MI-Supiana Available online at http://jurnal.permi.or.id/index.php/mionline DOI: 10.5454/mi.9.3.6ISSN 1978-3477, eISSN 2087-8575 Vol 9, No 3, September 2015, p 136-139 *Corresponding author; Phone: +62-81335278182, Email: diantbn@yahoo.co.id Typhoid fever, also known as abdominal typhoid, is caused primarily by Salmonella enterica. Cases of hepatic involvement with typhoid fever have been reported by Khosla (1990), which known as typhoid hepatitis. Coinfection of typhoid fever with hepatitis A and E also have been reported in previous studies (Bhat et al. 2008; Zhang et al, 2015; Tulara 2013). This coinfection makes the diagnosis and management a challenging task. Our previous paper reported that hepatitis B virus (HBV) was found in abdominal typhoid patients in Tuban. From 30 serum samples of abdominal typhoid patients, we found HBV DNA in 5 samples using nested Polymerase Chain Reaction technique (Nurtjahyani 2013). Hepatitis B virus (HBV) infection is commonly known worldwide to causes more than one million deaths every year (WHO 2015). This disease is also still a health problem in Indonesia. The prevalence of HBV infection in Indonesia is around 9.4% (Riskedas 2007). Hepatitis B virus is a small DNA virus belonging to the Hepa dna viridae family. The genome size of this virus is around 3.2 kilo bases (kb) with a diameter of 42-47 nm (Dane et al. 1970; Summers et al. 1975). The HBV virion (Dane particle) consists of an outer protein envelope and an inner protein core (Kott 2010). Hepatitis B Virus genome consisted of 4 ORFs (Open reading Frames) encoding for the Surface (S), Pre Core and Core (pre-C and C), X and Polymerase (P) proteins (Quarleri 2014). HBV has been classified into 10 genotypes: A, B, C, D, E, F, G, H, I, and J. The prevalence of HBV genotypes varies markedly between different regions. Genotype A is more abundantly found in the Northern and Middle Europe, compared to the North of America and Africa. Genotype B and C are found in Asia, genotype D is distributed and detected mostly in Mediterranea, whereas genotype E is found in the West Africa nations. Typhoid fever can cause liver disorder and may result in complication. Studies revealed hepatic involvement in thypoid known as typhoid hepatitis. Our previous paper reported the existance of hepatitis B virus (HBV) coinfection in the serum of patients with abdominal typhoid using nested-polymerase chain reaction (PCR). Based on its genomic sequence divergence, HBV has been classified into 10 genotypes (A-J),which used in the prediction of therapeutic response and correlates with the clinical outcome of chronic HBV infection. This study was conducted to determine the genotypes of HBV intyphoid patients coinfected with HBV in Tuban. Sera were obtained from 5 typhoid patients with positive HBV infection (proven by nested PCR). Study was performed by direct sequencing using BigDye V1.1 Terminator Cycle Sequencing kit and ABI Prism 310 Genetic Analyzer. Bioinformatic analysis had been done using the GenetixMac version 10 software to create the phylogenetic tree. Phylogenetic analysis showed 3 samples as genotype B and 2 two samples as genotype C. Key words: coinfection, genotype, hepatitis B virus Demam tifoid sering dikenal sebagai abdominalis tifus dapat menyebabkan gangguan hati dan penyakit komplikasi. Penyakit ini sering dikenal sebagai hepatitis tifoid. Dari penelitian sebelumnya, ditemukan adanya koinfeksi virus hepatitis B (VHB) pada pasien abdominalis tifus di kota Tuban dengan menggunakan teknik Polymerase Chain Reaction (PCR). Virus hepatitis B diklasifikasikan menjadi sepuluh genotipe (A-J) dan diketahui memiliki peran dalam keberhasilan terapi. Tujuan dari penelitian ini adalah untuk mengetahui genotip pada pasien tifoid yang koinfeksi dengan VHB di Tuban. Metode dalam penelitian ini adalah melakukan sekuencing langsung dari lima sampel pasien yang positif terinfeksi VHB. Analisis filogenetik dari hasil sekuensing langsung sampel berhasil mengidentifikasi tiga sampel dikategorikan sebagai VHB genotipe B dan dua sampel termasuk sebagai VHB genotipe C. Kata kunci: genotip, koinfeksi, virus hepatitis B Genotype of Hepatitis B Virus Coinfection in Typhoid Patients 1 2 SUPIANA DIAN NURTJAHYANI * AND RETNO HANDAJANI 1 Faculty of Teaching and Education Science, Department of Biology Science, Universitas Ronggolawe Tuban, Jalan Manunggal No. 61, Tuban, Jawa Timur, 62381, Indonesia; 2 Faculty of Medical and Tropical Disease Center, Universitas Airlangga, Mulyorejo, Surabaya, 60115, Indonesia SHORT COMMUNICATION Genotype F is specifically found to infect the Aboriginal Australia. Genotype G is found in United States and France, Genotype H is found to be restricted to Nothern Latin America, whereas Genotype I was identified in Vietnam and Laos, and Genotype J was found in Ryukyu Island in Japan (Lin 2010; Arankalle et al. 201; Zainal et al. 2009; Tatematsu et al. 2009). It is necessary to study the genotypes of HBV in typhoid patients coinfection in Tuban. It is reported that HBV genotypes correlated with the clinical characteristics of the infection, thus influencing the outcome of HBV infection, and the response to antiviral therapies (Okamoto et al. 1998; Norderet al. 1994; Stuyver et al. 2000; Arauz-Ruizet al. 1997; Lusida et al. 2008; Tatematsuet al. 2009; Orito et al. 2003; Akuta et al. 2005). Based on the previous studies, this study was conducted to determine the genotypes of HBV in typhoid patients with HBV coinfection in Tuban District. Written informed consent was obtained from all participants and this study was approved by the Ethical Committee of Koesma Hospital, Medika and Muhammadiyah Hospital Indonesia. Samples were 5 typhoid patients with positive HBV as proven by nested PCR from our previous study at 2013. Blood samples without anticoagulant were placed in sterile centrifuge tubes, allowed to stand for 30 min then centrifuged to obtain serum. The serum was transferred to 1.5 mL eppendorf sterile tube and stored at -80 °C. Method, Hepatitis B Virus was extracted from sera of typhoid patients with known positive HBV using ® DNAzol reagent kit (Invitrogen, USA) as described in the manual book. The extracted DNA was stored in elusion buffer at -80 °C until being used as a DNA source for nested-PCR. HBV DNA used as template in nested-PCR with 35 cycles. The PCR mix consisteds of 10× buffer solution, Tth DNA polymerase, and dNTPs. We used two sets of primers. The primers for the first-step PCR reaction were P1 (sense): 5’-GTGGTGGACTTCTCT CAATTTTC-3’ and P2 (antisense): 5’- CGGTA(A/T)A AAACTGGGCA(A/C)GAT-3’ (originally named P7 and P8 (Lindh, 1997)) which target the S genomic regions of HBV (542 nucleotides product including primers). The primers for the second-step nested-PCR reaction were HBS -1 (sense): 5’-CAAGGTATGTTGC C C G T T- 3 ’ a n d H B S - 2 ( a n t i s e n s e ) : 5 ’ - GCCCTAAAAACTCGAACC-3 (Telent 1997), with 261 nucleotides product including primer used a portion of the final solution from the first-step PCR reaction as the template in the second nested-PCR reaction. The detailed nested-PCR conditions were described in our previous paper (Nurtjahyani et al. 2013). The PCR products were visualized using 2% agarose gel electrophoresis in 0.5× TBE buffer solution containing ethidium bromide and documented on an ultraviolet light using digital camera (Mamiya brand). Nucleotide sequence of the HBV positive samples (based on nested-PCR) were further examined by sequencing usingone of the previous primers (P-1 or HBS1). Direct sequencing was performed using ® BigDye Terminator v1.1 Cycle Sequencing Kit (Thermo Fischer Cat.No.4337450) and a pro 0. 0.0226 0.0317 0.0054 0.0033 0.0091 0.0091 HBV-D M32138 222nt HBV-E AY090452 222nt HBV-E X75657 222nt HBV-F X75658 222nt 0.0000 HBV24-p7-222nt 0.0000 HBV45-p7-222nt 0.0000 HBV10-p7-222nt 0.0000 HBV-B D23678 222nt HBV-G AF160501 223nt HBV-A S50225 222nt HBV-C X75656 222nt HBV2-p1-222nt HBV49-p7-222nt C B 0.0371 0.0091 0.0091 0.0153 0.0244 0.0080 0.0033 0.0219 0.0067 0.0324 0.0137 0.0001 0.0137 0.0138 Fig 1 Phylogenetic tree of HBV from typhoid-HBV coinfection patients. Volume 9, 2015 Microbiol Indones 137 sequencing PCR technique (Applied Biosystems, USA). Nucleotide sequences of HBV S gene from serum samples were compared with the nucleotide sequences of S gene from HBVs with various genotypes that had been published in the international DNA data bank (DDBJ/EMBL/GeneBank). The Unweighted Pair Group Method using Arithmetic averages (UPGMA) method using Genetix MAC program version 10 was used to reconstruct a phylogenetic tree. The results, a phylogenetic tree was constructed based on the PCR amplication products using primers P1-P2 and HBS1-HBS2 (222 nucleotide), and the DNA from five samples as templates (Fig 1). The distribution of HBV genotypes in our five samples 60% (three samples) B and 40 % (two samples) C. Molecular phylogenetic analysis classifies HBV into ten major genotypes, A-J (Cao 2009). Studies showed that HBV genotypes correlateds with distinct geographical distributions and clinical characteristics. The genotypes also playroles in the outcome of the infection, and on the patient’s response to antiviral therapy (Erhardt et al. 2005; Fung and Lok 2004). Therefore is necessary to determine the genotypes in order to better understand the HBV coinfection and also to provide effective managementtherapy. Several cases of typhoid-hepatitis coinfection were reported in some countries (Ahmad et al. 2010; Bhat et al. 2009), however, our previous publication was the first to report the presence of HBV in thypoid patients (Nurtjahyani 2015). In our current report, genotyping of HBVs isolated from 5 patients with typhoid. HBV coinfection indicated that 3 samples belonged to HBV genotype B and 2 samples belonged to HBV genotype C. 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