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.     

A novel molecular method for HIV-1 proviral DNA detection: non-radioactively--reversed probe hybridization and nested PCR

A novel molecular method for HIV-1 proviral DNA detection comprising two main techniques: nested PCR, amplifying a target sequence of the ENV-gene of HIV-1, and nonradioactively-reversed probe hybridization for the detection of the amplified target sequence. The dual amplification of inserted HIV-1 proviral DNA in each DNA sample to be tested was performed by nested PCR in two steps: firstly with two outer primers covering the target sequence of the ENV-gene of HIV-1; secondly with two 5'-biotinylated primers specific to the target sequence. The biotinylated PCR product could be visualized as a single band of 141bps in length on agarose gel stained with ethidium bromide. For the confirmation of the primary result, a method of reversed probe hybridization, using a nylon membrane immobilized with the oligonucleotide probe specific to the target sequence, was established. The oligonucleotide probe was given a homopolymer tail with terminal deoxyribonucleotidyl-transferase; the tail was spotted onto a nylon membrane and bound covalently by UV irradiation. Owing to its length, the tail bound to the nylon, leaving the oligonucleotide probe free to hybridize. Hybridization of the amplified target sequence to the immobilized probe was accomplished by a simple colorimetric reaction involving the enzymatic oxidation of a colorless chromogen that yielded a purple color wherever hybridization occurred.     

HIV-1 viral DNA load in peripheral blood mononuclear cells from seroconverters and long-term infected individuals.

OBJECTIVE: To determine viral DNA load in peripheral blood mononuclear cells (PBMC) from HIV-1-infected individuals. DESIGN: HIV-1 copy numbers were determined using a quantitative polymerase chain reaction (PCR), the PCR-aided template titration assay (PATTY). PATTY utilizes an internal plasmid control DNA, which is amplified within the same tube and using the same primers as the PBMC target DNA. HIV-1 copy numbers were confirmed by limiting-dilution PCR analysis. RESULTS: PBMC viral load of 19 long-term (greater than 4 years) HIV-1-infected individuals ranged from 0.8 to 100 copies per 10(3) PBMC. Significantly higher copy numbers were found among p24-antigen-positive than among p24-antigen-negative individuals. In addition, the PBMC viral load of two HIV-1-infected individuals was monitored during the first 3 months after acute infection. For both patients, the HIV-1 copy numbers were shown to peak at the time of HIV-1-antibody seroconversion and decline subsequently (range, 0.6-10 copies per 10(3) PBMC). CONCLUSIONS: PATTY is a useful method for assessing the HIV-1 copy numbers in PBMC DNA. Viral DNA load peaks shortly after infection and reaches an individual specific level that is probably stable within a few months of infection. Viral DNA load in PBMC varies widely among long-term HIV-1-infected individuals.     

No evidence for recombination between HIV type 1 and HIV type 2 within the envelope region in dually seropositive individuals from Senegal

To investigate the frequency of recombination between HIV-1 and HIV-2 in vivo during dual infection, we performed a retrospective analysis of blood samples from 46 dual HIV-1/HIV-2-seropositive adults for evidence of recombination. HIV viral DNA from peripheral blood mononuclear cells (PBMC) was subjected to two separate nested polymerase chain reaction (PCR) assays using opposing HIV-1 and HIV-2 primer pairs selected to flank a approximately 650-base pair region including the V3 loop of the envelope gene. In the first assay, primers were chosen to amplify recombinants with HIV-1 on the 5' end and HIV-2 on the 3' end, and in the second assay, primers were chosen to amplify recombinants with the opposite orientation. All PCR experiments were run in parallel with positive controls consisting of partial-length env fragments bearing a single central HIV-1/2 recombination site, and appropriate primer-binding sites on each end. The limit of detection for both assays was <10 copies of recombinant product per 150,000 cell equivalents of input PBMC DNA. In all 46 dually seropositive patients in this study, PCR screening of PBMC failed to detect evidence of HIV-1/HIV-2 recombinants in the C2-V5 env region. Although genetic recombination between HIV-1 and HIV-2 may occur, we conclude that any such events within env are exceedingly rare, and do not result in the outgrowth of recombinant strains.     

Identification of mixed HIV-1/HIV-2 infections in Brazil by polymerase chain reaction.

Analysis of sera from hospitalized Brazilian patients by whole-virus lysate-based enzyme immunoassay and Western blot indicated that 0.4% were reactive to HIV-2 alone while 4% were reactive to both HIV-1 and HIV-2. When these sera were tested for HIV antibody by type-specific peptide enzyme immunoassays, dual seropositivity was confirmed in only 0.4% of patients. To define genetically the HIV strains within the population, we analyzed peripheral blood mononuclear cells from selected seropositive patients for the presence of HIV-1 and HIV-2 proviral DNA using the polymerase chain reaction (PCR). Independent primers/probes sets were used for the amplification and detection of viral sequences from the long terminal repeat (LTR), gag, and protease (prt) gene regions. Our findings confirmed the serologic evidence of HIV-2 in Brazil and determined the extent of mixed HIV-1 and HIV-2 infections. Detailed evaluation of the amplified viral protease sequences by endonuclease restriction analysis and DNA sequencing independently confirmed mixed HIV-1 and HIV-2 infections in the two patients seropositive for HIV-1 and HIV-2. The data further indicated that these isolates are distinct from the HIV laboratory standards. We interpret the combination of culture and PCR findings to demonstrate the presence of both HIV-1 and HIV-2 in Brazil.     

Detection of Francisella tularensis in infected mammals and vectors using a probe-based polymerase chain reaction

We investigated the use of a TaqMan 5' nuclease assay (5NA) directed against the Francisella tularensis outer membrane protein (Fop) gene and a polymerase chain reaction-enzyme immunoassay (PCR-EIA) directed against the tul 4 gene for detection of this organism in experimentally infected mice and in field-collected tick vectors. We also evaluated the use of specially formulated filter paper (FTA) for rapid sample preparation. The 5NA had a detection limit of 1 pg of genomic DNA (<100 colony-forming units) and could be completed within several hours. The PCR-EIA could detect 1 pg of genomic DNA and 10 attograms (ag) (22 copies) of cloned insert, but takes longer to perform. Both assays were genus-specific, and successfully detected F. tularensis in mouse tissues (5NA) and in tick extracts (PCR-EIA). The FTA paper provided inexpensive, rapid, template preparation for the tick extracts, mouse tissues, and DNA obtained from clinical specimens. These probe-based assays have the potential to provide rapid, real-time/high-throughput molecular diagnostics in field situations.     

套式PCR对HIV—l env基因检测的敏感度分析

采用套式PCR对含HIV—1膜蛋白基因的B亚型质粒和HIV—1感染者外周血单核细胞(PBMC)进行env基因部分片断的扩增,套式PCR可使常规PCR的敏感性提高100至1000倍,常规PCR只扩增出12份PBMC中的2份样品,套式PCR则扩增出全部样品中HIV—1膜蛋白基因片断.     

乙型肝炎病毒共价闭合环状DNA套式-实时荧光定量聚合酶链反应的建立

目的 建立检测HBV共价闭合环状DNA(cccDNA)的套式一实时荧光定量PCR法.方法 根据HBV cccDNA与松环DNA(rcDNA)结构上的差异,设计2对跨缺口的特异引物及1条位于负链缺口下游的特异TaqMan荧光探针.根据Plasmid-SafeTM ATP-Dependent Dnasc(PSAD)对rcDNA与cccDNA作用的不同,对模板DNA进行酶切纯化,降解reDNA,再进行套式PCR扩增,先用外引物和模板进行第一轮常规PCR,再用内引物、荧光探针和第一轮PCR产物进行实时荧光定量PCR,根据阳性参照标准品,得出待检标本定量值.结果 检测阳性参照标准品.得出该方法灵敏度可达2 lg拷贝/mL.用上述方法检测34份乙型肝炎患者血清HBV DNA阳性标本,25份血清HBVcccDNA阳性,28份外周血单个核细胞HBV cccDNA阳性.27份健康对照者血清HBV DNA阴性标本,6份HBV cccDNA阳性.对5份HBV cccDNA阳性标本扩增产物进行克隆测序,无碱基缺失、突变.与HBV不同基因型序列(A～G)比较,同源性为90.6%～99.1%,其中,与B、C基因型同源性为95.3%～99.1%,验证了方法的特异度.结论 套式-实时定量PCR法可检测乙型肝炎患者血清、PBMC中的HBV CCCDNA,且具有敏感、特异性.     

Quantification of integrated and total HIV-1 DNA after long-term highly active antiretroviral therapy in HIV-1-infected patients.

OBJECTIVE: To assess the impact of long-term virus suppression on the peripheral blood CD4 T cells integrated and total HIV-1 DNA loads in patients receiving highly active antiretroviral therapy (HAART). METHODS: A total of 10 HIV-1-infected patients receiving a triple combination therapy (two nucleoside analogues and one protease inhibitor) were longitudinally studied to compare integrated and total HIV-1 DNA loads. HIV-1 DNA quantification was performed using a quantitative nested polymerase chain reaction (PCR) on genomic peripheral blood mononuclear cell (PBMC) DNA obtained at baseline and at 48 weeks of HAART. RESULTS: All the study patients showed an early and sustained decrease in plasma HIV-1 RNA to below the limit of detection (200 copies/ml). Concordant with the plasma viral decline, a significant increase in the CD4 T cell count was observed (P = 0.007). A statistically significant fivefold decrease in total HIV-1 DNA was detected after 48 weeks of HAART (P = 0.005). However, no statistically significant change was noted after the therapy when the integrated HIV-1 DNA copy number was compared (P = 0.333). Taken together, these results suggest that in the patients analysed the integrated HIV-1 DNA does not decay rapidly after HAART. CONCLUSION: Within the study cohort the total amount of PBMC HIV-1 DNA decreased drastically after 48 weeks of HAART. Nevertheless, the integrated HIV-1 DNA did not significantly decay during this period. Although the data presented here are limited by the number of patients analysed, our findings suggest that 48 weeks of HAART does not significantly reduce the integrated HIV-1 proviral DNA load in the latently infected CD4 T cell reservoir.     

Development of a quantitative real-time polymerase chain reaction assay specific for Orientia tsutsugamushi

Two specific and sensitive polymerase chain reaction (PCR) assays were developed to detect and quantitate Orientia tsutsugamushi, the agent of scrub typhus, using a portion of the 47-kD outer membrane protein antigen/ high temperature requirement A gene as the target. A selected 47-kD protein gene primer pair amplified a 118-basepair fragment from all 26 strains of O. tsutsugamushi evaluated, but it did not produce amplicons when 17 Rickettsia and 18 less-related bacterial nucleic acid extracts were tested. Similar agent specificity for the real-time PCR assay, which used the same primers and a 31-basepair fluorescent probe, was demonstrated. This sensitive and quantitative assay determination of the content of O. tsutsugamushi nucleic acid used a plasmid containing the entire 47-kD gene from the Kato strain as a standard. Enumeration of the copies of O. tsutsugamushi DNA extracted from infected tissues from mice and monkeys following experimental infection with Orientia showed 27-5552 copies/microL of mouse blood, 14448-86012 copies/microL of mouse liver/spleen homogenate, and 3-21 copies/microL of monkey blood.     

Proviral HIV-1 dynamics and evolution in patients receiving efficient long-term antiretroviral combination therapy

Different experimental approaches have shown that, despite plasma viral loads under the threshold of detection, HIV-1 frequently continues to replicate in patients receiving potent antiretroviral therapy. However, whether this low-grade viral replication is sufficient for the generation of new major quasispecies has not been studied. Thus, in order to evaluate the extent of variation in the major proviral HIV-1 population, we monitored proviral DNA sequences in such patients over a time period of up to 30 months.
Methods   DNA was extracted from peripheral blood mononuclear cells (PBMC) and the V3 region was amplified by nested polymerase chain reaction (PCR) and directly sequenced. Additionally, both HIV-1 RNA and DNA levels and CD4⁺ T-lymphocyte counts were monitored.
Results   Analysing the V3 gene sequences of 17 patients, we observed a sequence evolution in nine patients. Interestingly, the majority of these changes (77%) occurred in the first interval following the initiation of therapy and despite signs of ongoing replication the proviral DNA levels continued to decrease in all patients.
Conclusions   Our data suggest that, although available data report that HIV-1 continues to replicate in patients with undetectable viraemia, the extent of viral replication in many of these patients is not sufficient to result in changes in the major viral population.     

Human immunodeficiency virus type 1-infected persons with residual disease and virus reservoirs on suppressive highly active antiretroviral therapy can be stratified into relevant virologic and immunologic subgroups

A significant percentage of human immunodeficiency virus type 1 (HIV-1)-infected persons treated with highly active antiretroviral therapy (HAART) will develop plasma HIV-1-specific virion RNA levels <50 copies/mL. HIV-1-infected persons receiving virally suppressive HAART were studied with a viral outgrowth assay of the patients' peripheral blood mononuclear cells (PBMC), and a quantitative polymerase chain reaction assay was used to analyze HIV-1 2-long terminal repeat (2-LTR) circular DNA in PBMC, which indicates new HIV-1 infections of cells in vivo. Viral outgrowth in vitro correlated inversely with the level of peripheral blood CD4(+) T lymphocytes. Detection and quantitation of 2-LTR circular DNA correlated strongly with viral outgrowth patterns and inversely with CD4(+) T lymphocyte counts. Relevant subgroups of HIV-1-infected subjects on suppressive HAART with residual viral disease and reservoirs can now be stratified.     

Rapid and quantitative detection of enzymatically amplified HIV-1 DNA using chemiluminescent oligonucleotide probes

A hybridization protection assay (HPA) that uses acridinium ester (AE) labeled oligonucleotide probes which are specific for a conserved gag gene region of human immunodeficiency virus type 1 (HIV-1) was developed to measure the amount of HIV-1 nucleic acid. Hybridization of the single-stranded probes with their target HIV-1 sequences protected the chemiluminescent AE group from subsequent alkaline hydrolysis. The chemiluminescence from the residual AE could be easily quantitated in a luminometer. The entire process comprising template dissociation, hybridization, alkaline hydrolysis, and chemiluminescence measurement can be completed in less than one hour and does not require the separation of hybridized probe from unhybridized probe. We demonstrated that HPA could quantitatively measure the amount of DNA amplified by polymerase chain reaction. A comparative study using amplified DNA from the peripheral blood mononuclear cells (PBMC) of HIV seropositive and seronegative persons showed that HPA was as sensitive as the previous methods using 32P-labeled DNA probes.     

Peripheral blood Dendritic cells are not a major reservoir for HIV type 1 in infected individuals on virally suppressive HAART

Dendritic cells (DCs) are potent antigen-presenting cells, and their physiological localization in tissues that interact with the external environment is important as a first barrier against pathogens such as human immunodeficiency virus type I (HIV-1). Several models have been proposed to explain the possible role of DCs as a reservoir for HIV-1 in patients on virally suppressive highly active antiretroviral therapy (HAART). However, the low yield of cell isolates has made this evaluation a difficult task. The present study analyzes whether peripheral blood DCs from HIV-1-infected individuals on virally suppressive HAART, with plasma HIV-1 RNA levels of less than 50 copies/ml, carry either HIV-1 provirus and/or HIV-1 virions. Peripheral blood DCs were isolated from a cohort of 10 HIV-1-seropositive men taking suppressive HAART. In five patients, plasmacytoid and myeloid dendritic cells were isolated to attempt to identify their respective roles in HIV-1 residual disease. Viral out-growth assays were performed in vitro, as well as gag and R/U5 polymerase chain reaction (PCR) amplification of viral RNA and DNA, respectively, from DC and peripheral blood mononuclear cell (PBMC) extracts. Fluorescence activated cell-sorting (FACS) data revealed cellular yields from 85.90 to 95.18%, of relatively pure DCs isolated from patients' PBMCs. Although HIV-1 RNA gag and DNA RU/5 were detected in all PBMC samples isolated from the patients, proviral DNA and viral RNA forms were not detected in any of the DC isolates. In addition, no replication-competent virus was demonstrated in DC coculture assays, while virus was isolated from each patients' CD8+ T-lymphocyte-depleted PBMC cocultures. Furthermore, HIV-1 gag proviral DNA was not detected in either plasmacytoid or myeloid DC subfractions. The current study suggests that in HIV-1-infected individuals treated with suppressive HAART, peripheral blood DCs do not carry HIV-1 proviral DNA or viral particles attached to their surface. These populations of peripheral blood DCs are likely not a major HIV-1 reservoir in patients on HAART with clinically undetectable plasma viral RNA.     

桂北地区HIV-1感染者HIV-1病毒亚型分布观察

目的观察桂北地区HIV感染者HIV-1病毒亚型分布。方法采集桂北地区80例HIV-1感染者的静脉血,提取其中单个核细胞的DNA,用巢氏PCR扩增病毒膜蛋白env基因的C2-V3区,对PCR纯化产物进行测序,并应用GCG软件等对序列进行分析。结果通过PCR扩增得到env基因序列55份,分属4个亚型,分别为B型1份,CRF07-BC型19份,CRF08-BC型23份,CRF01-AE型8份,C型4份。感染途径为静脉吸毒者16例,性34例,非法献血2例,不详3例。16例吸毒感染者中CRF08-BC型12例,CRF07-BC型4例。34例性行为感染者中CRF07-BC型19例,CRF08-BC型11例,CRF01-AE型4例。结论桂北地区HIV感染者存在HIV-1亚型多样。静脉吸毒感染者以CRF08-BC型为主。性行为感染者以CRF07-BC型和CRF08-BC型为主。     

A study to implement early diagnosis of HIV infection in infants born to infected mothers

A protocol for detecting HIV DNA from specimens collected on filter papers and the effect of storage temperatures on determination of HIV DNA from dried blood spots has been developed and optimized. Blood specimens collected from HIV-1 infected and normal persons were spotted onto blood collection cards (Whatman BFC 180). The HIV DNA was extracted by phenol-chloroform-isoamyl alcohol and was detected for C2V4 of HIV-1 env by nested polymerase chain reaction (nested PCR). One set was stored at -20 degrees C for 14 weeks, another at 37 degrees C for 1 week and then kept at -20 degrees C for 13 weeks and a third set at 25 degrees C for I week and then -20 degrees C for 13 weeks. The dried blood spots from each set were detected for the HIV DNA every 2 weeks for 14 weeks. The C2V4 region of HIV env DNA was determined from small amounts of the dried blood collected on the filter papers. The nested PCR procedure could detect as few as 5 copies of HIV proviral DNA, and HIV DNA could be detected from specimens with viral loads of 2x 10(4) copies/ml. HIV DNA could be detected from specimens collected at all temperatures tested for at least 14 weeks. Therefore, laboratory diagnosis of HIV infection can be done by PCR on dried blood spots. These techniques will be useful as a tool for studying the epidemiology of HIV infection among populations of interest such as mother to child infection using newborn screening specimens.     

Human parvovirus Bb19 detection in asymptomatic blood donors: association with increased neopterin concentrations

Serum neopterin concentrations were determined in 20,000 blood donations. For the 400 donations with neopterin concentrations above the 98 th percentile and another 1200 donations with neopterin concentrations in the lower range, results of human parvovirus (HPV) B19 tests were compared. Infectious specimens were identified by dot blot hybridization assay and polymerase chain reaction (PCR) that used the outer primers and detected 1 pg of HPV B19 DNA, corresponding to approximately 10(5) copies of the genome, in the specimens and by a nested PCR that detected 1-10 fg of DNA, corresponding to 10(2)-10(3) copies of the genome. Of 400 specimens with neopterin concentrations > or =12 nmol/L (98th percentile, current cutoff), 10 tested positive by dot blot hybridization assay (9 of these were confirmed by nested PCR). Among 1200 specimens with low neopterin concentrations (<12 nmol/L), no specimen containing HPV B19 DNA was detected. These findings suggest an association between elevated neopterin concentrations and HPV B19 infectivity.