Genetic variability of gag and env regions of HIV type 1 strains circulating in Slovenia

The aim of this study was to investigate the genetic diversity of HIV-1 strains circulating in Slovenia. Proviral DNA isolated from peripheral blood mononuclear cells (PBMCs) of 20 randomly selected HIV-1-infected individuals was classified into subtypes by sequence-based phylogenetic analysis of the env (C2V3) and gag (p24) regions of the viral genome. The phylogenetic tree based on env C2V3 sequences showed that 15 of the 20 samples were subtype B, two A1, one F1, one CRF01_AE, and one CRF02_AG. The phylogenetic analysis of the gag gene yielded identical results expect for one sample that had a discordant subtype; it was identified as subtype A1 in the env and AE in the gag region. Our study confirmed that although subtype B predominates, other subtypes and circulating recombinant forms (CRFs) are also present in Slovenia. The high intrasubtype genetic diversity of subtype B sequences suggests a multiple introduction of subtype B strains into Slovenia.     

Development of a one-tube multiplex reverse transcriptase-polymerase chain reaction assay for the simultaneous amplification of HIV type 1 group M gag and env heteroduplex mobility assay fragments

The emergence of intersubtype recombinant HIV-1 isolates has made it imperative to analyze different regions of HIV-1 genomes. For this purpose a one-tube multiplex RT-PCR, coamplifying first-round amplicons that allow amplification of gag and env heteroduplex mobility assay (HMA) fragments from different HIV-1 group M isolates, was developed, starting with plasma samples. The multiplex RT-PCR assay is sensitive: 115 of 136 (84.5%) samples were positive for both gag and env, positive amplification of the gag fragment was observed in 130 of 136 (95.6%) samples, while for the env fragment 119 of 136 (87.5%) tested positive. The multiplex RT-PCR in combination with gag and env HMA makes large-scale HIV-1 subtyping fast, simple, and more economical.     

应用多重PCR法鉴定蚊胃血血源的研揪

目的建立多重PCR法鉴定蚊胃血来源。方法依据常见蚊吸血对象(人、牛、猪和犬)的线粒体DNA细胞色素b序列的差异,设计种特异引物,建立多重PCR法,并应用该法检测现场按蚊标本。结果应用多重PCR法共检测249只按蚊,血源来自牛和猪的共91只和63只,未检出吸人血按蚊。立即处死并干燥保存的现场按蚊标本检测成功率最高,为92.50%。结论多重PCR法鉴定蚊胃血血源快速、灵敏,结果客观、可靠。     

Genetic variability affects the detection of HIV by polymerase chain reaction.

Nine isolates of HIV-1 obtained from Congolese AIDS patients were amplified by the polymerase chain reaction (PCR) using primer pairs and oligomer probes derived from the HIV-1 LAV-BRU (BRU) sequence. When compared to BRU, two isolates exhibited a significant decrease of PCR efficiency with a given primer pair. Moreover, the DNA amplified from two other isolates did not hybridize with the corresponding probe despite efficient PCR. Base substitutions were detected in the regions of proviral genomes involved in oligonucleotide annealing and were assumed to be responsible for the failure of both amplification and probing. Our data confirm that the genetic variability of HIV-1 may reduce the efficiency of PCR as a diagnostic procedure, especially in the case of African isolates.     

Detection of genomic mutations in katG, inhA and rpoB genes of Mycobacterium tuberculosis isolates using polymerase chain reaction and multiplex allele-specific polymerase chain reaction

ObjectiveIsoniazid (INH) and rifampin (RIF) are the most effective first line antibiotics against Mycobacterium tuberculosis. Mutations in several genes determine resistance of M. tuberculosis to INH, with the most common gene target of katG, and resistance to RIF is due to mutation in rpoB gene. The aim of present study was to assess the mutations in the regions related to RIF and INH resistance.MethodsWe characterized 80 clinical isolates of confirmed M. tuberculosis to analyze the most commonly observed INH and RIF mutations. PCR analysis and sequencing were used to detect mutations related to RIF and INH resistance. The multiplex allele-specific-PCR (MAS-PCR) was performed as a comparative assay and for evaluation of this method.ResultsThe sequencing of the 250-bp region of katG codon 315, revealed point mutations at 5 different codons in 13.7% of the M. tuberculosis isolates. The sequencing of the 270-bp central region of the rpoB gene revealed point mutations at 7 different codons in 12 (15%) of the M. tuberculosis isolates. The results obtained with MAS-PCR are in accordance with PCR-sequencing with high sensitivity and specificity for katG315, inhA15, and rpoB (531, 516, 526).ConclusionThe results of this study suggested that molecular techniques can be used as a rapid tool for the identification of drug resistance in clinical isolates of M. tuberculosis. Both DNA sequencing and MAS-PCR yielded high sensitivity for the detection of RIF and INH mutations and detecting multi-drug resistant tuberculosis cases.     

Detection of Campylobacter in duck using standard culture method and multiplex polymerase chain reaction

In 2005, total of 140 samples of duck meat and intestine from slaughterhouses in Nakhon Pathom Province, Thailand, were analyzed for Campylobacter spp. Twenty-eight samples (20%) were positive for Campylobacter spp using the standard culture method (SCM) with 21 samples of C. jejuni and the other 7 C. coli. Forty-four samples (31%) were positive using multiplex polymerase chain reaction, with 34 samples of C. jejuni and 10 of C. coli. This is the first report of Campylobacter contamination in duck in Thailand.     

Absence of HIV-1 DNA in high-risk seronegative individuals using high-input polymerase chain reaction.

Evidence of frequent HIV-1 infections in antibody-negative, high-risk individuals (so-called 'silent' infections) remains controversial. To evaluate whether these discrepant results may be the consequence of intermittent detection of rare infected cells (low viral load) preceding seroconversion, we developed a modification of the polymerase chain reaction (PCR) technique which enabled analysis of 10-fold greater amounts of cellular DNA per reaction than standard PCR (2 x 10(6) rather than 0.2 x 10(6) input cells). This technique allowed consistent detection of HIV-1 provirus in two seropositive individuals who had repeatedly tested negative by standard-input PCR. However, results were negative when high-input PCR was applied to 51 specimens from 39 selected high-risk seronegative individuals. These results suggest that variations in viral load preceding or in the absence of seroconversion probably do not explain discrepant evidence regarding silent HIV-1 infection.     

Multicentre quality control of polymerase chain reaction for detection of HIV DNA.

OBJECTIVE: Seven French laboratories tested the specificity and sensitivity of the polymerase chain reaction (PCR) for the detection of HIV-1 DNA. METHODS: Following its own PCR protocols, each laboratory independently tested blind two panels of 20 coded peripheral blood mononuclear cell samples collected from HIV-1-seropositive individuals and from HIV-1-seronegative individuals at high or low risk of HIV infection. For the first panel, laboratories were free to select type and number of primers; for the second, all were required to use the two primer pairs Pol 3/4 and MMy 9/10' (Nef 1). RESULTS: False-positive and false-negative results were observed in all laboratories (concordance with serology ranged from 40 to 100%). In addition, the number of positive PCR results did not differ significantly between high- and low-risk seronegatives. The use of crude cell lysates in DNA preparation produced the same PCR results as phenol-extracted DNA. Discrepancies between laboratories indicated that factors other than primer pairs contributed strongly to laboratory variability. CONCLUSIONS: Our results emphasize the importance of both positive and negative controls in PCR and demonstrate the value of multicentre PCR quality control.     

Mixed human immunodeficiency virus (HIV) infection in an individual: demonstration of both HIV type 1 and type 2 proviral sequences by using polymerase chain reaction

Sera from persons seroreactive to both human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2), by whole-virus (VEIA) enzyme immunoassays (EIAs) for each virus, were selected from a seroprevalence study of 944 persons in Abidjan, Cote d'Ivoire, West Africa, in 1987. These sera were subsequently tested for HIV-1 and HIV-2 antibody specificity by type-specific peptide EIAs (PEIA) and western blot (WB) analysis for both viruses. Peripheral blood monocytes (PBMCs) from representative individuals were cultured in the presence of phytohemagglutinin-stimulated normal donor PBMCs. These cultures were periodically monitored for HIV-1 and HIV-2 proviral sequences by using the selective DNA amplification technique polymerase chain reaction (PCR). As an outgrowth of this study, we report the case of a person dually reactive by various serological techniques in whom proviral sequences from HIV-1 and HIV-2 were detected by PCR. This is the first confirmed case of a mixed HIV-1 and HIV-2 infection in a single individual.     

The polymerase chain reaction in the diagnosis of vertically transmitted HIV infection

The presence of HIV-1 DNA sequences in DNA from peripheral blood mononuclear cells (PBMCs) was investigated in a two-stage polymerase chain reaction ('double' PCR) using four sets of nested primers. The PBMCs tested were obtained from 46 children born to HIV-seropositive mothers, seven 'control' children born to HIV-seronegative mothers and seropositive fathers, and 45 healthy adult blood donors who were HIV seronegative. Nine of the children had symptomatic HIV infection and other laboratory features characteristic of HIV infection: all nine were PCR-positive with each set of primers in each of their 22 blood samples tested. The remaining 44 children had no clinical or laboratory evidence of HIV infection, and each of their 50 samples was PCR-negative with each set of primers, as were all blood donor samples. PCR-positive samples were tested in more detail using two of the sets of primers, which spanned hypervariable regions in the env gene. Polyacrylamide gel electrophoresis of DNA amplified from these regions yielded patterns of amplified DNA length variation which were characteristic for each child, and which changed little with time (in serial samples obtained over periods of 3-7 months). This excluded contamination as a cause of PCR positivity. This is the first report of the use of a double PCR for the diagnosis of HIV infection. The results demonstrate the specificity of this PCR method in diagnosis, with failure to reveal in this cohort any cases of vertically transmitted HIV-1 infection in addition to those already confirmed by conventional laboratory techniques.     

Phylogenetic analysis of env, gag, and tat genes of HIV type 1 detected among the injecting drug users in West Bengal, India

A recent occurrence of HIV-1 seropositivity among a group of injecting drug users (IDUs) in Darjeeling, a hilly district in northern West Bengal, revealed overall 11.8% HIV seroprevalence. Our study based on env (C2-V3), gag (p24-p7), and tat (exon-1) genomic regions of HIV-1 detected among this population showed that Darjeeling IDU sequences belonged to subtype C. Interestingly, the IDU sequences from Darjeeling were again found to be closer to the C strains from Manipur, a northeastern state in India, which is linked to the Golden Triangle via the Manipur-Myanmar border, rather than the IDU C sequences from Nepal, a neighboring country of India. The outgroup reference strains from different sites of IDU-driven epidemics in the world like Russia, Vietnam, Thailand, and Spain belonged to the nonsubtype C group and formed separate clusters from the subtype C cluster in our analysis. These results indicate a rapid spread of HIV-1 by possible drug trafficking along international boundaries, which might also help in the invasion of HIV-1 among IDUs of Darjeeling through the Manipur-Myanmar border of India.     

Occult hepatitis B in Mexican patients with HIV, an analysis using nested polymerase chain reaction

Hepatitis B virus infection (HBV) with undetectable levels of HBsAg, has been named occult HBV infection and observed in immunosuppressed patients. The aim of this study was to determine the frequency of occult HBV infection in patients with HIV from the West of México, using a combination of serological markers and nPCR. Thirty eight HIV/AIDS patients, 32 men (84.2%) and 6 (5.8%) women, without liver damage related symptoms were studied. HBV coinfection was observed in 10 (26.3%) patients; while only 3 (7.9%) of them were positive to HBsAg. Thus, 7 (18.4%) occult HBV infected patients could be assessed in this population. One (10%) patient with occult HBV infection was positive to anti-HBs, in spite of the reinfection protection attributed to this serological marker. Anti-HBc was detected only in 2 (20%) patients with occult HBV infection. No significant association could be established between occult HBV infection and CD+4 cell count, biochemical, clinical parameters, AIDS stage, or any other risk factor. This study suggest that determination of HBV DNA utilizing highly sensitive techniques, as nPCR, should be performed to detect occult HBV infection, even in the absence of anti-HBc in HIV/ AIDS patients, in order to have a reliable diagnosis, prevent HBV dissemination and acute exacerbation of chronic hepatitis B or even fulminant hepatitis. To our knowledge this is the first study of occult HBV infection in Mexican patients with HIV. However, further studies are necessary in order to determine HBV genotypes and its relationship with evolution and clinical manifestation of the disease.     

应用多重聚合酶链反应技术快速鉴定牛分支杆菌卡介苗株

目的 建立快速鉴定牛分支杆菌卡介苗(BCG)菌株的方法。方法 依据最近研究发现的牛分支杆菌BCG菌株不存在命名为缺失区1(deleted region 1，RD1)的基因区，而其他牛分支杆菌菌株和其他结核分支杆菌复合群(MTC)菌种(结核分支杆菌、非洲分支杆菌和田鼠分支杆菌)存在RD1基因区的遗传学信息，应用多重聚合酶链反应(PCR)检测RD1基因区存在与否，以鉴别BCG菌株与其他牛分支杆菌菌株及其他MTC菌种。结果 所试5株BCG疫苗生产用标准菌株和近期国内发生的1例小儿BCG接种后全身播散性感染致死病例的2株BCG分离菌株，均缺失RD1基因区；其他牛分支杆菌菌株，包括3株牛分支杆菌标准菌株、5株分别从结核病牛或鹿分离的牛分支杆菌菌株，1株从结核病患痰标本分离的牛分支杆菌菌株，以及其他MTC菌种，包括结核分支杆菌H37Rv和H37Ra标准菌株、48株从结核病患痰标本分离的结核分支杆菌菌株、3株非洲分支杆菌标准菌株，均存在RD1区。结论子多重PCR技术用于牛分支杆菌BCG菌株的鉴定简便、快速、特异，适于在临床实验室应用。     

Identification of bacterial DNA in neutrocytic and non‐neutrocytic cirrhotic ascites by means of a multiplex polymerase chain reaction

Background: Even though bacterial cultures of ascitic fluid are negative in up to 65% of the cases of spontaneous bacterial peritonitis (SBP); bacterial DNA (bactDNA) has been frequently detected in episodes of SBP as well as in culture‐negative non‐neutrocytic ascites. Aims: To evaluate multiplex polymerase chain reaction (PCR) for pathogen identification in SBP and to determine the prevalence of ascitic bactDNA and its prognostic relevance in hospitalized patients with liver cirrhosis. Methods: Ascitic fluid from 68 consecutive patients who underwent diagnostic paracentesis was analysed for polymorphonuclear leucocyte (PMN) count, bacterial culture and bactDNA. BactDNA was identified by gel analysis after multiplex PCR of selectively enriched prokaryotic nucleic acids. Correlations of bactDNA status with PMN count, bacterial culture result and 3‐month mortality were determined for neutrocytic and for non‐neutrocytic ascites. Results: 11/68 patients presented with an elevated ascitic PMN count. BactDNA was detected in 5/5 culture‐positive neutrocytic samples, in 1/6 culture‐negative neutrocytic samples and in 8/56 culture‐negative non‐neutrocytic samples. Three‐month mortality did not differ with respect to ascitic bactDNA status (7/14 vs. 14/47, P=0.162). 3‐month mortality was increased in the presence of ascitic bactDNA for patients older than 65 years (4/5 vs. 4/14, P=0.046) and for patients with a model for end‐stage liver disease score >15 (7/10 vs. 9/30, P=0.025). Conclusions: Identification of ascitic bactDNA is an appropriate alternative to bacterial ascite culture for pathogen identification in patients at risk for SBP. Its prognostic relevance as a proposed marker of bacterial translocation for certain risk groups has to be further evaluated.     

Adenosine deaminase estimation and multiplex polymerase chain reaction in diagnosis of extra-pulmonary tuberculosis

SETTING: Extra-pulmonary tuberculosis (EPTB), including mycobacteriosis, contributes 15-20% of all tuberculosis (TB) cases. The diagnosis of EPTB remains elusive because of the inadequate sensitivity of routine and conventional bacteriological methods for the detection of Mycobacterium tuberculosis and related organisms in clinical specimens such as cerebrospinal fluid (CSF), pleural fluid and peritoneal fluid. OBJECTIVE: To develop a better diagnostic marker for EPTB. DESIGN: In our study, 179 cases of EPTB were analysed for acid-fast bacilli (AFB) smear, adenosine deaminase activity (ADA) and multiplex polymerase chain reaction (PCR). Although estimation of ADA is helpful, its sensitivity and specificity varies widely. On the other hand, a multiplex PCR using amplicons such as IS6110, dnaJ gene and hsp65 genes has high sensitivity (60-88%) and specificity (81-100%). RESULTS: On comparing AFB and ADA results with PCR, the PCR is clearly more effective than AFB (P < 0.001) and ADA estimation (P < 0.02) in CSF. The same result was observed with peritoneal fluid (P < 0.001 vs. P < 0.05) and pleural fluid (P < 0.001 and P < 0.05). CONCLUSION: The study shows that multiplex PCR remains the best tool and is a much better marker for diagnosing EPTB.     

Genetic analysis of the complete gag and env genes of HIV type 1 subtype C primary isolates from South Africa.

South Africa has one of the fastest growing HIV-1 epidemics, with an estimated 4.7 million people infected. To better understand the genetic diversity of this epidemic and its potential impact on vaccine development, we have cloned and sequenced the complete gag and env genes of 13 primary virus isolates. Phylogenetic analysis of our sequences and 69 complete env genes from the Los Alamos and GenBank databases revealed multiple subclusters within subtype C. The V3 loop region was relatively conserved in all our strains when compared with other subtypes, but the region immediately downstream was highly variable. No intersubtype recombinant forms were observed when comparing the gag and env sequences. Characterization of the complete gag and env genes enabled us to select specific strains for further vaccine development.