Restriction Fragment Mass Polymorphism Research Articles

Preliminary study of analyzing mucosal human papillomaviruses in cutaneous warts by restriction fragment mass polymorphism.

Cutaneous human papillomavirus (HPV) types 1, 2, 4, 7 and 57 are reportedly found in cutaneous warts. However, there are few reports that have investigated the prevalence of mucosal HPV types in cutaneous warts. The aim of this study is to investigate the prevalence of mucosal HPV types in patients with cutaneous warts and to determine any association between HPV types and patient characteristics. We analyzed 62 wart samples that were taken from patients who were diagnosed with cutaneous warts, and 30 normal skin samples were used as negative control. We recorded the following characteristics: sex, age, type of warts, duration of warts, number of warts and patient's immune status. A matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS)-based restriction fragment mass polymorphism (RFMP) assay was used for HPV genotyping. Of the total 62 wart samples, 50 samples (81.6%) were positive for HPV genotypes. All of the negative controls (30 samples) using normal skin showed negative reaction. Mucosal HPV types (49 samples, 84.4%) were highly detected, and high- or probable high-risk HPV types (39 samples, 67.2%) were more common than lower risk HPV types (10 samples, 17.2%). A statistically significant association was observed between sex, age, duration of warts and the risk of HPV types. To the best of our knowledge, this is the first study to use the RFMP assay to analyze cutaneous wart-associated mucosal HPV types. The high prevalence of high-risk and probable high-risk HPV in this study is of great significance.

Analysis of mucosal human papillomaviruses in cutaneous warts by restriction fragment mass polymorphism

Analysis of mucosal human papillomaviruses in cutaneous warts by restriction fragment mass polymorphism

Rapid Genotyping of Human Papillomavirus by Desorption Electrospray Ionization Mass Spectrometry

This study describes a new approach to rapidly detecting of human papillomavirus (HPV) DNA using desorption electrospray ionization mass spectrometry (DESI‐MS) without sample preparation such as extraction or purification. Our strategy utilizes the ambient ionization mass spectrometry for ionizing and detecting oligonucleotides from restriction fragment mass polymorphism (RFMP) assay, and is to identify which HPV genotype from combinations of multiply charged ions of oligonucleotides. In DESI‐MS, the spray solvent system operates at the stage of washing a complex mixture of polymerase chain reaction (PCR) products, so that the oligonucleotides can be detected without sample treatment. The study was verified to rapid diagnosis of cervical cancer by detecting and genotyping of HPV DNA based RFMP assay using DESI‐MS.

Comparison of GeneFinder human papillomavirus (HPV) Liquid Beads Microarray PCR Kit and Hybrid Capture 2 Assay for Detection of HPV Infection

Along with advances in methodological technologies, various assays for detecting high-risk human papillomavirus (HR HPV) have been introduced. The GeneFinder HPV liquid beads microarray PCR kit is one of the recently developed. Our aim was to compare the performance of GeneFinder to Hybrid Capture 2 for detection of HR HPV. A total of 900 cervical swab specimens were obtained. All specimens were submitted for HR HPV detection with Hybrid Capture 2 (HC2) and GeneFinder and then additionally analyzed the discordant or both positive results using restriction fragment mass polymorphism (RFMP) genotyping analysis. Hybrid Capture 2 detected 12.8% cases and GeneFinder detected 15.8% cases with 13 HR HPV types. Also, GeneFinder detected 27.4% cases for 32 detectable HPV types. The overall agreement rate was 93.2% with 0.724 kappa coefficient. Discordant results between these two assays were observed in 56 cases. HC2 showed sensitivity of 83.5% and specificity of 95.9%, while GeneFinder showed sensitivity of 85.4% and specificity of 91.9%. For HPV 16 or HPV 18 detection, GeneFinder showed 95.0% or 66.7% of sensitivity and 99.2% or 100%, respectively. Overall coinfection rate was 15.4% (38/247) in GeneFinder analysis. Considering the high agreement rate with HC2, high sensitivity and the ability to differentiate 32 HPV genotypes including HPV 16/18, GeneFinder could be used as a laboratory testing method for the screening of HPV infections. The use of GeneFinder may also contribute to future research associated with the significance of various HPV types and multiple coinfections.

Clonal evolution of multidrug resistant hepatitis B virus during entecavir rescue therapy.

Analysing the mutation pattern of multidrug resistance (MDR) is important in the treatment of chronic hepatitis B (CHB). In this study, the evolutionary pattern of MDR mutations was investigated in patients receiving entecavir (ETV) rescue therapy. Eight CHB patients with lamivudine (LAM)- and adefovir (ADV)-resistant mutations showing suboptimal response to ETV and to subsequent ETV-plus-ADV therapy were enrolled. The clonal evolution of the mutation pattern was investigated through direct sequencing, multiplex restriction fragment mass polymorphism (RFMP), and clonal analysis and the utility of these methods was compared. Among 160 clones at baseline, wild-type hepatitis B virus (HBV) was present in 62 (38.8%), LAM-resistant mutations in 92 (57.6%) and ADV-resistant mutations in 55 (34.4%). LAM-resistant mutations increased to 70.6% at the end of ETV therapy and increased to 74.4% at the 12th month of ETV-plus-ADV therapy. During the same time periods, ETV-resistant mutations were present in 46.3% and 38.8%, and ADV-resistant mutations were present in 3.1% and 9.4% respectively. When 256 nucleotides from 32 samples were examined for mutations, clonal analysis detected 93 mutations (36.3%), direct sequencing detected 36 mutations (14.1%) and RFMP detected 73 mutations (28.5%). The sensitivity (73.1%, 95% CI; 64.1-82.1%) and specificity (96.9%, 95% CI; 94.4-99.4%) of RFMP were high, showing a concordance rate of 88.3% with the results from clonal analysis. All mutations exceeding 40% of the total clones detected by clonal analysis were also detected by RFMP. The clonal evolution of the mutation pattern in MDR HBV showed the selection of LAM-resistant (±ETV-resistant) HBV during ETV rescue therapy, which may be the primary reason for patients' suboptimal response. Multiplex RFMP is a useful method for detecting MDR mutations in clinical practice.

Analysis of HBV genotype, drug resistant mutations, and pre-core/basal core promoter mutations in Korean patients with acute hepatitis B.

Acute hepatitis B, caused by hepatitis B virus (HBV) strains with drug resistant mutations or pre-core/basal core promoter (PC/BCP) mutations, is a public health concern, because this infection is often associated with poor disease outcome or difficulty in therapeutic choice. The HBV genotype, the prevalence of drug resistant mutations, and PC/BCP mutations in Korean patients with acute hepatitis B were studied. From 2006 to 2008, 36 patients with acute hepatitis B were enrolled prospectively in four general hospitals. Among them, 20 showed detectable HBV DNA (median value was 4.8 log copies/mL). HBV genotyping and analysis of HBV mutations that conferred resistance against lamivudine, adefovir, or entecavir and of PC/BCP mutations were performed using highly sensitive restriction fragment mass polymorphism (RFMP) analysis. All 20 patients were infected with HBV genotype C, which causes almost all cases of chronic hepatitis B in Korea. No patient showed mutations that conferred resistance against lamivudine (L180M, M204V/I), adefovir (A181T, N236S), or entecavir (I169M, A184T/V, S202I/G, M250V/I/L). However, four patients had BCP mutations, and two had PC mutations. Platelet counts were significantly lower in the four patients with PC/BCP mutations compared to those with wild type. In this study, all acute hepatitis B patients had genotype C HBV strains with no drug resistant mutations. However, 20% showed PC/BCP mutations. This highlights the need for further study on the significance of PC/BCP mutations.

Long-term outcomes and dynamics of mutants associated with lamivudine-adefovir rescue therapy in patients with lamivudine-resistant chronic hepatitis B.

Background/AimsTo investigate the association between the baseline profiles and dynamics of hepatitis B virus (HBV) DNA polymerase gene mutations and the long-term virological response of lamivudine (LAM)-adefovir (ADV) combination therapy in patients with LAM-resistant chronic hepatitis B.MethodsSeventy-five patients who received LAM-ADV combination therapy for more than 12 months were analyzed. Restriction fragment mass polymorphism assays were used to detect and monitor the dynamics of LAM- and ADV-resistant mutations.ResultsThe median duration of LAM-ADV combination therapy was 26 months (range, 12 to 58 months). The baseline mutation profiles, rtM204I (p=0.992), rtM204I/V (p=0.177), and rtL180M (p=0.051), were not correlated with the cumulative virological response, and the baseline HBV DNA level (p=0.032) was the only independent predictive factor for cumulative virological response. Tests for LAM- and ADV-resistant mutations were performed in 12 suboptimal responders in weeks 48 and 96. The population of rtM204 mutants persisted or increased in 8 of 12 patients, and rtA181T mutants newly emerged as a minor population in four patients until 96 weeks. Nevertheless, the viral loads progressively decreased during rescue therapy, and these dynamics did not correlate with virological response.ConclusionsThe baseline profile and dynamics of LAM-resistant mutations during LAM-ADV combination therapy are not associated with a virological response.

Multiplex detection of KRAS mutations by a matrix-assisted laser desorption/ionization-time of flight mass spectrometry assay

ObjectivesMutations of the Kirsten rat sarcoma viral oncogene (KRAS) gene are known to be important in the pathogenesis of a variety of cancers. Patients with mutant KRAS tumors do not respond to epidermal growth factor receptor (EGFR) inhibitors and fail to benefit from adjuvant chemotherapy. Testing for KRAS mutations is now being recommended as a tailored therapeutic strategy prior to anti-EGFR treatment; however, the low sensitivity of direct sequencing frequently leads to failure of detection of KRAS mutations in clinical samples. Design and methodsWe developed restriction fragment mass polymorphism (RFMP) assays, to detect KRAS mutations in codons 12, 13, and 61. We performed RFMP analysis for KRAS on DNA isolated from eight different KRAS mutant cell lines and 34 formalin-fixed paraffin-embedded (FFPE) lung cancer tissues. ResultsOverall, the RFMP assay was in good concordance with direct sequencing analysis in the detection of KRAS mutations. By using dilutions of KRAS mutant DNA in wild type DNA from mutation cell lines with a known KRAS status, we confirmed that the RFMP assay has a higher analytical sensitivity, requiring only 3% of cells in a sample to have mutant alleles, compared to direct sequencing, which had a detection threshold of ~25%. In the FFPE samples, RFMP successfully detected KRAS genotypes in codons 12, 13, and 61. ConclusionThe RFMP might be efficiently applicable for the detection of KRAS mutations in a clinical setting, particularly for tumor samples containing abundant non-neoplastic cells.

HBV Subgenotypes D1, D2, D-del! Are ‘Old‘ Genotyping Methods Interpreted Correctly?

HBV Subgenotypes D1, D2, D-del! Are ‘Old‘ Genotyping Methods Interpreted Correctly?

Restriction fragment mass polymorphism (RFMP) analysis based on MALDI-TOF mass spectrometry for detecting antiretroviral resistance in HIV-1 infected patients

Viral genotype assessment is important for effective clinical management of HIV-1 infected patients, especially when access and/or adherence to antiretroviral treatment is reduced. In this study, we describe development of a matrix-assisted laser desorption/ionization-time of flight mass spectrometry-based viral genotyping assay, termed restriction fragment mass polymorphism (RFMP). This assay is suitable for sensitive, specific and high-throughput detection of multiple drug-resistant HIV-1 variants. One hundred serum samples from 60 HIV-1-infected patients previously exposed to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs) were analysed for the presence of drug-resistant viruses using the RFMP and direct sequencing assays. Probit analysis predicted a detection limit of 223.02 copies/mL for the RFMP assay and 1268.11 copies/mL for the direct sequencing assays using HIV-1 RNA Positive Quality Control Series. The concordance rates between the RFMP and direct sequencing assays for the examined codons were 97% (K65R), 97% (T69Ins/D), 97% (L74VI), 97% (K103N), 96% (V106AM), 97% (Q151M), 97% (Y181C), 97% (M184VI) and 94% (T215YF) in the reverse transcriptase coding region, and 100% (D30N), 100% (M46I), 100% (G48V), 100% (I50V), 100% (I54LS), 99% (V82A), 99% (I84V) and 100% (L90M) in the protease coding region. Defined mixtures were consistently and accurately identified by RFMP at 5% relative concentration of mutant to wild-type virus while at 20% or greater by direct sequencing. The RFMP assay based on mass spectrometry proved to be sensitive, accurate and reliable for monitoring the emergence and early detection of HIV-1 genotypic variants that lead to drug resistance.

Detection ofABL1kinase mutations in Philadelphia-positive patients exhibiting an inadequate molecular response using restriction fragment mass polymorphism and its clinical significance: a single-center experience in Korea

ABL1 kinase mutations represent a major mechanism of imatinib resistance in Philadelphia-positive (Ph+) patients. There is a paucity of data on ABL1 kinase mutations in Ph+ patients in Korea. We used restriction fragment mass polymorphism (RFMP) analysis to detect ABL1 kinase mutations in blood or bone marrow specimens from 80 Ph+ patients. Fifty-seven patients met the criteria for inadequate molecular response (IMR). ABL1 kinase mutations were found in 2.6% of patients with chronic-phase chronic myelogenous leukemia (CML), 25.0% of accelerated-phase CML, 66.7% of blast-phase CML, and in 58.3% with Ph+ acute lymphoblastic leukemia. Twelve mutations were identified: 7 T315I, 2 E255V, 1 E255K, 1 F359V, and 1 Y253H. The majority of mutation-positive patients showed an unfavorable clinical course and often had an extra Ph or additional chromosomal abnormalities. Mutations were detected in two patients who had very low or absent BCR-ABL1 normalized ratios. Mutation analysis should be performed in Ph+ patients exhibiting an IMR to imatinib. RFMP analysis is helpful for revising therapeutic strategies because it can sensitively detect clinically relevant ABL1 kinase mutations with high frequencies.

Comparison of the clinical performance of restriction fragment mass polymorphism (RFMP) and Roche linear array HPV test assays for HPV detection and genotyping

BackgroundThe need for accurate genotyping of human papillomavirus (HPV) infections is becoming increasingly important as HPV is the primary cause of cervical cancer worldwide. The matrix-assisted laser desorption ionization time-of-flight mass spectrometry-based restriction fragment mass polymorphism (RFMP) assay provides accurate, broad-spectrum, high-throughput genotyping of HPV. ObjectivesWe evaluated the clinical performance of the RFMP assay compared to a commercially available Roche linear array HPV genotyping test (LA) for detecting and genotyping of HPV. Study designThe RFMP assay and the LA were compared for detecting and genotyping HPV among a cohort of 244 liquid-based cytology samples. ResultsOverall, 216 specimens (93.1%, κ=0.86) generated concordant results for the presence or absence of high-risk HPV (HR-HPV) by the two assays. The RFMP assay and the LA assay generated concordant, compatible, and discordant genotyping results for 79.3, 9.9, and 10.8%, respectively. The diagnostic sensitivity and specificity of RFMP and LA for the cervical lesions of squamous cell carcinoma (SCC) were similar, at 92.9 and 85.0% (RFMP) and 92.9 and 83.8% (LA), respectively. In addition, the odds ratio for SCC with HR-HPV positivity estimated by the RFMP assay (73.7, 95% CI: 8.9–3173.3) was higher than the LA assay (67.0, 95% CI: 8.2–2887.0). ConclusionsThe RFMP and the LA assays were highly comparable with regard to detection and genotyping analysis of HPV. The sensitivity and specificity of RFMP assay for the detection of HR-HPV in various levels of cervical lesions seems to be valuable in the monitoring of HPV-associated cervical cancer.

Randomized Trial of Entecavir plus Adefovir in Patients with Lamivudine-Resistant Chronic Hepatitis B Who Show Suboptimal Response to Lamivudine plus Adefovir

A substantial proportion of patients with lamivudine-resistant hepatitis B virus (HBV) show suboptimal virologic response during rescue combination treatment with lamivudine plus adefovir. In this randomized active-control trial, 90 patients with serum HBV DNA levels of >2,000 IU/ml after at least 24 weeks of treatment with lamivudine-plus-adefovir therapy for lamivudine-resistant HBV were randomized to combination treatment with entecavir plus adefovir (ETV+ADV, n = 45) or continuation of lamivudine plus adefovir (LAM+ADV, n = 45) for 52 weeks. At baseline, patients' mean serum HBV DNA level was 4.60 log(10) IU/ml (standard deviation [SD], 1.03). All 90 patients completed 52 weeks of treatment. At week 52, the proportion of patients with serum HBV DNA levels of <60 IU/ml, the primary endpoint, was significantly higher in the ETV+ADV group than in the LAM+ADV group (n = 13, 29%, versus n = 2, 4%, respectively; P = 0.004). The mean reduction in serum HBV DNA levels from baseline was significantly greater in the ETV+ADV group than in the LAM+ADV group (-2.2 log(10) IU/ml versus -0.6 log(10) IU/ml, respectively; P < 0.001). At week 52, additional mutations causing resistance to adefovir or entecavir were analyzed in all patients with detectable HBV DNA by restriction fragment mass polymorphism assays and detected in none of the ETV+ADV group but in 15% of patients in the LAM+ADV group (P = 0.018). Safety and adverse event profiles were similar in the two groups. In conclusion, entecavir-plus-adefovir combination therapy provides superior virologic response and favorable resistance profiles, compared with the continuing lamivudine-plus-adefovir combination, in patients with lamivudine-resistant HBV who fail to respond to lamivudine-plus-adefovir combination therapy.

Mutational complex genotype of the hepatitis B virus X /precore regions as a novel predictive marker for hepatocellular carcinoma

This study explored the combined effect of number and pattern of mutations in the X/precore regions of the hepatitis B virus (HBV) genome, mutational complex genotype (MCG), on hepatocellular carcinoma (HCC) development. Sequence variations were determined by direct sequencing and multiplex restriction fragment mass polymorphism analysis in 150 age-, sex- and hepatitis B e antigen (HBeAg) status-matched patients with and without HCC. In addition, a longitudinal study and an external validation of MCG were conducted. All were HBV subgenotype C2. Eight high-frequency mutations (G1613A, C1653T, T1753V, A1762T, G1764A, A1846T, G1896A and G1899A) were significantly associated with HCC. Whereas C1653T, T1753V, G1764A and A1846T were independent mutational factors for HCC, the significance of these individual mutations was negligible when analyzed with all clinico-virological variables. The total number of mutations was the only independent viral factor for HCC, irrespective of HBeAg status. There was a significant dose-risk relationship between the number of mutations and HCC, in which high risks for HCC were associated with mutation numbers ≥ 6. Pattern analysis of the mutations revealed disparity in distribution among the top seven high-risk mutation combination patterns, which accounted for 40 and 2.7% of HCC and non-HCC cases, respectively. The predictive accuracy of the high-risk mutations for HCC was similar to that of α-fetoprotein. Longitudinal and external validation studies also supported the association of mutation number with HCC development. MCG in the HBV X/precore regions is a risk indicator for HCC, and might serve as a new guide to the HCC screening scheme for chronic HBV carriers.

Prevalence and Distribution of Human Papillomavirus Infection in Korean Women as Determined by Restriction Fragment Mass Polymorphism Assay

The development of a prophylactic vaccine that targets human papillomaviruses (HPV) 6, 11, 16, and 18 to prevent cervical cancer has increased interest in the ethnic and geographical distributions of HPV genotypes. We investigated HPV prevalence and type distribution by restriction fragment mass polymorphism (RFMP) testing a total of 60,775 specimens (aged 18-79 yr, median 44) taken from liquid-based cytology. Overall HPV positive rate of total patients was 34.2%. Among the positive patients, 87.7% was single type infections, and 12.3% was multiple HPV types. HPV-16 was the most prevalent genotype observed in 2,307 (26.0%), followed by type 52 in 2,269 (25.5%), type 58 in 1,090 (12.3%), type 18 in 633 (7.1%), type 56 in 436 (4.9%). The pattern of high risk-HPV positive rate according to age showed U-shape with a peak in HPV prevalence among women less than 30 yr of age, and a second peak among the older females aged 70 to 79 yr. The leading four high-risk HPV genotypes were HPV-16, HPV-52, HPV-58, and HPV-18 in descending order. In conclusion, this study provides the most representative prevalence and type-specific distribution of HPV among Korean women, and demonstrates that the epidemiology of HPV infection is different from that of other regions of the world.

Pre‐existing YMDD mutants in treatment‐naïve patients with chronic hepatitis B are not selected during lamivudine therapy

Although the rate at which mutations in the tyrosine-methionine-aspartate-aspartate (YMDD) motif of hepatitis B virus polymerase form is high during prolonged lamivudine (LAM) therapy, these mutations sometimes occur naturally in treatment-naïve patients with chronic hepatitis B. The prevalence of natural YMDD mutants differs geographically, and its clinical significance during LAM therapy is unknown. This study aimed to investigate whether pre-existing YMDD mutants were selected during LAM therapy. It included 14 treatment-naïve patients who were treated with LAM for at least 9 months. LAM resistance was evaluated before and at 3-month intervals during treatment. Mutations were analyzed by direct sequencing, restriction fragment mass polymorphism (RFMP) assays, and a single-step multiplex polymerase chain reaction (PCR) test using dual-priming oligonucleotide (DPO) primers. DPO-based multiplex PCR showed two YMDD mutations in two patients before LAM therapy; rtM204V and rtL180M + rtM204V/I. Further, two patients had an rtL180M mutation without an accompanying rtM204V/I mutation. No mutant was detected in any patient by direct sequencing or the RFMP assay before LAM therapy. A virological response was observed at 3 months in all patients with pre-existing YMDD mutants. All mutations disappeared after 3 months of LAM therapy, and during the follow-up period, no re-emergence was detected by any of the three methods. Further, the viral load was suppressed optimally. In conclusion, pre-existing YMDD mutants were cleared early during the course of LAM therapy, which produced a consistent virological response, and the mutants were not selected by LAM therapy.

Analytical and clinical performances of a restriction fragment mass polymorphism assay for detection and genotyping of a wide spectrum of human papillomaviruses

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry-based restriction fragment mass polymorphism (RFMP) assay was adapted to human papillomavirus (HPV) genotyping. The analytical sensitivity and the clinical utility were evaluated by testing defined HPV genome equivalents and a total of 426 specimens composed of normal cytology, atypical squamous cells of undetermined significance, low grade squamous intraepithelial lesion, high grade squamous intraepithelial lesion and invasive squamous cell carcinoma. The RFMP assay was able to detect 38.4-114.6 genomic equivalents of a wide variety of HPV types. The RFMP assay detected 34 different HPV genotypes in cervical samples of which 8% were found to be multiple-type infections. The high-risk HPV positivity rate according to the histological diagnosis was 7.9% (8/101), 31.7% (38/120), 50% (55/110), 86% (37/43), 96.2% (50/52) in normal, atypical squamous cells of undetermined significance, low grade squamous intraepithelial lesion, high grade squamous intraepithelial lesion and squamous cell carcinoma subgroups, respectively. Diagnostic sensitivities/specificities for the cervical lesions of squamous cell carcinoma and high grade squamous intraepithelial lesion or worse histology were found to be 96.2%/92.1% and 91.6%/92.1%, respectively. The sensitivity, accuracy, wide range of genotype identification and high-throughput capacity with cost-effectiveness of the test consumables make the RFMP assay suitable for mass screening and monitoring of HPV-associated cervical cancer.

Analysis of TPOX short tandem repeat locus with matrix-associated laser desorption/ionization time-of-flight-based restriction fragment mass polymorphism assay

Short tandem repeat (STR) loci are routinely analyzed by capillary electrophoresis. However, this method has several disadvantages, including long operational time, low throughput, and inaccuracy. As a result of the introduction of matrix-associated laser desorption/ionization time-of-flight (MALDI–TOF) and electrospray ionization (ESI), mass spectrometry has become an alternative method for genotyping polymorphic STR loci. Here we established a restriction fragment mass polymorphism (RFMP) assay for genotyping STR locus, TPOX, by typeIIS restriction endonuclease cleavage of polymerase chain reaction (PCR) amplicon followed by MALDI–TOF mass spectrometry. The resulting TPOX genotypes from this assay were in good agreement with the results from direct DNA sequencing and GeneScan assays. Our results showed that the RFMP assay is an accurate and high-throughput method for analyzing long DNA fragments such as STR markers. Further research with multiple STR loci may allow this assay to be used for diverse applications such as forensics, paternity tests, and detection of genetic disorders.

Comparison of Multiplex Restriction Fragment Mass Polymorphism and Sequencing Analyses for Detecting Entecavir Resistance in Chronic Hepatitis B

Drug resistance is a major limitation to the long-term efficacy of controlling chronic hepatitis B (CHB). There is a growing need to analyse multiple mutations associated with drug resistance because sequential or combinational use of antivirals is increasingly being used in treatment. In this study, we introduced a multiplex restriction fragment mass polymorphism (RFMP) assay for detecting mutations conferring entecavir and lamivudine resistance, and compared its performance with direct or clonal sequencing assays. Multiplex PCR was performed with mixed primers designed to interrogate rt184, rt202, rt204 and rt250. The PCR products were digested with restriction enzymes and the resulting fragments were analysed by mass spectrometry. A total of 251 serum samples, taken serially from 45 patients who received entecavir treatment after confirmed diagnosis of lamivudine resistance and inadequate adefovir dipivoxil response, were analysed by the multiplex RFMP assay. The multiplex RFMP assay correctly identified known viral sequences with sufficient analytical sensitivity to detect as few as 100 IU/ml of HBV and with superior ability to determine haplotypes composed of neighbouring variations. Complex mutational patterns and relative abundances determined by multiplex RFMP assay were in good concordance with results obtained by direct or clonal sequencing analyses. Defined mixtures were successfully and consistently identified at 2% relative concentration of mutant versus wild-type virus by the assay. The multiplex RFMP assay is an accurate and sensitive means to detect entecavir and lamivudine resistance mutations, simultaneously. The method is expected to enable early and efficient diagnosis of multiple drug resistance mutations for optimal management of CHB.

Performance evaluation of the Abbott RealTime HCV Genotype II for hepatitis C virus genotyping

The Abbott RealTime hepatitis C virus (HCV) Genotype II (Abbott Molecular Inc.) for HCV genotyping, which uses real-time PCR technology, has recently been developed. Accuracy and sensitivity of detection were assessed using the HCV RNA PHW202 performance panel (SeraCare Life Sciences). Consistency with restriction fragment mass polymorphism (RFMP) data, cross-reactivity with other viruses, and the ability to detect minor strains in mixtures of genotypes 1 and 2 were evaluated using clinical samples. All performance panel viruses were correctly genotyped at levels of >500 IU/mL. Results were 100% concordant with RFMP genotypic data (66/66). However, 5% (3/66) of the samples examined displayed probable genotypic cross reactivity. No cross reactivity with other viruses was evident. Minor strains in the mixtures were not effectively distinguished, even at quantities higher than the detection limit. The Abbott RealTime HCV Genotype II assay was very accurate and yielded results consistent with RFMP data. Although the assay has the advantages of automation and short turnaround time, we suggest that further improvements are necessary before it is used routinely in clinical practice. Efforts are needed to decrease cross reactivity among genotypes and to improve the ability to detect minor genotypes in mixed infections.