Genotyping Kit Research Articles

Tasa de mutaciones genotípicas y resistencia a antirretrovirales en un hospital general

Objectives The objective is to describe the resistance mutation rate in protease and reverse transcriptase genes and sensitivity to different antiretrovirals in our environment. Methods We performed an observational descriptive study in which we examined the samples provided at the clinical immunology laboratory between April 2004 and April 2009. We analysed both the resistance tests and the sensitivity to different drugs in patients with therapeutic failure using trugene hiv01 genotyping kits ®. Results We registered samples from 242 patients, 61 of which had no detectable resistance. The most prevalent mutations according to drug families were: for nucleoside analog reverse transcriptase inhibitors T215A/C/D/F/L/N/S/Y (24.10%), M184G/I/V/W (14.66%), M41J/L/R/T/W (11.24%) and K219E/G/H/N/R/T/W (10.24%). The highest levels of resistance corresponded to stavudine and lamivudine/emtricitabine, and tenofovir produced the least resistance in our environment. The non-analogues were K103N/R (23.98%), V179D/E/I/M/T (10.82%), A98E/G/S (10.53%) y K101E/P/Q/R (9.06%). Nevirapine presented greater resistance than efavirenz. Protease inhibitors were L10F/I/V (15.95%), M36I/L (13.81%), A71I/T/V (13.10%) and 154L/S/V (7.38%). The combination darunavir/ritonavir combination was that which presented the least resistance, and tipranavir/ritonavir and lopinavir/ritonavir the most resistance. Conclusions Antiretroviral resistance and sensitivity to retroviral treatment in our environment was similar to results from other studies in Spain, but differed in the high level of resistance to lamivudine/emtricitabine and lopinavir/ritonavir.

Interleukin‐6 Receptor Gene Polymorphism Modulates Interleukin‐6 Levels and the Metabolic Syndrome: GBCS‐CVD

Interleukin-6 (IL-6) is a key pleiotropic cytokine that modulates the inflammatory response. Single-nucleotide polymorphisms (SNPs) within associated genes may contribute to the metabolic syndrome (MES). We examined the role of the IL-6 (rs1524107-C/T) and IL-6 receptor (IL-6R, rs8192284-A/C, Asp358Ala) SNPs in modulating IL-6 levels and the syndrome. A total of 1,979 older Chinese subjects aged 50-92 years from Guangzhou Biobank Cohort Study (GBCS) were recruited. SNPs were detected using Taqman SNP genotyping kits. IL-6 was measured using enzyme-linked immunosorbent assay. The genotype frequencies were 4.9, 33.9, and 61.3% for the IL-6 CC, CT, and TT, and 12.0, 44.9, and 43.1% for the IL-6R CC, AC, and AA, respectively. Both SNPs were in Hardy-Weinberg equilibrium. The IL-6 SNP was not associated with IL-6 levels or the MES, but was dose-dependently associated with fibrinogen levels, P = 0.049. IL-6 levels significantly decreased with increasing proportions of the IL-6R A-allele 9.8 ± 4.9, 9.3 ± 4.8, and 8.4 ± 4.3, respectively, P = 0.001. Conversely, the A-allele was associated with elevated triglyceride, P = 0.009, C-reactive protein, P = 0.047, and potentially with fasting glucose levels, P = 0.077. There was an increasing prevalence of the MES in those carrying the IL-6R CC, AC, and AA genotypes at 18.1, 21.5, 25.2%, respectively, P = 0.010. The SNP was a significant independent predictor of the MES after adjusting for general obesity, age, gender and lifestyle, and socioeconomic parameters, P = 0.023. These data, which are in accord with studies from white populations suggest the IL-6R SNP may play a role in the pathogenesis of the MES possibly through modulating IL-6 levels.

Optimized protocol for detection of HIV-1 drug mutations in patients with low viral load

The TRUGENE HIV-1 Genotyping Kit for HIV-1 drug resistance testing is limited by the need for samples with HIV-1 RNA loads of at least 1000 copies/mL. In order to enable sequencing of clinical samples with viral loads under 1000 copies/mL, an optimized automated sample preparation protocol on the VERSANT kPCR Sample Preparation (SP) Module was evaluated. In order to prove the concept of successful sequencing of low-titer clinical samples with the optimized protocol, a dilution series of a routine clinical sample was analyzed. Furthermore, 57 routine clinical samples with viral loads below 1000 HIV-1 RNA copies/mL were tested. Finally, samples obtained from two patients with low viral loads were tested retrospectively for HIV-1 drug resistances and results were compared with those of the preceding and the subsequent sample. The dilution containing 92 HIV-1 RNA copies/mL was the lowest yielding an analyzable sequence with the optimized protocol. When routine clinical samples with viral loads between 100 and 1000 HIV-1 RNA copies/mL were tested, a sequence was obtained in 90.5%. Samples with low viral load of two patients that could not be analyzed with the routine protocol showed identical drug mutations in both, the low viral-load and the subsequent samples. Together with the optimized automated sample preparation protocol, the TRUGENE HIV-1 Genotyping Kit allows successful sequencing of the majority of samples with HIV-1 RNA loads between 100 and 1000 copies/mL. Detection of resistance mutations in low viral-load samples may lead to an earlier optimized antiretroviral therapy.

Population genetic study in Hungarian canine populations using forensically informative STR loci

Allele frequencies for 10 short tandem repeats (STRs) were determined using the StockMarks ® Dog Genotyping Kit (Applied Biosystems) from a pool of 668 unrelated dogs, consisting of 79 different breeds or breed variants from the Hungarian canine population. For the comparative statistical analysis, four pure bred, one mixed group – all individuals except from the four breeds – and considering to unequal representation of breeds the group of all pooled individuals (“All breeds”) were distinguished. The forensically informative values – Hardy–Weinberg equilibrium (HWE), observed heterozygosity ( H obs), polymorphism information content (PIC), power of discrimination (PD), power of paternity exclusion (PE), linkage disequilibrium (LD) and fixation index ( F) were determined. The Hungarian pure bred dog populations could be distinguished by comparing the allele frequency values using G-statistics and calculating the F ST indices with pair-wise comparisons of inter-population molecular variance (AMOVA). The results showed that these 10 loci can be adequate for individual identification in forensic cases even in relatively inbred dog populations.

Abstract 150: HPV and aberrant DNA methylation status in paired saliva and tumor samples in HNSCC

Abstract To examine the utility of non-invasive detection of high risk HPV and aberrant DNA methylation in matched saliva and tumor DNA in squamous head and neck cancer (HNSCC). In a cohort of 35 primary HNSCC with matched tumor (cases) and saliva specimens, HPV status was initially determined using the Linear Array HPV Genotyping kit and confirmed by PCR for HPV subtypes 16, 31, 33 and 45. Aberrant methylation of 24 tumor suppressor genes was interrogated using the methylation specific multiplex ligation probe amplification (MS-MLPA) assay. Of the 35 cases, 20 were negative for HPV in the saliva and tumor, 9 were positive for HPV in both and in 6 cases HPV was positive in the tumor specimen but not detected in the corresponding saliva. In the 9 cases that were positive for HPV in the tumor and matched saliva, 6 were HPV 16 positive and 1 case each was positive for HPV 31, 33 and 45. The sensitivity and specificity for HPV in this cohort were 100% and 60%, respectively. Promoter hypermethylation frequency in tumor samples was 60% (21/35) as compared to 14% (5/35) in saliva. The most frequently methylated genes in tumors included RARB-10/21, APC- 9/21, TIMP3 and CDKN2B- 7/21, CDKN2A and IGSF4- 5/21 tumor samples. The most frequently methylated genes in saliva samples included TIMP3, ESR1 and IGSF4 (3/5 saliva samples), followed by APC, CDKN2B and DAPK1 (2/3). Two of the 35 tumor and saliva pairs had commonly methylated genes, APC in one pair, and TIMP3, CDKN2B, ESR1 and IGSF4 in the other. There was no association between HPV and methylation. PCR analysis of HPV DNA in salivary rinses allows for detection of HPV and promoter methylation status in HNSCC and is a feasible noninvasive alternative to more invasive procedures. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 150.

Highly efficient multiplex PCR of noninvasive DNA does not require pre‐amplification

Among the key issues determining success of a study employing molecular genetics tools in wildlife monitoring or research is a large enough set of highly informative genetic markers and a reliable, cost effective method for their analysis. While optimized commercial genotyping kits have been developed for humans and domestic animals, such protocols are rare in wildlife research. We developed a highly optimized multiplex PCR that genotypes 12 microsatellite loci and a sex determination locus in brown bear (Ursus arctos) faecal samples in a single multiplex PCR and a single sequencer run. We used this protocol to genotype 1053 faecal samples of bears from the Dinaric population, and obtained useful genotypes for 88% of the samples, a very high success rate. The new protocol outperformed the multiplex pre-amplification strategy used in a previous study of 473 faecal samples with a 78.4% success rate. On a subset of 182 samples we directly compared the performance of both approaches, and found no advantage of the multiplex pre-amplification. While pre-amplification protocols might still improve PCR success and reliability on a small fraction of low-quality samples, the higher costs and workload do not justify their use when analysing reasonably fresh non-invasive material. Moreover, the high number of multiplexed loci in the new protocol makes it comparable to commercially developed genotyping kits developed for domestic animals and humans.

Hepatitis C virus genotype frequency in Isfahan province of Iran: a descriptive cross-sectional study

BackgroundHepatitis C is an infectious disease affecting the liver, caused by the hepatitis C virus (HCV). The hepatitis C virus is a small, enveloped, single-stranded, positive sense RNA virus with a large genetic heterogeneity. Isolates have been classified into at least eleven major genotypes, based on a nucleotide sequence divergence of 30-35%. Genotypes 1, 2 and 3 circulate around the world, while other genotypes are mainly restricted to determined geographical areas. Genotype determination of HCV is clinically valuable as it provides important information which can be used to determine the type and duration of therapy and to predict the outcome of the disease.ResultsPlasma samples were collected from ninety seven HCV RNA positive patients admitted to two large medical laboratory centers in Isfahan province (Iran) from the years 2007 to 2009. Samples from patients were subjected to HCV genotype determination using a PCR based genotyping kit. The frequency of HCV genotypes was determined as follows: genotype 3a (61.2%), genotype 1a (29.5%), genotype 1b (5.1%), genotype 2 (2%) and mixed genotypes of 1a+3a (2%).ConclusionGenotype 3a is the most frequent followed by the genotype 1a, genotype 1b and genotype 2 in Isfahan province, Iran.

Monitoring of drug resistance in therapy-naïve HIV infected patients and detection of African HIV subtypes in Hungary

Mutations in the HIV-1 pol gene associated with resistance to antiretroviral drugs in therapy-naïve Hungarian individuals transmitted as primary infection by their foreign sexual partners originated from African, Asian and other European countries had been analyzed. Drug resistance genotyping of HIV RT and PR genes were performed where mutations of 72 codons - among them 64 specific resistance codons representing 6 nucleoside reverse transcriptase inhibitor (NRTIs), 2 non-nucleoside reverse transcriptase inhibitor (NNRTIs) and 6 proteinase inhibitor (PRIs) drugs - had been analyzed by Truegene HIV-1 Genotyping kit and OpenGene Sequencing System. Viral variants harboring resistance mutations in the po l gene were detected in 14% of the subjects. The highest rate of resistance to a single class of inhibitors was detected towards PR inhibitors (12%), followed by NRTI (8%) and NNRTI (5%). On the contrary, 25% of viruses transmitted by homosexual activity contained mutations led to resistance to NNRT. Viruses from 11 percent of cases were resistant to 2 classes of inhibitors, and 7 percent to three classes of inhibitors. Based upon sequence data non-B subtypes and CRFs were detected in more than 71% of cases. HIV-1 C (10.7%), HIV-F1 (7.2%) and HIV-1 G (3.6%) were detected as the more frequent subtypes. Among the HIV-1 recombinant viruses CRF02_AG variants were found more frequently (28.5%) followed by CRF06_cpx (17.8%) indicating penetration of non-B subtypes and recombinant African variants into Hungary, which raises serious clinical and public health consequences.

Transmission of HIV‐1 Drug‐Resistant Variants: Prevalence and Effect on Treatment Outcome

Human immunodeficiency virus type 1 (HIV-1) drug resistance is an important threat to the overall success of antiretroviral therapy (ART). Because of the limited sensitivity of commercial assays, transmitted drug resistance (TDR) may be underestimated; thus, the effect that TDR has on treatment outcome needs to be investigated. The objective of this study was to investigate the prevalence of TDR in HIV-infected patients and to evaluate the significance of TDR with respect to treatment outcome by analyzing plasma viral RNA and peripheral blood mononuclear cell proviral DNA for the presence of drug resistance mutations. In a prospective study, we investigated the level of TDR in 61 patients by comparing the results of a sensitive multiplex-primer-extension approach (termed HIV-SNaPshot) that is capable of screening for 9 common nucleoside reverse-transcriptase inhibitor and nonnucleotide reverse-transcriptase inhibitor mutations with those of a commercial genotyping kit, ViroSeq (Abbott). Twenty-two patients were found to carry mutations. More patients with TDR were identified by the HIV-SNaPshot assay than by ViroSeq analysis (33% vs 13%; [P=.015). There was no significant difference in the time from initiation of ART to virological suppression between susceptible patients and those carrying low- or high-level resistance mutations (mean +/- standard deviation, 128 +/- 59.1 vs 164.9 +/- 120.4; P=.147). Furthermore, analyses of CD4 cell counts showed no significant difference between these 2 groups 1 year after the initiation of ART (mean, 184 vs 219 cells/microL; P=.267). We found the prevalence of TDR in recently infected ART-naive patients to be higher than that estimated by ViroSeq genotyping alone. Follow-up of patients after treatment initiation showed a trend toward there being more clinical complications for patients carrying TDR, although a significant effect on treatment outcome could not be demonstrated. Therefore, the clinical relevance of low-abundance resistant quasispecies in early infection is still in question.

Efficacy evaluation of imatinib treatment in patients with gastrointestinal stromal tumors: A meta-analysis

To perform a meta-analysis to derive a more precise estimation of imatinib treatment for different genotypes of gastrointestinal stromal tumors (GIST). Studies were identified by searching PubMed and Embase. Inclusive criteria were patients with exon 9-mutant, exon 11-mutant or wide type (WT) GIST, receiving chemotherapy of imatinib for clinical trial, and efficacy evaluation was cumulative response (CR) including complete response and partial response. The odds ratios (OR) for CR in stem cell factor receptor (KIT) mutation patients vs WT genotype patients, KIT exon 11-mutant genotype patients vs KIT exon 9-mutant genotype patients and KIT exon 9-mutant genotype patients vs WT genotype patients were calculated with 95% confidence interval (CI) for each study as an estimation of the efficacy of imatinib. Five studies including 927 patients were involved in this meta-analysis. The overall OR (KIT group vs WT group) was 3.34 (95% CI: 2.30-4.86, P < 0.00001, P(heterogeneity) = 0.04). The overall OR in KIT exon 11 group vs KIT exon 9 group was 3.29 (95% CI: 2.17-5.00, P < 0.00001, P(heterogeneity) = 0.33). The overall OR in KIT exon 9 group vs WT group was 1.23 (95% CI: 0.73-2.10, P = 0.44, P(heterogeneity) = 0.42). Most patients with different genotypes of GIST and KIT exon 11-mutant will benefit from the individualized treatment of imatinib.

Polymorphisms of vascular diseases-related genes in Guangzhou Biobank Cohort Study

To study the polymorphisms of cerebrovascular and cardiovascular disease genes using Taqman single nucleotide polymorphism (SNP) genotyping kits. A total of 2000 subjects were recruited from the Guangzhou Biobank Cohort Study (GBCS), and 15 SNPs were detected using Taqman SNP genotyping kits and an ABI 7900HT real time PCR system. The data were tested for the Hardy-Weinberg equilibrium, and then compared with the data of the Chinese population from the International HapMap Project (HapMap_HCN). (1) All genotype data of the 15 SNPs were consistent with the Hardy-Weinberg rules. (2) The significant differences were observed among two SNPs, rs4220 and rs5368 and the HapMap_HCN (rs4220 28.2% vs 17.8%; chi(2) = 4.891, P = 0.028; rs5368 22.1% vs 32.2%, chi(2) = 5.137, P = 0.024). Comparing other gene bank data, such as AFD-CHN-PANEL, the Allele Frequency Database (ALFRED) and JBIC-allele, it would be most likely that our observations represent differences between the Northern and Southern populations in China. Such Biobank study provided a useful platform for the study of the role of genetic and environmental determinants on cerebrovascular and cardiovascular disease.

SOD2 V16A SNP in the Mitochondrial Targeting Sequence is Associated with Noise Induced Hearing Loss in Chinese Workers

Objective:To investigate whether single nucleotide polymorphisms (SNPs) in the Mn-superoxide dismutase gene (SOD2) underlie the susceptibility to noise-induced hearing loss (NIHL).Methods:Audiometric data from 2400 Chinese Han workers who exposed to occupational noise were analyzed. DNA samples were collected from the 10% most susceptible and the 10% most resistant individuals, and five SNPs (SOD2 rs2842980, rs5746136, rs2758331, rs4880 and rs5746092) were genotyped by Taqman SNP Genotyping Kits. The SNP main effects and interactions between noise exposure and SNP were analyzed using logistic regression. Haplotypes were analyzed by using Haploview software.Results:The CT genotype of rs4880 (SOD2 V16A SNP) was associated with a higher risk of NIHL (covariates-adjusted OR, 2.18; 95% CI, 1.34–3.54,P= 0.002). Haplotype analysis revealed that the frequency of AGCCG at the five SNP loci was significantly higher in the susceptible group (P= 0.020). With AGCTG as the reference, the OR (95% CI) was 2.63 (1.14, 6.06). The rs4880 polymorphisms imposed larger effects when the carriers were exposed to higher levels of noise, indicating the interaction between SNP and noise exposure.Conclusions:Our results suggest that SOD2 V16A SNP in the mitochondrial targeting sequence is associated with noise induced hearing loss in Chinese workers, and this effect was enhanced by higher levels of noise exposure.

SOD2 V16A SNP in the mitochondrial targeting sequence is associated with noise induced hearing loss in Chinese workers.

Objective: To investigate whether single nucleotide polymorphisms (SNPs) in the Mn-superoxide dismutase gene (SOD2) underlie the susceptibility to noise-induced hearing loss (NIHL). Methods: Audiometric data from 2400 Chinese Han workers who exposed to occupational noise were analyzed. DNA samples were collected from the 10% most susceptible and the 10% most resistant individuals, and five SNPs (SOD2 rs2842980, rs5746136, rs2758331, rs4880 and rs5746092) were genotyped by Taqman SNP Genotyping Kits. The SNP main effects and interactions between noise exposure and SNP were analyzed using logistic regression. Haplotypes were analyzed by using Haploview software. Results: The CT genotype of rs4880 (SOD2 V16A SNP) was associated with a higher risk of NIHL (covariates-adjusted OR, 2.18; 95% CI, 1.34–3.54, P = 0.002). Haplotype analysis revealed that the frequency of AGCCG at the five SNP loci was significantly higher in the susceptible group (P = 0.020). With AGCTG as the reference, the OR (95% CI) was 2.63 (1.14, 6.06). The rs4880 polymorphisms imposed larger effects when the carriers were exposed to higher levels of noise, indicating the interaction between SNP and noise exposure. Conclusions: Our results suggest that SOD2 V16A SNP in the mitochondrial targeting sequence is associated with noise induced hearing loss in Chinese workers, and this effect was enhanced by higher levels of noise exposure.

Affordable in-house antiretroviral drug resistance assay with good performance in non-subtype B HIV-1

The introduction of antiretroviral (ARV) therapy in resource-poor settings is effective in suppressing HIV-1 replication and prolonging life of infected individuals. This has led to a demand for affordable HIV-1 drug resistance assays, since treatment failure due to development of drug resistance is common. This study developed and evaluated an affordable “in–house” genotyping assay to monitor HIV-1 drug resistance in Africa, particularly South Africa. An “in-house” assay using automated RNA extraction, and subtype C specific PCR and sequencing primers was developed and successfully evaluated 396 patient samples (viral load ranges 1000–1.6 million RNA copies/ml). The “in-house” assay was validated by comparing sequence data and drug resistance profiles from 90 patient and 10 external quality control samples to data from the ViroSeq™ HIV-1 Genotyping kit. The “in-house” assay was more efficient, amplifying all 100 samples, compared to 91 samples using Viroseq. The “in house” sequences were 99.2% homologous to the ViroSeq sequences, and identical drug resistance mutation profiles were observed in 96 samples. Furthermore, the “in-house” assay genotyped 260 of 295 samples from seven African sites, where 47% were non-subtype C. Overall, the newly validated “in-house” drug resistance assay is suited for use in Africa as it overcomes the obstacle of subtype diversity.

Tokyo-172 BCG Vaccination Complications, Taiwan

Tokyo-172 BCG Vaccination Complications, Taiwan

초위성체 DNA표지인자를 이용한 국내 육우집단의 품종특성 및 개체식별 체계설정

소 품종 판별을 위해 DNA 마커 정보는 품종을 구별하거나, 형질을 구분하는데 있어 꾸준히 이용되고 있다. 본 연구에서는 Finnzymes (DIAGNOSTICS)사의 Bovine Genotypes Kit Ver1.1/2.1을 농촌진흥청 국립축산과학원이 보유한 호주산 및 미국산 수입우 DNA 샘플 148두/국내산 육우 DNA 샘플(Holstein) 170두와 정읍지역 한우 DNA 샘플 177두에 적용하여 한우품종 식별력을 분석 하였다. Bovine Genotype Kit 1.1은 11개의 ISAG MS 마커로 이루어져 있으며, 여기에 5개 MS 마커가 추가된 ver2.1 Kit를 사용하여 집단별 유전자형 데이타를 구축하였고, MS Tool kit 분석 및 Phylip program 분석을 수행하여 Phylogenetic tree를 작성하였고, Genotype 분석 프로그램인 GeneClass 2.0 (INRA/France)을 이용하여 품종 식별력을 추정하였다. 분석 결과 95% 이상의 정확성을 가진 한우 식별력은 100%로 나타났고, 호주산 수입우 95.3%, 국내산 육우는 90%의 높은 식별력을 각각 나타내었다. 따라서 Finnzymes 사의 상용화된 16종의 MS 마커는 한우집단의 유전적 특징을 객관적으로 구분하여 수입쇠고기/젖소고기/한우쇠고기에서 간편하게 한우개체 및 품종식별에 활용될 수 있는 가능성과 특히 국내에서 비육된 육우(젖소)를 수입산 쇠고기로부터 식별할 수 있는 장점이 있을 것으로 사료된다. DNA marker information is used to identify or distinguish cattle breeds or individual animal. The purpose of this study was to apply Bovine Genotypes Kit Version 1.1/2.1 to bovine DNA samples (National Institute of Animal Science) taken from Australian / American beef (n=148), Holstein beef (n=170) and Hanwoo cattle (n=177) bred in Jeongeub, Jeonbuk, Korea, so that it could distinguish Hanwoo breed. The Bovine Genotype Kits consist of 16 ISAG MS markers, which were used to build a database of genotypes in each group. Genotyping results were analyzed using MS Tool kit and Phylip program to create phylogenetic tree. The GeneClass 2.0 was used to estimate breed identification. These analyses found that this kit had 100% capacity to distinguish Hanwoo beef, 95.3% capacity to differentiate Australian / American beef and 90% capacity to identify Korean Holstein steer beef. Hence, it is expected that 16 commercial microsatellite markers is useful to categorizegenetic characteristics of Hanwoo breed and also identify Hanwoo individuals and the origin of beef. In particular, it is expected that these markers will be advantageous in discriminating domestic Holstein beef from Australian / Americanbeef.

Colorimetric microwell plate reverse-hybridization assay for detection and genotyping of hepatitis C virus

This study describes a colorimetric method for detecting and genotyping hepatitis C virus (HCV) in which four different oligonucleotide probes are fixed onto microwell plates and hybridized separately with biotinylated PCR amplification products derived from clinical samples. The first probe capable of hybridizing with all seven known HCV genotypes was used for overall detection, and the remaining probes were used to recognize specifically genotypes 1–3. When combined with an improved silica-based RNA extraction method, the sensitivity of the test was 50 IU/mL. Eighty-five of the 86 samples analyzed (98.8%) yielded results in agreement with reference detection methods. The remaining sample was HCV-RNA positive in the COBAS Amplicor qualitative assay, but was negative using the reverse-hybridization method. The usefulness of the new genotyping test was confirmed by comparison with direct sequencing of PCR products: 98% of samples tested (54/55) were in agreement using the two methods (21, 7 and 27 from genotypes 1–3, respectively). The single discrepancy might have been due to a mixed HCV infection. The new method is an alternative to the use of commercially available genotyping kits and should be particularly convenient in developing countries where genotypes 1–3 represent a high proportion of HCV isolates.

Usefulness of Real-time Semi-quantitative PCR, JAK2 MutaScreenTM Kit for JAK2 V617F Screening

Real-time PCR for quantification of JAK2 V617F has recently been introduced and used to evaluate the importance of mutant allele burden in both diagnosis and disease progression in myeloproliferative diseases (MPDs). We evaluated the usefulness of JAK2 MutaScreen kit that uses a real-time semiquantitative PCR method and has been designed to screen JAK2 V617F mutant allele burden. Forty MPD patients were included in this study. We screened JAK2 V617F and determined the mutant allele burden using JAK2 MutaScreen kit. The mutant allele burden was estimated by six-scaled standards of JAK2 V617F mutant allele (2%, 5%, 12.5%, 31%, 50%, and 78%). For evaluation of test performance, an allele-specific PCR (AS-PCR) was carried out in all samples by using Seeplex JAK2 Genotyping kit. We assessed the clinical differences in distinct disease entities of MPDs according to JAK2 V617F mutant allele burden. JAK2 V617F mutation was detected in 30 cases, including 10 of 11 cases (91%) of polycythemia vera (PV), 13 of 20 cases (65%) of essential thrombocythemia (ET), and 2 of 3 cases (67%) of chronic idiopathic myelofibrosis (CIMF). The concordance rate between the two tests was 95% (38/40). JAK2 V617F mutant allele burden was greater than 50% in 17 cases, and 10 of them (59%) were PV. In contrast, mutant allele burden was less than 50% in 13 cases and 11 of them (85%) were ET. JAK2 MutaScreen kit that utilizes a real-time semi-quantitative PCR method is a useful tool for diagnosing MPDs precisely. It can be used to assess the grade of mutant allele burden as well as to screen JAK2 V617F simultaneously.

TS and MTHFR gene polymorphisms in patients (pts) with recurrent or metastatic squamous cell carcinoma of the head or neck (SCCHN) treated with pemetrexed (P) and bevacizumab (B)

e17011 Background: P inhibits multiple enzymes in folate metabolism. We examined polymorphisms in thymidylate synthase (TS) and methylenetetrahydrofolate reductase (MTHFR) in patients with SCCHN treated in a phase II clinical trial with P and B (ASCO 2008; A6069). Methods: All pts were treated with P 500 mg/m2 and B 15 mg/kg, given IV every 21 days until progression. Primary endpoint was time to progression (TTP). DNA was isolated from whole blood samples using commercially available kits. Polymorphisms examined were MTHFR (C677T, A1298C and G1793A) and TS (TS2R3R, TSG2RG and TSmut6). The MTHFR SNPs were detected using TaqMan based SNP genotyping kits from Applied Biosystems, run on the ABI Prism 7700 Sequence Detection systems v 1.7 (Foster City, CA). The TS promoter repeat and promoter SNP polymorphisms and the 3’ untranslated region 6 bp deletion polymorphism were determined using published methods to detect PCR product size and RFLP-PCR assays respectively. Results: 22 pts were genotyped from 34 enrolled. There was no significant difference in characteristics between pts with and without genotype data. For the MTHFR polymorphism C677T, there was a trend towards decreased disease control rate (DCR) (CR/PR/SD) (p = 0.058, Jonckheere-Terpstra trend test) and worse TTP (p = 0.04) transitioning from variant CC to CT to TT; comparing TT genotype versus CT and CC combined, pts with TT had inferior DCR (p = 0.03) and TTP (p = 0.0003); homozygotes with TT had a median TTP of 2.6 months (mo) 95% CI (1.4, NA) versus 5.6 mo (4.2, 11.4) for pts with CT or CC variants. For the MTHFR A1298C SNP, there was no significant difference in DCR between variants, median TTP for homozygotes pts with AA was 4.1 mo (2.6, NA) vs. 6.7 mo (5.1, NA) in pts with AC or CC variants (p = 0.084); median overall survival for AA was 10.2 mo (7.6, NA) and for AC or CC 17.6 mo (17, NA) (p = 0.045). The MTHFR G1793A and TS polymorphisms did not impact DCR, TTP or overall survival. There was no association between any polymorphism and the incidence of grade &gt;2 toxicities. Conclusions: Polymorphisms in MTHFR are potentially associated with antitumor efficacy of P-based therapy in recurrent or metastatic SCCHN. These results warrant validation in larger studies with P in SCCHN. No significant financial relationships to disclose.

Experience of Antiretroviral Treatment in Georgia

HIV infection is the major public health, social and economic problem in Georgia. Although the HIV epidemic is in its nascent phase in the country, the potential risk for development of a wide spread HIV epidemic is very high. The aim of this study is to evaluate the effectiveness of ARV treatment principles in Georgia, including treatment and monitoring methods. The study included 985 people living with HIV/AIDS in Georgia registered at Infectious Disease, AIDS and Clinical Immunology Research Center since 2004. To ensure universal access to ARV therapy all HIV/AIDS individuals included in the study were investigated by special algorithm, all identified patients requiring ARV therapy were offered treatment and monitored during therapy on treatment effectiveness and side effects. HIV-1 RNA in plasma was measured by quantitative Polymerase Chain Reaction. For determination of percentages and absolute count of T-lymphocyte subpopulations single-platform immunophenotyping technique using the Becton-Dickinson FACSCalibur flow cytometer was applied. For resistance testing TRUGENE HIV-1 Genotyping Kit with the OpenGene DNA Sequencing System (Siemens) was used. Reasons of treatment failure and mortality rate among ARV treated patients were analyzed. Treatment was offered to 398 HIV/AIDS patients. 397 patients started treatment, 1 patient refused. Out of 397 HIV/AIDS patients treated 21 patients discontinued, 54 patients died and 322 patients are currently on ARV treatment. Out of the treated patients 281 adults and 11 children are receiving first-line treatment, 27 adults and 2 children are on second-line treatment and 1 adult is receiving salvage regimen. Treatment failure was defined in 52 cases. Among them immunological failure was observed in 7 cases, clinical failure in 1 case and virologic failure in 44 cases. Prevalence of drug resistance among virologic failure cases accounted for 73% and inadequate adherence for 27% cases. Out of drug resistance cases 3% has three-class drug resistance, 84%--two-class drug resistance and 13% found to be resistant to one class. In ARV naive patients the prevalence of drug resistance to any class was 4.33%. The majority of death cases among ARV treated patients was due to non-AIDS related or incurable conditions, while deaths due to AIDS related conditions were mainly associated with delayed referral of patients in already advanced stage of disease. It's worth to mention that the highest number of death cases was due to liver failure in HIV/HCV and/or HBV co-infected patients.