J Arthropod-Borne Dis, December 2020, 14(4): 363–375 Z Kakooei et al.: Treatment Failure in … 363 http://jad.tums.ac.ir Published Online: December 31, 2020 Original Article Treatment Failure in Cutaneous Leishmaniasis Patients Referred to the School of Public Health, Tehran University of Medical Sciences during 2008–2017 Zahra Kakooei1; Homa Hajjaran1; *Behnaz Akhoundi1; Sorour Charehdar1; Samira Elikaee1; Zahra Shafeghat1; Hamid Hassanpour1,3; Mohammad Taghi Satvat1; Elham Kazemi-Rad1; *Mehdi Mohebali1,2 1Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Center for Research of Endemic Parasites of Iran (CREPI), Tehran University of Medical Sciences, Tehran, Iran 3Department of Medical Parasitology, School of Medicine, Ilam University of Medical Sciences, Ilam, Iran *Corresponding authors: Dr Behnaz Akhoundi, E-mail: behnazakhoundi@yahoo.com, Dr Mehdi Mohebali, E- mail: mohebali@tums.ac.ir (Received 05 Nov 2019; accepted 03 Dec 2020) Abstract Background: Cutaneous leishmaniasis (CL) is a vector borne disease predominantly found in tropical and subtropical countries, including Iran. For more than 6 decades, pentavalent antimonials have been used successfully worldwide for the treatment of leishmaniasis, but over the past few years, clinical resistance to these medications has increased. In this study, we evaluated CL patients who did not show any desirable responses to the anti-leishmanial treatment within a 10- year period (2008 to 2017). Methods: All patients from different parts of Iran suspected of having cutaneous leishmaniasis, who were referred to the laboratory of leishmaniosis in Tehran University of Medical Sciences from 2008–2017 were parasitological examined. Results: During this period, a total of 1480 suspected CL patients were referred to the laboratory of leishmaniosis. Samples from 655 patients (70.8%) suspected of having CL were positive microscopically. The failure rate in patients treated with anti-leishmaniasis medications for a minimum of three complete treatment periods was 1.83% (12 cases). There was no association between the number and size of skin lesions and patient characteristics. Also, the route of drug administration had no significant effect on the number and size of lesions. Conclusion: In the present study, treatment failure was found in some confirmed CL patients treated with meglu- mine antimoniate. Over the past few years, it seems that had been increased in resistance to these medications. So, a review of the correct implementation of the treatment protocol and/or a combination therapy may be helpful in prevent- ing an increase in the rate of treatment failure. Keywords: Cutaneous leishmaniasis; Anti-Leishmania drug; Treatment failure; Iran Introduction Protozoan parasites of the genus Leishma- nia cause a wide spectrum of clinical mani- festations known as leishmaniases. Cutaneous leishmaniasis (CL) is the most common forms of this disease in the world, with more than 350 million people at risk. There is an esti- mated incidence of 0.9–1.7 million new cases each year (1-2). In Iran, two forms of CL have been reported: zoonotic cutaneous leishmani- asis (ZCL) and anthroponotic cutaneous leish- maniasis (ACL). The main pathogen of ZCL in Iran is Leishmania major (L. major), whereas ACL is mainly due to Leishmania tropica (L. tropica) infections (3). For more than 6 dec- ades, pentavalent antimonial (SbV) compounds, such as meglumine antimoniate (Glucantime®) and sodium stiboglucunate (Pentostam®), have been successfully used as the first-line treat- ment for all forms of leishmaniasis (4). In Iran, the national treatment protocol for cutaneous leishmaniasis recommends intramuscularly ad- ministered 20mg SbV5/kg body weight per Copyright © 2020 The Authors. Published by Tehran University of Medical Sciences. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International license (https://creativecommons.org/licenses/by- nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:behnazakhoundi@yahoo.com mailto:mohebali@tums.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, December 2020, 14(4): 363–375 Z Kakooei et al.: Treatment Failure in … 364 http://jad.tums.ac.ir Published Online: December 31, 2020 day. The recommended treatment duration is 14 days for ZCL and 21 days for ACL (5). How- ever, over the past few years, there has been in- creasing concern about the resistance of para- sites to Glucantime® (6). Anti-Leishmania re- sistance and treatment failure is a major chal- lenge in new and old-world countries (7-9). Resistance of L. tropica to Glucantime® was first reported in Iran in 2006 (10). Previously identified risk factors for the failure of treat- ment with Glucantime® are body weight above 68kg, previous anti-Leishmania treatment, hav- ing ≥ 3 skin lesions, and failure to complete a course of treatment (11). In this study, we eval- uated cases of treatment failure in CL patients who were referred to the leishmaniosis labor- atory of Tehran University of Medical Scienc- es (TUMS) between 2008–2017 (10 years) (12, 13). Materials and Methods Patients All patients suspected of having cutaneous leishmaniasis, who were referred to the leish- maniosis laboratory of TUMS between 2008– 2017 were considered eligible for inclusion in this study. Parasitological confirmation was per- formed after gaining accurate information about the place of living of patients. After sterilizing the skin around the lesions/nodules with Eth- anol 70%, a small incision was made in the margin of the lesion with a disposable lancet, and some tissue and exudates were removed by scraping. The scrapings from the margins of the lesions were air dried, fixed in absolute methanol, and stained with Giemsa 10%. The specimens were then examined for amastigotes demonstration by light microscopy with high magnification (14). Design The following data were obtained and rec- orded for each patient: age, sex, history of travel to endemic areas of leishmaniasis, number of lesions, size of the largest lesion, duration of infection, and adverse events. In this study, we focused more on CL patients who failed to respond to treatment, and who remained posi- tive for smear prepared from the lesions after receiving at least two complete treatment cours- es. The necessary criteria for investigating cases of relapse, treatment failure, and clinical re- sistance were as follows: Relapse: patients who received a topical or systemic treatment courses, whose outcome was improvement but the symptoms (any ac- tive lesion) reappeared in the original site of the lesion (15). Treatment failure: cases in which the le- sion remained active after four weeks of com- plete topical or systemic treatment course (15). Clinical resistance: cases of relapse and treat- ment failure, in which active lesions persisted for weeks after at least two complete courses of systemic treatment (15). According to the criteria above, 40 patients overall did not show any desirable therapeutic response to the following regimen of anti-Leish- mania drugs: - Glucantime®: systemic injection (20mg SbV5/ kg for two weeks): (No= 8) - Glucantime®: local injection 1–2ml of Glu- cantime® intralesionally injection around each skin lesion weekly for 4–6 weeks): (No= 0) - Glucantime®: systemic and local injection (20mg per kg for two weeks): (No= 3) - Miltefosine®: oral (2.5mg per kg daily for 28 days) and Glucantime®: (No= 4) - Antibacterial compounds(Antibiotics): (No= 25) Data analysis The data were analyzed using SPSS soft- ware version 24. Chi-square and Fisher's exact test were used, and p≤ 0.05 was considered significant for differences between groups. Results During the 10-year study period, 1480 in- dividuals suspected of having CL were re- ferred to the leishmaniosis laboratory. Of http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 363–375 Z Kakooei et al.: Treatment Failure in … 365 http://jad.tums.ac.ir Published Online: December 31, 2020 these, 655 cases were confirmed positive for leishmaniasis by microscopic examination of samples (amastigotes were seen in microscopic examination). The maximum number of posi- tive cases was recorded in 2018 (116 patients, 17.7 %), whereas the minimum number of positive cases was recorded in 2013 and 2016 (45 cases in each year, 6.9%) (Fig. 1). Out of the 655 positive cases, 464 (70.8%) were males and 191 (29.2%) were females. The youngest patients with confirmed infection was 2 months old and highest age among the positive cases was 80 years old (Fig. 2). About the nationality of the patients, 572 pa- tients (87.3%) were Iranians and 83 (12.7%) were from Afghanistan. Overall, the majority of the patients, 397 individuals (60.6%), lived in Tehran but had a history of trip to endemic regions. Karaj (the capitol of Alborz Province) had the second highest number of cases (48 pa- tients, 7.3%). On the other hands, the Highest number of lesions were one to four (Fig. 3) and also the most common site of the lesions was on the hands with 42/3 % (304) (Fig. 4). Among the patients who were referred to the leishmaniosis laboratory, 139 cases (21.2%) had relapse and were referred to the laboratory for re-examination. In the next step, for follow- up and understanding the phase of the disease, the patients were followed up. In 2008 and 2009, 20 patients with positive microscopic test were lost to follow-up due lack of contact information. During 2010–2017, 40 cases were initially identified as treatment failure, but only 34 patients could be followed up due to lack of contact information. Out of the 34 patients who were followed up, 22 patients improved by modifying the treatment regimen, such as reinjection of Glucantime®, use of supple- mentary drugs such as Miltefosine® capsule, use of ointments, and other traditional drugs. Among the remaining 12 cases, even with mod- ification of diet and increase in drug dosage, 7 cases did not show any desirable response to treatment (58.3%) and five cases had treat- ment failure (41.7%) (Table 1, Fig. 5). Among these 12 individuals with treatment failure, the youngest and oldest were 16 and 62 years old, respectively. Among the patients with treatment failure, the shortest duration of disease was six and the longest was 312 months, respec- tively. 66 65 55 45 90 50 45 64 116 60 0 20 40 60 80 100 120 140 n u m b e r Year 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 Fig. 1. Distribution of confirmed CL cases referring to leishmaniasis laboratory Tehran University of Medical Sciences during 2008–2017 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 363–375 Z Kakooei et al.: Treatment Failure in … 366 http://jad.tums.ac.ir Published Online: December 31, 2020 Table 1. The characteristics of patients with no responses to anti-Leishmania drugs referring to leishmanisis laboratory Tehran University of Medical Sciences during 2008–2017 No Age (year) Living place Travel place during two past years Duration of persis- tence of lesion Site of lesion Type of administered drug and the type of injection 1 62 Tehran Firoozkuh 3 years leg 21 systemic Glucantime® 2 22 Iranshahr Unknown 6 years leg, ankle, toes 200 systemic Glucantime® 60 local Glucantime® 56 oral Miltefosine® 3 47 Hesarak Ka- raj Qom 4 years hand 76 systemic Glucantime® 4 30 Qom Kashan 3 years hand 100 systemic Glucantime® 5 27 Neyshabour Unknown 13 years Face, nose 300 systemic Glucantime® 100 local Glucantime® 70 oral Miltefosine® 6 36 Tabriz Isfahan 3 years Ear, eyes 25 systemic Glucantime® 25 local Glucantime® oral Miltefosine® 7 30 Dehloran Unknown 13 years back 23 systemic Glucantime® 8 20 Karaj Afghanistan 6 years face 200 systemic Glucantime® 100 local Glucantime® 9 16 Mashhad Unknown 9 years In nasal mucosa 84 systemic Glucantime® 90 oral Miltefosine® 10 25 Tehran Afghanistan 26 years The entire body 21 systemic Glucantime® 11 16 Tehran Afghanistan 1 year face 21 systemic Glucantime® 12 43 Unknown Pakistan 6 months face 42 systemic Glucantime® 0.6% 4.9% 5.9% 3.2% 85.3% 0 10 20 30 40 50 60 70 80 90 ˂1Y 1-5Y 5-10Y 10-15Y >15Y Fig. 2. Frequency of confirmed CL patients referring to leishmaniasis laboratory Tehran University of Medical Scienc- es by age groups during 2008–2017 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 363–375 Z Kakooei et al.: Treatment Failure in … 367 http://jad.tums.ac.ir Published Online: December 31, 2020 42.3% 25.9% 22.3% 9.6% 0 5 10 15 20 25 30 35 40 45 Hand Head Foots Body Fig. 3. Distribution of skin lesions on the bodies of the confirmed CL cases referring to leishmaniasis laboratory Teh- ran University of Medical Sciences during 2008–2017 304 115 62 33 141 46.4 17.6 9.5 5 21.7 0 50 100 150 200 250 300 350 1 2 3 4 >10 NUMBER PERCENT Fig. 4. Distribution of the number of lesions in confirmed CL patients referring to leishmaniasis laboratory Tehran University of Medical Sciences during 2008–2017 Fig. 5. Skin lesions with no responses to anti-Leishmania drugs in two CL patients referred to leishmaniasis laboratory Tehran University of Medical Sciences http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 363–375 Z Kakooei et al.: Treatment Failure in … 368 http://jad.tums.ac.ir Published Online: December 31, 2020 Discussion In this study, 40 cases were initially con- firmed as treatment failure, but only 34 could be followed up. Out of this, 22 improved by changing therapeutic regimen such as reinjec- tion of Glucantime®, use of supplementary drugs such as Miltefosine® capsule, use of ointments, and other unknown drugs (12, 13, 15). However, in the remaining 12 cases, even by changing the therapeutic regimen and rais- ing the drug dose, they did not show any de- sirable response and treatment failure was ob- served (Table 1). Furthermore, in the present study, among treatment resistant patients, the longest course of the disease was 312 months (26 years), high- lighting the importance of factors such as im- mune system of the patient, drug kinetics, and existence of resistant strains (11). There have been various reports on the incidence of drug resistance in different foci of leishmaniasis in Iran. Specifically, treatment failure rate in an- throponotic cutaneous leishmaniasis due to L. tropica infection has been reported to be 10.8 % in Mashhad and 11.1% in Bam. In Isfahan, one of the main foci of ZCL, drug resistance was reported as 11.6% in 2005 and in 2013, 3.7% failure rate for topical injection, 4.7% for systemic injection, and 3.4% for concurrent use of both treatment methods were reported (14, 17). One of the mechanisms leading to the re- duced response clinical forms of leishmaniases to the antimony pentavalent compounds is the development of acquired resistance to the drug. Although the antimony pentavalent compounds have been used for several decades as the first- line treatment for all clinical forms of leish- maniasis, unfortunately the therapeutic effect of these compounds has been jeopardized with the emergence of resistance strains in most en- demic regions (16). Specifically, in India, Su- dan, Latin America, Europe, and Middle East, drug resistance is an important threat to effec- tive treatment of the clinical forms of Leishman- iasis. For example, in India, over 60% of cases of visceral leishmaniasis caused by Leishmania donovani do not respond to treatment, which can be due to different reasons such as the development of drug resistance by the para- site, immunologic changes of the patient, in- effective treatment regimen mainly due to lack of patient compliance (23, 24). In this regard, different studies have cor- roborated the idea that the resistance to the an- tibiotics could be acquired. To investigate the existence of acquired drug resistance in Bihar region in India, Lira et al. isolated L. donovani from drug responsive patients and those who were confirmed as treatment failure. Using the presence of intra-macrophage amastigote (in- vitro) in isolates as criteria for resistance, they found that the isolates from patients who had responded to treatment were three times more sensitive to Sodium Stibogluconate drug in comparison to drug-resistant isolates. These results confirm the existence of acquired re- sistance in India (25). In other studies conducted in France, ac- quired resistance in clinically resistant Leish- mania infantum isolates was confirmed in drug resistance tests under in-vitro conditions (26). Also, studies in Latin American countries such as Columbia using in-vitro tests indicated that some cases of treatment failure in new-world cutaneous and mucocutaneous leishmaniasis have been due to development of resistant strains (24-28). Similarly, in Iran, studies in 2004 and 2006 on 185 patients with ACL in Mashhad reported treatment failure in 20 cas- es (10.8%). In these studies, drug resistance of isolates was confirmed under in vitro conditions through the culture of macrophages. The isolates which were clinically resistant to Glucantime® were also resistant in drug sensitivity tests, and re- quired higher doses for their elimination. In the anthroponotic form of the disease, the probabil- ity of spread of drug resistant L. tropica strains http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 363–375 Z Kakooei et al.: Treatment Failure in … 369 http://jad.tums.ac.ir Published Online: December 31, 2020 across human populations is very high, and it’s of significant public health importance (17). Also, acquired resistance of L. infantum and L. major with zoonotic cycles have been re- ported in Iran, and have been confirmed under in-vitro conditions. The progression of anti- monial resistance in anthroponotic forms such as in India resulting from L. donovani and in Iran due to L. tropica infections suggests that in the future, resistance to other anti-leishman- iasis drugs such as Miltefosine® and Ampho- tericin B may also develop in case of exten- sive usage (11). Considering the fact that drug resistance is one of the major challenge in the successful treatment of the disease, identification of the mechanisms involved in drug resistance can be useful in improving therapeutic strategies. Furthermore, identification of suitable mark- ers for monitoring and detecting drug resistant cases and predicting the course of development of resistance in endemic regions could be help- ful in this regard. Among the most important reasons of resistance are genetic, protein, and enzyme factors, as well as intracellular factors such as signaling pathways and apoptosis. The initial mechanisms in the development of drug resistance include reduction of drug concen- tration in the parasite through a decreased up- take or increase in drug excretion through cell pumps, deactivation of drug, and inhibition of drug activation in the cell. A recent research indicated that other than these typical mecha- nisms, other factors such as apoptosis and sig- naling pathways are also involved in the de- velopment of natural drug resistance. Accord- ingly, today various methods such as real-time RT-PCR, microarray, and proteomic methods are used to detect the factors and genes affect- ing clinical resistance phenomenon (27, 28). In Iran, a study conducted by Kazemi Rad et al. (2013) on L. tropica to detect genes whose expression is different between sensitive and resistant L. tropica using cDNA-AFLP tech- nique confirmed that 13 genes play a major role, the most important of which were as fol- lows: Aqua Glycero Porine (AQP1), affects drug uptake; Multi Drug Resistance Protein A (MRPA), involved in entrapping the drug; Phos- pho Glycerate Kinase (PGK), involved in car- bohydrate metabolism; ubiquitin, involved in degrading oxidized proteins; Amino Acid Per- mease (AAP3), involved in uptake of arginine amino acid; protein kinase (PK), involved in signaling pathways; mitogen activated protein kinase (MAPK); and protein tyrosine phospha- tase (PTP), involved in phosphorylation path- way. Also, in their study, using Quantitative Real Time PCR (QRT PCR) method, it was found that in resistant isolates, there was an increased expression of AAP3, ubiquitin, PGK, PTP, and MRPA, whereas AQP1 and MAPK had diminished expression (29, 30). In another study conducted by Zaeran et al. (2015) on L. major resistance and sensitive to Glucantime® using two-dimensional electro- phoresis method performed for determining and comparing expression of proteins, it was found that out of 2967 protein points, 89 points in resistant L. major had altered expression com- pared with sensitive L. major; 60 proteins had increased and 29 proteins had diminished pro- tein expression. Also, they found that 11 pro- tein points which did not exist in the sensitive L. major were expressed by resistance L. ma- jor. These changes of expression may be one of the major causes of resistance in L. major (31). Conclusion Considering the increased rate of drug re- sistance cases and numerous reports in this re- gard in different endemic regions of Iran, com- plete or hybrid treatment should always be taken into consideration by the treatment team in both private and governmental healthcare cen- ters in order to tackle the acquired resistance of the parasite and relapse of disease. On the other hand, researchers should seek to devel- op new drugs with high efficacy in order to combat problems such as incomplete treatment http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 363–375 Z Kakooei et al.: Treatment Failure in … 370 http://jad.tums.ac.ir Published Online: December 31, 2020 and the presence of resistant strains in the in- digent population. There are several reports on different aspects of leishmaniasis in the coun- try. These reports will provide a guideline for disease control (32-66). 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