by alzerin staning. given the available evidence, we conclude that am-mscs and ucb-mscs have suitable access, low immunization and lack of medical ethics problems are one of the appropriate sources for differentiation in to osteogenic and adipogenic cells. also, they can be considered as good choices for treatment of mesenchymal tissue injuries and tissue engineering. keywords: amniotic membrane, umblical cord, osteocyst, osteonectin, runx2 1. introduction stem cells are predictable the mainly positive agent for the treatment of degenerative and ischemic diseases as of their self-renewal and multiline age differentiation capacity [1]. mesenchymal stem cells (mscs) were first discovered in 1968 by friedenstein as a supporter fibroblast-like population in the bone marrow competent of differentiating into bone [2]. it was then revealed that mscs can be isolated from different tissues for instance adipose tissue, peripheral *corresponding author: nooshin barikrow, phd department of molecular and cellular sciences, tehran medical sciences branch, islamic azad university, tehran, iran tel/fax: +98 21 88991117 email: nooshinbarikrow@gmail.com http://orcid.org/0000-0001-9367-3885 received: february, 08, 2021 accepted: november, 22, 2022 blood, placenta, amniotic membrane (am), and umbilical cord (uc) [3, 4]. the human amniotic membrane (ham) is the innermost fetal layer considered as an imperative source of mscs for the treatment of several disorders [5, 6]. furthermore, ham is a highly plentiful tissue and willingly available having a single layer of epithelial cells, underlying fibroblasts, and an avascular collagenous stroma [7]. umbilical cord blood (ucb) is considered one of the youngest © the author(s) 2022 https://jcbior.com/ tabasi et al. 2 accessible sources of adult stem cells. as well hematopoietic stem cells, ucb has been shown to have endothelial progenitor cells with mscs [8, 9]. human umbilical cord derived mscs isolated from the cord’s structures; arteries, vein, cord lining and wharton’s jelly. various studies have shown that amniotic membrane mesenchymal stem cells (am-mscs) and ucb-mscs are both multipotent in nature differentiating into various cell types such as adipocytes, osteoblasts, hepatocyte, chondrocytes, haemopoetic cells, cardiac and neural cells [10-13]. umbilical cord stem cells have also been proved promising in probable executive of several diseases and situation [14-16]. through the past decades, the efficiency of mscs such as ham-dmscs and ucb-mscs has been widely investigated both in basic and clinical experiments [17, 18]. due to their anti-inflammatory properties, low immunogenicity and less ethical issues related with their use compared with further sources of stem cells, ham-dmscs and ucb-mscs are alternative resource in the field of tissue engineering [19, 20]. these varieties of mscs have been studied in clinical trials for currently deadly diseases, for instance bone and cartilage defects, stroke, myocardial infarction, graftversus-host disease (gvhd) and autoimmune diseases [21]. these cells have a homing aptitude, meaning that they can transfer into injured sites, and they acquire the ability to differentiate into confined components of injured sites and the ability to secrete chemokines, growth factors and cytokines, that facilitate in tissue regeneration [22]. these cells are identified to create trophic factor for tissue repair/regeneration [23]. it has been clarified that many various genes are involved in differentiation of osteoblast cells such as alkaline phosphatase (alp), runx2, andosteonectin. alp is an enzyme which its expression and activity have been evaluated in some mammals like humans to conclude cell fate in developmental studies or even within clinical trials. this enzyme is considered as key markers in the recognizing of embryonic stem cell. runt-related transcription factor 2 (runx2) is a member of the runx family, which is crucial for differentiation of osteoblast and chondrocyte maturation [24]. expression of runx2 in mesenchymal cells has an induction effect on osteoblast differentiation which leads to bone formation [25, 26]. in addition, osteonectin, also known as secreted protein acidic and rich in cysteine (sparc) is a calcium-binding matricellular protein considered as an important non-collagenous protein expressed in mineralized and non-mineralized tissue [27, 28]. thus far, because of mscs relieve of isolation, culture expansion, multipotential differentiation and immunomodulatory properties, they have the potential for concern in regenerative medicine and have therapeutic purpose [29]. furthermore as mentioned above, mscs used in the treatment of bone lesion. thus, in this study, two available and noninvasive extra-embryonic sources (am-mscs and ucb-mscs) were selected. also the differentiation potential of these cells to differentiate into osteocytes was compared.in addition, they can be considered as good choices for treatment of mesenchymal tissue injuries and tissue engineering. initially we isolated mscs from am and ucb sources. in the next step, differentiation of am-mscs and ucb-mscs into adipogenic and osteogenic cells were carried out in induction mediums. then, the expression of alp, runx2, and osteonectin genes were evaluated in osteogenic cells differentiated from am-mscs and ucb-mscs. 2. materials and methods 2.1 isolation, mscs from amniotic membrane amniotic membrane samples were collected from mothers who gave informed consent at milad hospital (iran). they were then transferred to the laboratory to isolate the mscs from amniotic membrane. a twostep method was used to isolate mscs: first the amniotic tissue was treated with trypsin to remove the epithelial cells, which was followed by treatment with dnase and collagenase iv (gibco, uk) for 5 minutes at 37˚c. the mscs extracted from amniotic membrane are adherence cells [30, 31]. am-mscs were cultured with dmem (low glucose) medium and 10% fbs, 1% penicillin streptomycin solution at 37˚c in 5% co2. 2.2 isolation, mscs from ucb in this study, mscs were isolated from ucb after receiving an approval form. cord blood mononuclear cells (mncs) were extracted according to the instructions [32]. briefly, mncs division was obtained by ficoll (sigma-aldrich, usa) followed by concentration gradient centrifugation and in a 225 cm flask containing 6 ml of dmem culture medium with tabasi et al. 3 10% fbs (invitrogen, usa), 1% penicillin and 1% streptomycin (cinnagen, iran) and then cells were cultured at 37 ° c. the, 48 hours after culture, nonadherent cells were removed by altering the culture medium. replacement of cell culture medium with an interval of 3 days removed non-adherent cells. finally, mesenchymal stromal cells from the third passage were provided for final experiments. 2.3 differentiation to osteoblasts and adipocytes the mscs were differentiated to osteoblasts and adipocytes. the cells in passage 5 were cultured in sixwell plates. the osteogenic differentiation culture containing dmem and 10% fbs, ascorbic acid 50 µg/ml, β-glycerolphosphate 5 mm and dexamethasone 10 mm, and the adipogenic differentiation culture containing dmem and 10% fbs, ascorbic acid 50 µg/ml, indomethacin 60 mm, and dexamethasone 10 mm were used (all from sigma-aldrich, usa). the cells were incubated for 14 days at 37 ̊ c and 5% co2. the differentiation to osteoblasts was observed during staining with alizarin red and the differentiation of adipocytes was revealed by staining with oil red o (all from sigma-aldrich, usa) [33]. for oil-red-o staining, the differentiation medium was first removed and the cells were washed with pbs for stabilization. cold ethanol 71% was added to the cells. after 1 hour, the ethanol was removed and the cells were washed 2 to 3 times with distilled water. then 1ml oil-red-o 2% solution was added to the cells and incubated for 50 minutes at room temperature. after draining the staining solution, the cells were washed 4 times with distilled water. finally in order to prevent the cells from drying out, 1 ml of distilled water was added to them and the cells were stained with were evaluated (all from sigma-aldrich, usa). for alizarin staining, first the differentiation medium was removed and the cells were washed with pbs. cold ethanol was added to the cells. after 1 hour, remove the ethanol and wash the cells 2 to 3 times with distilled water, then 1 alizarin red% 2 solution was added to the cells and for 3040 minutes at room temperature was incubated. after draining the staining solution, the cells were washed 4 times with distilled water. finally in order to prevent the cells from drying out, 1 ml of distilled water was added to them. 2.4 flow cytometry flow cytometric immune phenotyping was used to determine cell identity and surface markers. based on references, after washing with pbs and trypsinizing, we dissolved about 105 cells in 1 ml pbs and 100 μl of this suspension was transferred to a tube; 11 μl of monoclonal antibody conjugated to fluorecein isothiocyanate (fitc) and phycoerythrin was transferred to the microtube. after washing with pbs and trypsinized, the cells were stained with monoclonal antibodies against, percp-29, fitc-cd44, fitc-cd45 and pe-cd34 for 30 min on ice (bd bioscience, san jose, ca, usa), where percp-igg1, pe-igg1, and fitc-igg1 were used as controls. then, they were washed with pbs and analyzed using flow cytometer (facs calibur; becton-dickinson) for stained cell [34]. moreover, flomax and flowjo (flowjo, llc) softwares were applied for analyzing flow cytometrydata. 2.5 rna isolation and real-time pcr total rna was extracted by yt4500 total rna purification mini kit (yekta tajhiz azma, iran) according to manufactured protocol from the samples. then, cdna was synthesized using total rna and cdna synthesis kit (stem cell technology co., iran). to conclude the concentration of dna, fluorescence color syber green (sigma-aldrich, usa) was used. gapdh was used as the internal control [35]. finally, real-time pcr (steponeplus, korea) was employed to measure the expression of runx2, osteonestin and alp genes. primer sequences are presented in table 3. 2.6 statistical analysis all experiments were performed with at least three repetitions. in this study, standard error is used to describe the data. using spss software, version 23 is used to compare the means of two groups independent of the test and to compare the means of more than two groups of the test. the results are considered statistically significant if p value <0.05. graphs and tables are drawn using excel software. 3. results 3.1 growth characterization of am-mscs and ucb-mscs in morphological study by invert microscope it was clarified that mscs derived from amniotic membrane (supplementary figure 1a) and ucb tabasi et al. 4 (supplementary figure 1b) were spindle-shaped cells. these kind of cells presented typical properties like having a large rounded nucleus containing nucleolus, and long and short cellular tails. moreover they were adhesive cells which could maintain a strong mitotic competency to passage 3. 3.2 surface markers of am-mscs and ucbmscs the isolated am-mscs and ucb-mscs were characterized with their surface antigens using flow cytometry. flow cytometry analysis showed that mscs derived from amniotic membrane and ucb was positive for mesenchymal associated markers (cd29 and cd44) and negative for the hematopoietic cell antigens (cd34 and cd45). the results displayed that in am-mscs cd29, cd44, cd34 and cd45 were expressed 97.4%, 99.6%, 1% and 0.5%, respectively. as well, the expressions of these markers in ucb-mscs were 95% (cd29), 99% (cd44), 1% (cd34) and 5% (cd45). 3.3 adipogenic differentiation from am-mscs and ucb-mscs to evaluate adipogenic differentiation from ammscs and ucb-mscs, cells of passage 3 were cultured in induction medium and differentiated into adipogenic cells (supplementary figure 3). on the other hand, the mscs cultured in basic culture medium were considered as controls and did not show any differentiation (supplementary figure 3a and c). as shown by arrows, red points were lipid vesicles identifying adipogenic differentiation from am-mscs (supplementary figure 3b) and ucb-mscs (supplementary figure 3d). 3.4 osteogenic differentiation from am-mscs and ucb-mscs am-mscs and ucb-mscs of passage 5 were used to culture in osteogenic induction medium, which differentiated into osteogenic cells (supplementary figure 4). nevertheless any differentiation shown in the control mscs cultured in basic culture medium (supplementary figure 4a and c). after 14 days alizarin red staining was used to represent the osteogeniccells by inverted microscopy. this kind of staining could illustrate osteogenic differentiated from am-mscs (am-ost) (supplementary figure 4b) and ucb-mscs (ucb-ost) (supplementary figure 4d) with calcium deposition. 3.5 gene expression by rt-pcr analysis in osteogenic differentiated cells quality of extracted rna in osteogenic differentiated cells from am-mscs and ucb-mscs were evaluated by nano drop. moreover, agarose gel was run to check the quality of rna (supplementary figure 5). expression of alp, runx2, and osteonectin in these osteogenic differentiated cells was analyzed by rt-pcr compared to gapdh transcript level (as internal control) (supplementary figure 6). as presented in supplementary figure 6a, expression of runx2 displayed a dramatic enhancement in amost (5-fold) and ucb-ost (6-fold) in comparison to the control. alp gene expression in am-ost and ucb-ost significantly enhanced 10 and 6.8 times respectively greater than that in mscs control (supplementary figure 6b). in addition, the greatest increase was found in expression level of osteonectinin am-ost and ucb-ost, which showed 13and 9-fold increase, respectively, compared to the equivalent control (supplementary figure 6c).since the expression of these genes were equal in am-mscs and ucb-mscs, we considered am-mscs as the control. 4. discussion mscs are multipotent cells and capable to discriminate into connective tissues for instance bone, cartilage, tendon, fat and muscle. they show to be very influential role for regenerative medicine and tissue engineering, not just for their multilineage discrimination possible [35]. but frequently for their paracrine activity, exerted during the release of soluble agent [36] for instance anti-inflammatory cytokines, trophic molecules and anti-apoptotic that stimulate tissue renovate and counteract inflammation [37]. these cells are regular and reside in different tissues for self-repair and tissue homeostasis. they can be remote from bone marrow, periosteum, adipose tissue, trabecular bone, synovium, skeletal tissue, blood, spleen, brain, kidney, liver, lung, muscle, bone marrow, thymus, pancreas and deciduous teeth [38]. in this study potential differentiation of am-mscs and ucb-mscs into adipogenic and osteogenic cells were compared. additionally, the expression of alp, runx2, and osteonectin genes were evaluated in tabasi et al. 5 osteogenic cells differentiated from am-mscs and ucb-mscs. before studies showed that am-mscs were able to reverse the bone deficiency caused by oxidative stress and inflammation [39]. furthermore, ucb-mscs transplants were used in patient with bone marrow failure syndromes, hematologic malignancy, or inherited metabolic disorders, which led to treat them [40]. several researches have represented that particular surface antigens, such as cd44, cd29, cd90, and cd105, are identified mscs that originate from ucb, am, bone marrow and adipose tissue [41, 42]. consist with wang et al. and chamberlain et al., our results showed that mscs were positive for mesenchymal associated markers cd29, cd51, cd44, cd73, and cd105, showed lacked expression of cd34 and cd45 as hematopoietic markers [7, 32]. these mscs could differentiate into osteoblasts one of the most important cells correlated to bone repair. also adipocytes diffrentiaed from mscs could represent a new therapeutic target for multiple diseases from osteoporosis to leukemia [43]. former researches have revealed that aging or harmful mscs have damaged osteogenic differentiation ability and improved adipogenic differentiation capacity, which is a probable cause of osteoporosis in the elderly [44, 45]. the differentiation of mscs in vitro mostly depends on the culture situation. growth factors, for example the tgf-β family, result in chondrogenic demarcation [46], whereas mscs cultured with isobutyl methyl xanthine, dexamethasone, and indomethacin will discriminate down an adipogenic lineage [47]. osteogenic differentiation of mscs in vitro is induced by the occurrence of dexamethasone, β-glycerol phosphate and ascorbic acid [48]. alternatively, ascorbic acid 50 µg/ml, indomethacin 60 mm, and dexamethasone are needed in culture medium for induction of adipogenic demarcation from mscs [49]. signaling cascades which expand mscs into adipogenic and osteogenic lineage differentiation rely on two type transcription factors: ppar𝛾and runx2, respectively. also, ppar𝛾 as the master regulator of adipogenesis has anti-osteoblastogenic effects. these transcription factors are devoted for moderating the effects of diverse cytokines in purpose of adipogenic versus osteogenic mscs differentiation [50, 51]. on the other hand, in this study we used particular induction medium for adipogenic and osteogenic differentiation from am-mscs and ucb-mscs, which lead to emerge individual adipogenic and osteogenic lineages. in this study, beside cells differentiation, the expression of some genes, runx2, alp, and osteonectin, involve in osteogenic differentiation from mscs were examined. and all genes showed enhanced expression in osteogenic differentiated cells.runx2 is a member of the runx family, which has the dnabinding domain runt. this factor is necessary for differentiation of osteoblast and chondrocyte maturation. expression of runx2 in mesenchymal cells has an induction effect on osteoblast differentiation which leads to bone formation. thus, runx2 formed a condensed cell layer of osteoblast progenitors by enhancing their generation and inducing their differentiation into osteoblast lineage cells [52-54]. runx2-dependent osteoblast differentiation is associated with bone morphogenetic proteins (bmp) signaling. actually, bmps participate a role in induction of numerous target genes expression particularly runx2 by activating smad proteins and also further signal transduction pathways. in addition, runx2 increases the cells responsiveness to bmps [55, 56]. through differentiation of osteoblast, runx2 shows low expression in mesenchymal cells and the upregulated expression in preosteoblasts. furthermore, its transcript level attains the highest level in immature osteoblasts, and finally is reduced in mature osteoblasts; which were in agreement with qin et al [57]. alp is an enzyme which its expression and activity have been evaluated to conclude cell fate in developmental studies or even within clinical trials. hydrolysis of phosphate esters is mentioned as a general activity of alp [58]. this enzyme is considered as key markers in the recognizing of embryonic stem cell. it has been known some isoenzymes of alp are specific for definite tissue in humans, which are expressed by embryonic cells but also carcinoma cells. another isoenzyme is ubiquitous, which is not tissuespecific and appears in bone, kidney, and liver [59, 60]. some of alps are responsible in the mineralization of hard tissue by providing free phosphate for the creation of hydroxyapatite crystals and hydrolysing pyrophosphate (an inhibitor of bone matrix formation). also, they interfere in the metabolism of vitamin b6, and also, 𝛾-aminobutyric acid (gaba, as a tabasi et al. 6 neurotransmitter). these alp activities are dominant in the developing skeleton; therefore their suppression causes various defects in the mineralization of hard tissue and the expansion of the nervous system [61, 62]. the main modulating pathways regulating osteoblastic differentiation and alp expression are the bmp/runx2 (cbaf1, aml3)/osterix system, and the wingless type (wnt) signaling cascade, which correlate to each other [63-65]. some studies of the bmp/runx2 system have indicated that a number of patterning gene products were involved in both inductions of osteogenesis in mscs and controlling expression of some isoenzymes of alp via chromosomal remodeling [66]. an additional regulation of alp expression is applied through the actions of 1, 25-(oh) 2 -vitamin d, and parathyroid hormone and retinoic acid, which interact with the main modulatory systems [67, 68]. in various studies, osteoblastic differentiation and mineralization were elucidated by determining alp expression by rtpcr, and mineralized matrix pattern by alizarin red s staining which is consist with our study [69]. the results presented here are consistent with previous reports [67, 70]. osteonectin was a different protein which its gene expression was evaluated in this study. this protein identified as sparc or basement membrane protein 40 is a 32 kda calcium-binding matricellular protein is secreted by osteoblasts in bone during bone development [71, 72]. usually, expression of this protein is related to fibrillar collagens such as collagen type i. it has been proposed that osteonectin binds collagen and hydroxyapatite crystals in the osteoid (pre-mineralized bone matrix), and releases calcium ions rising mineralization of the collagen matrix in bones. osteonectin can be originate in osseous tissue for instance in active osteoblasts and bone marrow progenitor cells, hypertrophic chondrocytes, odontoblasts (dentin-forming cells), and osteoid, which show high expression of this protein [73]. various studies on animals have revealed that defection in osteonectin could cause various phenotypic abnormalities and decrease in bone mineral density [74, 75]. these consequences could lead to osteopenia is considered as a forerunner to osteoporosis [76]. furthermore, these evidences have clarified that osteonectin has an effect on osteoblast and osteoclast activity; and induction of osteoblast differentiation in precursors with osteonectin defects would be impaired [77, 78]. results showed that osteonectin protein level increased at early stages of differentiation and then declined as cells mature and began to express mature osteoblastic marker. our results were in agreement with these reports. in conclusion, our results presented here indicate that mscs isolated from am and ucb were constructive for mesenchymal associated markers. this finding supported that mscs could be obtained from these two various resources, am and ucb, which are more potent than other sources of mscs which their numbers decrease by increasing the age. in this study, the capacity of am-mscs and ucb-mscs differentiation to osteogenic cells was confirmed by enhanced expression of runx2, alp and osteonectin gene and deposition calcium shown by alzerin staning. given the presented evidence, we conclude that ammscs and ucb-mscs which have convenient access, low immunization and lack of medical ethics problems are one of the appropriate sources for differentiation in to osteogenic and adipogenic cells. moreover, they can be considered as good choices for treatment of mesenchymal tissue injuries and tissue engineering. it seems that further studies in this field are needed. supplementary files supplementary file 1. authors' contributions the authors were equally involved in the article. all authors read and approved the final version of manuscript. conflict of interests the authors declare that they have no conflicts of interest. ethical declarations all ethical principles were performed in accordance with ethical standards in the 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