Development of multiplex pyrosequencing for HLA-B*57:01 screening using single nucleotide polymorphism haplotype.

WHAT IS KNOWN AND OBJECTIVE: Abacavir (ABC) is a commonly used nucleoside reverse-transcriptase inhibitor with potent antiviral activity against HIV-1. The US Food and Drug Administration and international HIV treatment guidelines recommend HLA-B*57:01 screening before initiating treatment with ABC. The current standard method for HLA-B*57:01 screening is limited by its high-cost, time-consuming and labour-intensive procedure with the requirement of a specialized laboratory. Our study aims to develop a more reliable screening test by selecting rs3093726 as an additional single nucleotide polymorphism (SNP) to combine with rs2395029 for multiplex pyrosequencing development. It offers high-accuracy, cost-effective and rapid detection. METHODS: Multiplex pyrosequencing was developed for HLA-B*57:01 screening using rs2395029 and rs3093726 as a surrogate marker and tested in 130 Thai subjects in parallel with singleplex pyrosequencing of each SNP and the standard sequence-based method. RESULTS AND DISCUSSION: Multiplex pyrosequencing showed 100% concordance when compared with both singleplex pyrosequencing and standard sequence-based method. This method showed 100% of negative predictive value (NPV), positive predictive value (PPV), specificity and sensitivity. WHAT IS NEW AND CONCLUSION: Multiplex pyrosequencing is a powerful tool for HLA-B*57:01 screening using the rs2395029 and rs3093726 haplotype genotyping as surrogate marker for this HLA-B. The assay provides accurate, cost-effective and rapid detection of this haplotype. It can be applied for ABC hypersensitivity screening of the Thai population before initiating treatment with ABC.

Coreceptor tropism determined by genotypic assay in HIV-1 circulating in Thailand, where CRF01_AE predominates.

OBJECTIVES: Chemokine (C-C motif) receptor 5 (CCR5) inhibitors are a novel class of antiretroviral agents that are promising for treatment of patients who harbour the HIV-1 R5 strain. Data on coreceptor tropism in non-B HIV-1 subtypes are limited. We studied coreceptor tropism in HIV-1 circulating in Thailand, where CRF01_AE predominates, using a genotypic assay. METHODS: We compiled V3 sequences of HIV-1 strains circulating in Thailand during 2010-2012. Coreceptor tropism was predicted based on V3 sequences using geno2pheno version 2.5 (http://coreceptor.bioinf.mpi-inf.mpg.de). RESULTS: One hundred and fifty-five HIV-1-infected patients were enrolled in this study. Ninety-nine patients (63.9%) were antiretroviral-naïve, and the remainder had virological failure. The median (interquartile range) CD4 cell count and HIV-1 RNA were 220 (74-379) cells/µL and 75,374 (14,127-226,686) HIV-1 RNA copies/mL, respectively. Of the sequences obtained from these patients, 119 (76.8%) were CRF01_AE and 22 (14.2%) were subtype B. At a false positive rate of < 5%, 61 (39.4%) HIV-1-infected individuals were predicted to harbour the X4 phenotype. X4 viruses were detected more frequently in the treatment-failure group compared with the treatment-naïve group (30.3 vs. 55.4%, respectively; P = 0.002). Those with CRF01_AE had a higher proportion of X4 viruses compared with non-AE subtypes (47.9 vs. 11.1%, respectively; P < 0.001). By multivariate logistic regression, CRF01_AE and treatment failure were independently associated with predicted X4 phenotype [odds ratio (OR) 7.93; 95% confidence interval (CI) 2.57-24.50; P < 0.001, and OR 3.10; 95% CI 1.50-6.42; P = 0.002, respectively]. CONCLUSIONS: CRF01_AE and treatment failure are associated with the predicted X4 phenotype. In regions where CRF01_AE predominates, use of CCR5 inhibitors must be considered with caution. The phenotypic assay and its correlation with genotypes should be further investigated in CRF01_AE.

Draft Genome Sequence of the Mycobacterium tuberculosis Strain 43-16836, Belonging to the Indo-Oceanic Lineage, Isolated From Tuberculous Meningitis in Thailand.

We present the draft genome sequence of Mycobacterium tuberculosis strain 43-16836, belonging to the Indo-Oceanic lineage, isolated from a tuberculous meningitis patient in Thailand. The genome is 4,381,942 bp long with 4,316 protein-coding genes and contains new single nucleotide polymorphisms (SNPs), including SNPs of genes that may encode cell wall components and possibly influence virulence.

Genotyping of HPA-1 to -7 and -15 in the Thai population using multiplex PCR.

BACKGROUND: Different polymerase chain reaction (PCR) techniques for human platelet antigens (HPA) genotyping have been implemented, in order to diagnose the clinical syndromes of patients with thrombocytopaenia and provide effective HPA-matched platelet donors. OBJECTIVES: The aim of this study is to develop an in-house multiplex PCR for HPA-1 to -7 and -15 genotyping in the Thai population. METHODS: One hundred DNA samples of known HPA genotyping by the PCR with sequence-specific primers (PCR-SSP), as previously described, were tested with the multiplex PCR. Additionally, 300 DNA samples of group O donors were tested for HPA-1 to -7 and -15 genotyping using multiplex PCR. RESULTS: The comparison of HPA-1 to -7 and -15 genotype results between multiplex PCR and PCR-SSP technique was in 100% concordance. Interestingly, HPA-2b2b genotype was found in two samples; however, other low-incidence genotypes such as HPA-1b1b, HPA-5b5b, HPA-6b6b and HPA-7b7b were not found in this study. Moreover, 30 samples were randomly tested twice for HPA genotyping using the multiplex PCR and demonstrated reproducible results. CONCLUSIONS: This study shows that the in-house multiplex PCR is simple, cost-effective and suitable for HPA genotyping for routine laboratories in other developing countries. Nevertheless, a large-scale evaluation of this technique through multicentre analysis is suggested.

Cytomegalovirus viremia after kidney transplantation in Thailand: predictors of symptomatic infection and outcome.

BACKGROUND: While prevention of cytomegalovirus (CMV) infection after kidney transplantation (KT) has become a standard practice in Western countries, this approach is not always feasible in Thailand. In order to argue for the need for CMV prevention, the knowledge on the incidence and impact of the CMV infection following KT is highly desirable. METHODS: We retrospectively reviewed medical records of adult patients who underwent KT at our transplant center between January 2006 and December 2010. Patients who developed CMV viremia within 1 year after transplantation were studied for the incidence, risk factors, and outcome of symptomatic infection. The threshold value of blood CMV DNA load indicating symptomatic infection was also analyzed. RESULTS: Symptomatic CMV infection occurred in 18 (4.6%) patients within a median time of 12.1 (range, 3-30) weeks after KT. At initial presentation, coexisting opportunistic infection was common (44%) and gastrointestinal tract was the major type of organ involvement (44%). Between groups of patients with symptomatic and asymptomatic CMV infection, the mean (± standard deviation) level of blood viral load were significantly higher in the first group [4.2 (± 0.5) vs 3.3 (± 0.4) log copies/mL]. From multivariate analysis, associated factors of symptomatic infection included acute rejection [odds ratio (OR) 7.32, P = 0.001], and acute tubular necrosis (OR 3.44, P = .019). Death (13%) and graft failure (13%) were significantly higher among the symptomatic infection group than those in the no-infection group (P = .005 and .03, respectively). CONCLUSION: Despite a low incidence rate, symptomatic CMV infection clearly resulted in significant morbidity following KT. In Thailand, the prevention of CMV infection should be prioritized among high-risk KT populations.

Adenovirus disease after kidney transplantation: course of infection and outcome in relation to blood viral load and immune recovery.

Information on the clinical spectrum and management of adenovirus infection after kidney transplantation is limited. From April 2007 to April 2010, 17 kidney transplant recipients were diagnosed with adenovirus disease. The median time to infection was 5 (range, 2-300) weeks after transplantation. Of the 17 patients, 13 (76.5%) presented early, within 3 months posttransplant, and four (23.5%) presented late, more than 3 months after transplant. Besides urinary tract, involvement of other organs was common (63.6%) among patients with adenovirus viremia. Despite reduction of immunosuppression, six patients subsequently had a rise in the level of blood viral load, mostly within a week after diagnosis. However, only three (27.3%) patients with early infection developed disease progression. Compared to the late infection group, patients with early infection had significantly lower absolute lymphocyte counts at week 1 (p = 0.01) and 3 (p = 0.002) after diagnosis. Four patients received intravenous cidofovir. At 6-month follow-up, 10 (90.9%) patients had reversible graft dysfunction. Only one (5.7%) died from bacterial sepsis. Adenovirus disease is a significant complication following kidney transplantation. Early case recognition with reduction of immunosuppression is critical. Serial blood adenovirus viral loads and assessment of lymphocyte recovery are also useful in monitoring the course of infection.

Clinical performance of three rapid diagnostic tests for influenza virus in nasopharyngeal specimens to detect novel swine-origin influenza viruses.

BACKGROUND: Influenza is an important public health problem. The aim of this study was to evaluate and compare the sensitivity and specificity of three rapid diagnostic tests (SEKISUI, QuickVue Influenza A + B, and SD BIOLINE) for novel swine-origin influenza viruses (S-OIV) and seasonal influenza. MATERIALS AND METHODS: A total of 210 nasopharyngeal swabs from unique clinical specimens were previously tested by real-time reverse transcription polymerase chain reaction (RT-PCR) assay and tested again in this study. RESULTS AND DISCUSSION: Of these, 164 (78%) were influenza A-positive and 46 (22%) were influenza A-negative by RT-PCR. From 115 positive swabs, 80 (69.6%), 66 (57.4%), and 46 (40.0%) showed S-OIV by SEKISUI, QuickVue Influenza A + B, and SD BIOLINE, respectively. Specific positive and negative predictive values of these three commercial rapid tests were all 100%. Therefore, positive rapid influenza virus diagnosis does not require an RT-PCR confirmatory test. Conversely, only negative rapid influenza virus diagnosis should be evaluated. The SEKISUI test would be a useful diagnostic tool for screening clinical samples for influenza. Concerning the various specimen types, among 25 patients with RT-PCR-proven S-OIV infection, influenza was identified in sputum (21/25; 84.0%) and nasopharyngeal swab (15/25; 60.0%) specimens, but in only 36.0% (9/25) of throat swab specimens. Sputum and nasopharyngeal swab specimens were the most predictive of influenza virus infection, while throat swab specimens were the least predictive of influenza virus infection.

Adenovirus infection after kidney transplantation in Thailand: seasonal distribution and potential route of acquisition.

BACKGROUND: Data on the epidemiology and route of acquisition of adenovirus (ADV) infection after kidney transplantation are limited. From April 2007 to March 2010, there were 17 cases of ADV infection: namely, 2 from April to December 2007; 8 from January to December 2008; 4 from January to December 2009; and 3 from January to March 2010. Most cases occurred in October and November (n = 8; 47.1%), followed by February to April (n = 6; 35.3%) and July (n = 3; 17.6%). METHODS: From April 2007 to August 2009, the diagnosis of ADV infection was made based on patient symptoms. From September 2009 to March 2010, in addition to symptoms, the diagnosis was complemented by urine surveillance for ADV using real-time polymerase chain reaction (PCR) prospectively performed every 1-2 weeks among recipients of living-related kidney, starting at week 2 posttransplantation for a total of 8-12 weeks. Before transplantation, recipients and donors were screened for ADV in urine and also using nasal swab. RESULTS: Only 1 of the 24 patients displayed a positive ADV PCR in the urine surveillance study. A local investigation during a cluster of cases in October 2008 showed 2 patients who developed ADV after sharing a room in the transplant unit. Although nosocomial transmission was probable, the majority of cases were scattered over time rather than clustering in 1 time period. CONCLUSION: These findings suggested that ADV infection cases occurred after exogenous exposure. In a resource-limited country, early diagnosis of ADV is justified for patients with compatible symptoms complemented by intense infection control to prevent nosocomial transmission from a confirmed case.

Prevalence of antiretroviral drug resistance in treated HIV-1 infected patients: under the initiative of access to the NNRTI-based regimen in Thailand.

To determine the prevalence of antiretroviral resistance in treatment-failure HIV-1 infected individuals, under the initiative of the non-nucleoside reverse transcriptase inhibitor (NNRTI)-based regimen in Thailand, plasma samples were collected from 1,376 HIV-1 infected patients, who were failing in their current HAART therapy during 2000-2004. They were stratified into 2 intervals: group one (1), 558 HIV-1 infected patients (2000-2002; before the initiative of access to HAART), and group two (2), 818 HIV-1 infected patients (2003-2004; after the initiative of access to HAART). Genotypic resistance testing was performed. The frequency of antiretroviral drug resistance in treatment-failure HIV-1 infected patients has significantly increased over time from 68.5% (382/558) during 2000-2002 to 74.9% (613/818) during 2003-2004 (P<0.01). Resistance to NNRTI during 2003-2004 (59.2%) was much higher than that during 2000-2002 (36.9%; P<0.001). However, the frequency of nucleoside reverse transcriptase inhibitor (NRTI) drug resistance was not significantly higher (P=0.153). We showed that this correlated with an increase in the NNRTI-based regimen prescribed during 2003-2004, especially the Thai-produced combination pill, GPO-VIR. Our finding also showed that a high level of genotypic drug resistance is associated with GPO-VIR (40.8% lamivudine, 40.6% stavudine, 43.8% nevirapine). In order to avoid the rapid emergence of resistant viruses in a resource-poor setting, a close surveillance of antiretroviral drug resistance is feasible and should be considered.

Surveillance of genotypic resistance mutations in chronic HIV-1 treated individuals after completion of the National Access to Antiretroviral Program in Thailand.

BACKGROUND: Due to the establishment of the National Access to Antiretroviral Program for People who have AIDS (NAPHA), approximately 80,000 Thai HIV-1 infected patients received antiretroviral drugs through the NAPHA program, which was completed at the end of 2005. The development of drug resistance is required for access to ARV drugs. The objective of this study was to determine the prevalence of antiretroviral drug resistance in Thai HIV-1 treated individuals after completing the NAPHA program. PATIENTS AND METHODS: Viral genotypic resistance testing was carried out for 1,880 HIV-infected patients experiencing treatment failure, who enrolled during 2000-2005. All patients were in a follow-up treatment with ARV drugs available in clinical practice. The genotype was performed with the TRUGENE HIV-1 kit to assess resistant mutations to reverse transcriptase inhibitors and to protease inhibitors. RESULTS: The frequency of ARV drug resistance has significantly increased after the National Access To Antiretroviral Program was implemented. The reverse transcriptase genes M184V/I (919/1,880; 48.9%) and K103S/H (416/1,880; 22.1%) were the most frequent in nucleoside reverse transcriptase and non-nucleoside reverse transcriptase, respectively. In the protease genes, minor mutations or polymorphisms were found in the majority. Thymidine analogue mutations were presented and increased over time. This study showed a sharp increase in the prevalence of mutations associated with the GPO-VIR combination; nevirapine (948/1,880; 50.4%), lamivudine (889/1,880; 47.3%), and stavudine (703/1,880; 37.4%) after the program was completed. CONCLUSION: With the increased availability of antiretroviral therapy in a resource-constrained country, antiretroviral drug resistance should be closely monitored. HIV-1 drug resistance testing to enable the salvage therapy will remain a priority in Thailand. Furthermore, resistance testing should also become routine before prescribing treatment, and the consequences of continuing to provide a failing regimen must be considered.

Delayed progression to AIDS in volunteers treated with long-term HIV-1 Immunogen (REMUNE) therapy in Thailand.

OBJECTIVES: To observe the long-term effects of an immune-based therapy HIV-1 Immunogen (REMUNE; Immune Response Corp., Carlsbad, CA, USA) as a first course of treatment designed to sustain the immune system and thus delay the initiation of therapy with antiretroviral drugs and/or delay disease progression. METHODS: In this open-label, multi-institute extended phase II P2101B study, disease progression, CD4 and CD8 T-cell counts, HIV-1 RNA levels, and genotypic antiretroviral drug resistance were examined in 223 asymptomatic HIV-1-infected Thai volunteers receiving REMUNE every 12 weeks over 132 weeks. A subset of subjects was randomly selected by the physicians to receive antiretroviral drugs for 10 months. RESULTS: Patients treated with REMUNE demonstrated a low rate of clinical disease progression (0.72 per 100 person-years), higher CD4 and CD8 T-cell counts, higher body weight before and after treatment in the same patient, and stable viral load with no serious adverse events. We found no genotypic evidence of drug resistance in subgroups of patients on REMUNE monotherapy or REMUNE plus antiretrovirals (ARTs). CONCLUSIONS: This Thai study, like previous US and European studies, confirms that therapeutic immunization of HIV-infected volunteers modifies disease progression, as evidenced by stabilization of CD4 and CD8 T-cell counts, body weight, and viral load. As the majority of asymptomatic patients demonstrated an objective response to immunization, this study suggests that REMUNE may be utilized prior to initiation of antiviral drug therapy when CD4 cell counts are still above the current ART guidelines. Further work should be carried out to examine its potential use in combination with ART in order to reduce the increasingly common occurrence of drug resistance.

Genotypic subtyping of the HIV-1 polymerase gene in 30 naåve patients from Thailand.

To investigate the subtype classification of the circulating virus strains among infected Thai patients with human immunodeficiency virus type 1 (HIV-1). A random population of patients who were HIV-1 antibody positive after two independent screening assays was selected. HIV RNA from plasma samples was reverse-transcribed and amplified with specific primers that annealed to conserve regions of the HIV-1 pol gene. Amplified products were sequenced directly by using an automated sequencer. The sequencing products represent about 1.2 kb of the pol gene from each patient and they were phylogenetically analyzed and compared to the corresponding pol sequences of the published HIV-1 sequences of known genotypes. Genotype E was found in 25 of 30 patients (83.3%), and 5 patients (16.7%) were HIV-1 genotype B. The result confirmed that HIV-1 subtype E is still predominant in Thailand. Genotype B is found frequently, but there have been no examples of genotype A. In concordance with the serotypic assay, which was previously reported using the V3-peptide enzyme immunoassay (V3-PEIA), the genotypic assay of subtype E was high, at 80% and 83.3% in serotyping and genotyping, respectively. These findings of two subtypes with low heterogeneity indicate that Thailand may be a desirable site for evaluating candidate HIV-1 antiretroviral drugs and vaccines. The mixture of subtype E and B' strains also offers the opportunity to study phenotypic differences between the two subtypes.

Survey of reverse transcriptase from the heterosexual epidemic of human immunodeficiency virus type 1 CRF01_AE in Thailand from 1990 to 2000.

Genetic diversity of the HIV-1 envelope gene has shown a steady increase over time in the Thai and other regional epidemics. A serial survey of subtype CRF01_AE polymerase gene (RT) diversity in Thailand was performed, using 48 novel and 15 reported sequences covering the period 1990--2000. These sequences were gathered from individuals whose sole risk factor for infection was heterosexual contact. By contrast to envelope, diversity was low and, despite a 40% increase early in the epidemic, has remained static since 1996. These results indicate that epidemic HIV-1 may be constrained within defined limits of genetic diversity at least in some genomic regions.

Database of nine tetrameric STR loci--D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317 and D7S820 in Thai population.

Allele distributions for the nine STR loci included in the AmpFlSTR Profiler Plus kit were evaluated in a Thai population of 300 unrelated individuals.

Thai population data on nine tetrameric STR loci - D3S1358, vWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317 and D7S820.

Allele frequencies for the nine STR loci included in the AmpFlSTR Profiler kit were determined in a Thai population of 100 unrelated individuals.

p53 status and human papillomavirus infection in Thai women with cervical carcinoma.

Loss of p53 function has been implicated in a wide variety of human malignacies. Many studies suggest that in cervical carcinoma p53 function is inactivated either by gene mutation or by complex formation with E6 oncoprotein product of high-risk human papillomavirus (HPV). The aim of this study was to determine the status of HPV infection and p53 gene mutation as well as their correlation in cervical carcinomas. Formalin-fixed paraffin-embedded tissues of 12 cervicitis, 21 cervical intraepithelial neoplasia grade 3 (CIN 3) and 17 squamous cell carcinomas were determined for the presence of HPV using polymerase chain reaction (PCR) amplification and dot blot hybridization. The status of p53 mutations in exons 5-8 was evaluated by polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) and confirmed by direct nucleotide sequencing. HPV infections were detected in all CIN 3 and squamous cell carcinomas (100%). Mutations of p53 were present in 3 of 38 HPV-positive samples: one with an ATG-->TTG transversion (Met-->Leu) in codon 237 of exon 7; and the others with a TGC-->TGG transversion (Cys-->Trp) in codon 242 of exon 7, and a CGT-->CCT transversion (Arg-->Pro) in codon 273 of exon 8, respectively. Our findings show that the frequency of p53 mutation is low in primary cervical carcinoma and that the p53 gene mutation and HPV infection are not mutually exclusive events in the development of cervical cancer. Thus, other genetic events independent of p53 inactivation may also significantly contribute to the carcinogenesis of the uterine cervix.

Preliminary report for the application of nucleic acid sequence-based amplification in detection of human cytomegalovirus (HCMV) late pp67 mRNA for diagnosis of HCMV infection.

Human cytomegalovirus (HCMV) late pp67 mRNA expression by nucleic acid sequence-based amplification (NASBA) in patients, clinically diagnosed as possible HCMV, probable HCMV disease, and no disease, was evaluated. The RNAs were isolated from 11 whole-blood samples of 11 patients for the specific amplification of the pp67 mRNA. NASBA results were compared to results from PCR assay and serological assay. The HCMV pp67 mRNA could be found in 3 of 11 patients, whereas, HCMV-DNA PCR was positive in 6 of 11 patients. PCR assay for HCMV-DNA in plasma has proved to correlate with clinical diagnosis of HCMV infection. Only 2 patient samples of NASBA positive results coincided with HCMV-DNA PCR. However, the diagnosis of clinically relevant HCMV infection by NASBA was seen. Anti-CMV IgG titers of 1:1,600 or over 1:1,600 were found in 2 of 3 NASBA positive cases and 5 of 6 HCMV-DNA positive cases, whereas, anti-CMV IgM were all negative. These results showed the correlation of HCMV infection detected by NASBA, PCR assay and anti-CMV IgG of the titers up to 1:1,600. Additionally, a low antibody titer of the HIV patient could be diagnosed by NASBA or PCR. In conclusion, pp67 mRNA NASBA appears to be a promising diagnostic tool in analysis of HCMV infection and/or disease. Its diagnostic value should be defined in the specific group for the follow-up of immunocompromised patients, such as organ transplant recipients in future prospective studies.

Forensic identification by DNA fingerprinting and mitochondrial DNA typing.

Short tandem repeats (STRs), that represent an important source of highly polymorphic markers in human genome, and mitochondrial DNA (mtDNA) typing, that its sequences were conserved within the same maternal lineage, facilitated by use of the polymerase chain reaction (PCR) provide a powerful tool for forensic identification. We report the analysis of 9 STR loci and mtDNA typing of a muscle biopsied sample with 2 months postmortem by comparison with the genotype of the relative. The DNA profile showed common alleles with that of the relative but only 12 from 20 alleles (60%) were identifiable. Then, we performed mt DNA sequencing of the hypervariable region I (HV I) and obtained 100 per cent homology with that of the relative. In conclusion, personal identification can be performed precisely by the data of DNA profile and mtDNA typing compared to the genotype of the relative.

Paternity testing by PCR-based STR analysis.

We present application of polymerase chain reaction (PCR)-based short tandem repeat (STR) system for use in paternity testing. The process involves a single tube multiplex PCR of 9 STR loci on different chromosomes, in conjunction with Amelogenin sex test and internal size standards, followed by using an automated DNA sequencer to detect amplified products. The results showed that this system provided unambiguously reliable results. In addition, the method is useful for routine use in that it is robust and reproducible and provides a reliable means of paternity testing.

Comparison of DNA extraction from blood stain and decomposed muscle in STR polymorphism analysis.

Forensic samples that are often degraded and limited in quality cause DNA typing analysis by conventional methods unsuitable. We performed a single tube-multiplex PCR on 9 STR loci (D3S1358, vWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317, and D7S820) and the X-Y homologous gene amelogenin of DNA extracted from six week postmortem blood stain and decomposed muscle by using QIAGEN QIAamp blood or tissue procedure. An automated genetic analyzer based on fluorescent dye technology was used to detect STR allele patterns. The DNA profile of blood stain sample obtained a complete and unambiguous pattern, whereas, that of muscle DNA extracted from QIAamp tissue and Chelex plus QIAamp blood protocols showed detected STR alleles for 70 per cent and 50 per cent of all tested alleles, respectively. The degraded muscle DNA could not yield amplified products of large size STR alleles; CSF1PO, D13S317 and D7S820. However, the analysis which relied upon the PCR-based STR polymorphism analysis and automated genetic analyzer system offers an ideal strategy for forensic identification.