Detection of human immunodeficiency virus type 1 DNA in dried blood spots by a duplex real-time PCR assay

A dried blood spot (DBS) is a well-accepted means for the collection, transport, and storage of blood samples for various epidemiologic, serologic, and molecular assays for human immunodeficiency virus (HIV) studies. It is particularly important for mother-to-infant-transmission studies of affected individuals living in remote areas. We have developed a real-time PCR method to detect HIV type 1 (HIV-1) DNA in dried blood spots. A cellular gene, RNase P, was coamplified with the HIV-1 DNA in the same tube to monitor the DNA extraction efficiency and the overall assay performance. Our assay is a one-tube, single-step closed-system assay and uses a dUTP/uracil DNA glycosidase anti-PCR contamination control. The HIV-1 primers and probe were derived from a conserved region of the long terminal repeat. The detection of RNase P is attenuated by lowering the forward and reverse primer concentrations so that its amplification will not overwhelm the HIV-1 amplification and yet will provide a semiquantitative measurement of the quality of the isolated DBS DNA. We examined 103 HIV-1-seropositive and 56 seronegative U.S. adults and found that our assay has a sensitivity of 98.1% (95% confidence interval [CI], 95.5% to 100%) and specificity of 100% (95% CI, 99% to 100%). The positive and negative predictive values are 100% and 96.6%, respectively. This duplex PCR assay may be useful in identifying HIV-1-infected persons, particularly infants born to seropositive mothers in remote areas of the world.     

Quantitation of human immunodeficiency virus type 1 DNA by competitive nested PCR assay

A quantitative competitive nested PCR assay was developed for quantifying HIV-1 proviral DNA in clinical samples. A competitor DNA was constructed from a conserved region of the HIV-1 gag gene by deleting a sequence of 18 base pairs. We quantitated HIV-1 proviral DNA copy number in clinical samples. Peripheral blood mononuclear cells (PBMCs) from 35 HIV-infected patients with a CD4 count range of 4-728 cell/mm3 were analyzed by this method. The copy numbers of HIV-1 DNA detected ranged between 518 to 67,340 copies per 10(6) CD4+ T-cells. The copy numbers correlated inversely with the CD4 counts.     

干血斑滤纸片中人免疫缺陷病毒1型前病毒基因的检测

目的建立套式引物聚合酶链反应（ＰＣＲ），用于检测滤纸干血斑中的人免疫缺陷病毒１型（ＨＩＶ１）前病毒ｐｏｌ基因ＤＮＡ片段。方法采集ＨＩＶ１感染者的全血约５０μｌ滴在经ＥＤＴＡ－蛋白酶Ｋ预处理并干燥的滤纸片上，室温下干燥，将滤纸片密封于塑料袋中，在室温及４℃下保存１～６４周后，分别将滤纸片置０５ｍｌ试管中直接进行ＨＩＶ１前病毒ｐｏｌ基因的外侧引物ＰＣＲ检测，然后进行内侧引物的ＰＣＲ检测。结果经ＥＤＴＡ－蛋白酶Ｋ预处理的滤纸片在４℃下保存４０周、在室温下保存２４周仍可检出ＨＩＶ１前病毒目的基因。根据ＰＣＲ产物的琼脂糖凝胶电泳溴化乙锭染色带形并参比实验设立的标准对照可直接判断结果。结论该方法具有快速、特异、敏感的特点，敏感性可以达到检出１０个靶ＤＮＡ分子。样品采集后可通过邮件传递至中心实验室，特别适合于ＨＩＶ１感染的确证及筛检     

Simple DNA extraction method for dried blood spots and comparison of two PCR assays for diagnosis of vertical human immunodeficiency virus type 1 transmission in Rwanda

Dried blood spots (DBS) on filter paper facilitate the collection, transport, and storage of blood samples for laboratory use. A rapid and simple DNA extraction procedure from DBS was developed and evaluated for the diagnosis of human immunodeficiency virus type 1 (HIV-1) infection in children by an in-house nested-PCR assay on three genome regions and by the Amplicor HIV-1 DNA prototype assay version 1.5 (Roche Molecular Systems). A total of 150 samples from children born to HIV-1-infected mothers were collected in Kigali, Rwanda, in parallel as DBS and as peripheral blood mononuclear cell (PBMC) pellets. The results obtained on DBS by the two PCR assays were compared to the results of nested PCR on PBMCs. Of 150 PBMC samples, 10 were positive, 117 were negative, and 23 were indeterminate for HIV-1 infection. In DNA extracted from filter papers and amplified by using the in-house nested PCR, 9 of these 10 positive samples (90%) were found to be positive, and 1 was found to be indeterminate (only the pol region could be amplified). All of the negative samples and all of the 23 indeterminate samples tested negative for HIV-1 infection. When we used the Amplicor DNA test on DBS, all of the 10 PBMC-positive samples were found to be positive and all of the 23 indeterminate samples were found to be negative. Of the PBMC-negative samples, 115 were found to be negative and 2 were found to be indeterminate. We conclude that this simple rapid DNA extraction method on DBS in combination with both detection methods gave a reliable molecular diagnosis of HIV-1 infection in children born to HIV-infected mothers.     

Standardized nested polymerase chain reaction-based assay for detection of human immunodeficiency virus type 1 DNA in whole blood lysates.

The routine detection of human immunodeficiency virus type 1 (HIV-1) proviral DNA in clinical samples requires a standardized, simple, and sensitive test. To identify the HIV-1 proviral DNA in blood, we used a solid-phase assay based on the affinity capture and the gamma counting of the amplified product after a nested polymerase chain reaction (AMPLICIS test). In order to simplify the general process, whole-blood lysates rather than peripheral blood mononuclear cell lysates were used for the amplifications. The solid-phase capture and counting of the final amplified products allowed us to define precise interpretive criteria to determine the positivity level of the test. Three new primer sets located in the gag and pol structural genes and in the tat regulatory gene of HIV-1 were studied. The results obtained in 54 seropositive and 120 seronegative individuals demonstrated the ability of the AMPLICIS test to be used for HIV-1 provirus detection: 53 of 54 of the seropositive specimens were found to be positive with at least two primer sets. We also assessed the usefulness of this test for the estimation of the HIV-1 DNA load by the end point dilution method with serial dilutions of blood lysates from 26 HIV-1-seropositive patients.     

使用多重巢式PCR对我国HIV-1主要流行株亚型鉴定方法的建立

目的 建立一套新的亚型鉴定方法，仅仅使用巢式PCR，一次扩增，即可对我国HIV-1主要流行株B、C和CRF01-AE进行亚型鉴定。方法 从HIV阳性样本中提取核酸，使用能覆盖HIV-1型M组gag区的引物进行第一轮扩增，第二轮扩增则使用分别检测B、C、CRF01-AE亚型的三套特异性引物进行扩增，三套引物放在同一个反应管中。反应产物经琼脂糖电泳后观察，不同亚型的位置不同，以此来判断亚型。另外设计一套引物，专门检测我国重组株CRF07-BC和CRF08-BC。所有样品均经过基因测序、系统进化树分析，以进行结果验证。结果 在检测的119份样品中，经基因测序和系统进化树分析证实B亚型样品43份(欧美B11份，泰国B32份)，C、CRF01-AE、A和D亚型样品分别为54份、17份、3份和2份。其中C亚型的样品，有52份属于CRF07-BC和CRF08-BC。而经过上述多重巢式PCR方法检测到的B亚型样品为35份(81．4％)，C亚型46份(85．2％)和CRF01-AE13份(76．5％)。另外，检测CRF07-BC和CRF08-BC重组株的引物特异性地检测到43份(82．7％)样品。上述结果与基因分析结果吻合，各个亚型之间无交叉，一种亚型的特异性引物只对该亚型有反应，而对其他亚型无反应，特异性达到100％。虽然有时会有非特异扩增带，但一般不影响结果判断。结论 我们建立了一套简单快速的H1V-1亚型鉴定方法，不需基因测序，即可检测我国主要流行株B、C、CRF01-AE、CRF07-BC和CRF08-BC。该方法具有高度特异性和敏感性，可以作为初筛方法在我国及其他国家HIV-1实验室推广使用。     

Ultrasensitive human immunodeficiency virus type 1 p24 antigen assay modified for use on dried whole-blood spots as a reliable, affordable test for infant diagnosis

The ultrasensitive human immunodeficiency virus (HIV) p24 antigen assay was modified for use on pediatric dried whole-blood spots on Whatman no. 1 filter paper. The modified assay was found to be reliable and accurate, making it an affordable tool for pediatric HIV diagnosis in developing countries.     

Defining the human immunodeficiency virus type 1 transmission genetic bottleneck in a region with multiple circulating subtypes and recombinant forms

The Mbeya region of Tanzania has a genetically complex HIV epidemic with multiple subtypes and recombinant forms circulating, together with a high frequency of dual infections with more than one subtype. This study aimed to determine whether this impacted the HIV-1 transmission bottleneck. A total of 210 env sequences from 22 participants were generated from recently infected women from Mbeya using the single genome amplification approach. Participants were infected with subtypes C (n = 9), A (n = 4), or D (n = 1), and recombinants AC (n = 4), CD (n = 2), AD (n = 1), or ACD (n = 1). Sixteen participants (73%) were infected with a single variant; five (23%) with multiple variants; and one (4%) was dually infected. Thus the frequency of single variant infections was similar to cohorts located in genetically restricted subtype B or C epidemics, suggesting that multiple circulating subtypes and unique recombinant forms do not have a significant impact on the transmission bottleneck.     

Rapid detection of human immunodeficiency virus type 1 subtype e infection by PCR

The CRF01_AE (subtype E) strain of human immunodeficiency virus type 1 (HIV-1), originally reported in Thailand, spread rapidly to and showed prevalence in several countries in Southeast Asia, including Taiwan. This strain was also found in other regions of the world. Based on sequence analysis of the vpu gene, a nested PCR assay including an outer primer pair and a subtype E-specific inner primer pair was developed in this study for rapid detection of subtype E viruses. It was tested with 397 HIV-1-positive samples of known subtypes. For these samples, the sensitivity of detection of subtype E viruses was 100% (127 of 127), and the specificity was 97.8% (264 of 270). Although six samples of either subtype A or G showed a positive PCR, most of the cross-reactivity could be reduced by raising the annealing temperature from 54 degrees C to 63 degrees C. When tested with 195 HIV-positive samples of unknown subtypes, the assay had a sensitivity of 98.0% and a specificity of 98.6%. This is a simple, convenient, and sensitive method for rapid detection of subtype E viruses, especially in regions in which viruses of subtypes B and E are predominant.     

Stability of dried blood spot specimens for detection of human immunodeficiency virus DNA by polymerase chain reaction.

Blood sampling on filter paper has many advantages for the detection of perinatal human immunodeficiency virus (HIV) infection by the polymerase chain reaction (PCR). However, if the method is to be widely used, an assessment of its performance under field conditions is required. To simulate conditions in the field, 50-microliters aliquots of whole blood containing low levels of HIV proviral DNA (4 to 1,024 copies per 100,000 nucleated cells) were spotted onto filter paper; dried; and subjected to heat, humidity, and prolonged storage at room temperature. After exposure, the DNA was recovered and amplified with primers to human leukocyte antigen DQ alpha- and HIV-specific sequences. Treatment at 37 degrees C and 60% humidity for 7 days, storage for 12 weeks at 22 degrees C, and freeze-thawing twice had no adverse effect on PCR reactivity when compared with the results obtained with reference spots stored at -20 degrees C. The lower limits of HIV detection in all tests ranged from 4 to 16 HIV copies per 100,000 cells. Fixation in 70% ethanol improved the amplification of low levels of HIV DNA and reduced biohazard risks. These findings suggest that dried blood spots will provide a powerful new resource for testing for HIV by PCR, especially in remote areas where refrigeration and immediate sample processing are unavailable.     

Development and validation of an immunoassay for identification of recent human immunodeficiency virus type 1 infections and its use on dried serum spots

The objective was to develop and to validate an immunossay to identify recent human immunodeficiency virus type 1 (HIV-1) infections that can be used on dried serum spots (DSS). A single, indirect enzyme-linked immunosorbent assay was developed to quantify antibodies toward four HIV-1 antigens: consensus peptides of the immunodominant epitope of gp41 (IDE), consensus V3 peptides, recombinant integrase, and recombinant p24. The parameters of the logistic regression used to classify the samples were estimated on a training sample (210 serum samples) using resampling techniques to get stable estimates and then applied to a validation sample (761 serum samples). The IDE and V3 peptides were the best able to discriminate between the antibodies present in serum from recently (< or =6 months) infected individuals and those with long-lasting infection. Combined quantification of antibody binding to these two synthetic antigens allowed us to identify recent infections with an area under the receiver operating characteristic curve of 0.949 and a sensitivity of 88.3%, with a specificity of 97.6% in patients with long-term infection (but not AIDS) and 86.0% in patients suffering from AIDS with a threshold of 0.50 in the validation sample. This simple immunoassay can be used to identify recently HIV-1-infected patients. Its performance is compatible with its use in population-based studies including DSS.     

Detection of cytomegalovirus DNA on dried blood spots collected from infants infected with HIV: An in-house method adaptable in resource-limited settings

In countries with limited resources, infants infected with HIV are highly exposed to CMV co-infection which probably represents a major risk factor for disease progression in this population. This study aimed to evaluate the performance of a low cost CMV DNA extraction method from DBS and the feasibility of its implementation in laboratories of 4 countries with limited resources. DNA was extracted from DBS with a cationic resin (chelex 100) and amplified with an “in house” real time CMV PCR. Dilutions of a quantified whole blood sample were spotted on paper to evaluate the 95% detection limit. A DBS quality control panel was analyzed in all laboratories. CMV PCR was compared between DBS and liquid whole blood (gold standard) in 2 populations: 418 transplanted patients and 59 infants infected with HIV (median age of 2 months). The CMV PCR 95% detection limit in DBS was 3.87 log₁₀ copies/mL. Its positive and negative predictive values for CMV diagnosis in infants infected with HIV were 100% and 87.5% respectively. Quality control panels gave consistent qualitative results in all laboratories. This assay had high predictive values for CMV diagnosis in infants infected with HIV and its implementation in resource-limited countries with limited resources is feasible.     

Evaluation of a prototype Amplicor PCR assay for detection of human immunodeficiency virus type 1 DNA in blood samples from Tanzanian adults infected with HIV-1 subtypes A, C and D.

BACKGROUND: In previous evaluations, the standard Amplicor HIV-1 DNA PCR test (Roche Diagnostic Systems) has been reported to have low sensitivity for the detection of some non-B HIV-1 subtypes. It has therefore become necessary to determine the performance of commercially available as well as prototype HIV-1 PCR assays for HIV-1 DNA detection in samples from various geographical settings, in order to assess their ability to detect the different HIV-1 genotypes. OBJECTIVES: To determine the performance of the prototype Roche Amplicor version 1.5 PCR test in comparison to that of the standard Roche Amplicor PCR test for the detection of HIV-1 DNA in blood samples from HIV-1 seropositive pregnant Tanzanian women infected with various HIV-1 subtypes. STUDY DESIGN: This was a cross-sectional study done on 161 blood samples collected from 106 HIV-1 seropositive and 55 seronegative asymptomatic pregnant women attending antenatal clinic in Dar es Salaam, Tanzania. METHODS: Cell pellets for PCR were prepared from EDTA blood by the Amplicor whole blood PCR sample preparation method. Plasma was used for HIV serology by enzyme linked immunosorbent assays. Subtyping was done by the heteroduplex mobility assay (HMA) using cell pellets and/or plasma. RESULTS: The sensitivities of the prototype PCR and the standard assays were 99.1% (105/106) and 97% (99/102), respectively. All samples from 55 HIV-1 seronegative women were negative by both PCR assays. Among the 101 samples subtyped by HMA, 48 (47%) were subtype A, 30 (30%) subtype C, 20 (20%) subtype D and 3 (3%) were indeterminate. In the standard DNA PCR assay, a statistically significantly higher proportion of subtype A samples had a low level of reactivity as measured as optical density compared with the subtypes C and D samples while in the prototype assay all three subtypes showed a high level of reactivity. CONCLUSIONS: The Amplicor version 1.5 DNA PCR test has a high sensitivity for the detection of HIV-1 DNA in blood samples from Tanzanian adults. Since performance of this assay does not appear to be influenced by differences in HIV-1 subtypes A, C and D, it has the potential for use in the detection of HIV-1 DNA in samples from geographic areas where these subtypes are prevalent.     

Quantification of human immunodeficiency virus type 1 RNA from dried plasma spots collected on filter paper.

To assess dried plasma spots (DPSs) as a source of material for virus quantification, human immunodeficiency virus type 1 (HIV-1) RNA levels were quantified in matched DPS and liquid plasma samples from 73 infected patients, including 5 neonates and 4 adult patients with acute HIV-1 infection. Quantifications were performed by commercially available assays (NASBA [nucleic acid sequence-based amplification] or Amplicor, or both). There was a strong correlation between HIV-1 RNA levels in plasma and DPSs. More importantly, there was no decline in HIV-1 RNA levels in DPSs stored for as long as 2 weeks at 20 degrees C. Similarly, storage of DPSs for 3 days at 37 degrees C resulted in no decrease in viral RNA levels. For patients with primary infection, the DPS method allowed for the measurement of RNA levels in plasma during the initial spike in the level of viremia and in the subsequent period of suppressed viral replication. DPS quantification was equally informative in the neonatal setting, with all five newborns showing HIV-1 RNA loads of greater than 4.991 log10 copies/ml. We conclude that the viral RNA levels in DPSs are equivalent to those measured in fresh-frozen plasma. The ease and economy of DPS sampling, the minute volumes required, and the unexpected stability of dried RNA suggest that the use of DPSs will be particularly valuable for small-volume neonatal samples and large, population-based studies in which cold storage and transportation present special problems, as is often the case in developing countries. The ability to measure viral changes during primary infection suggests that the method will be useful for assessing vaccine efficacy in large field trials.     

A novel polymerase chain reaction method for detection of human immunodeficiency virus in dried blood spots on filter paper.

A method for detection of proviral human immunodeficiency virus DNA in dried blood spots on filter paper by direct polymerase chain reaction (PCR) has been developed. To develop the method, a standard system was used which was prepared from cells each containing a single integrated provirus and titrated with normal donor blood. This rapid procedure provides virtually quantitative yields of nuclear DNA and exploits most of the standard methodology described for blood specimens. A nested PCR using SK38-SK39 gag as the internal primer pair was also designed; this PCR detected a single copy of provirus per filter at near theoretical frequency with SK19 probe. The utility of the procedure was demonstrated with clinical specimens. Blood spot filters from human immunodeficiency virus-infected and uninfected individuals were readily and unequivocally discriminated. The method is designed for ultimate use with large (1.5-ml) sample preparation tubes that are compatible as PCR tubes with thermal cyclers. This will permit convenient, direct single-tube PCR of dried blood specimens on filters. It should be adaptable to analysis of dried blood spots for a variety of infectious or genetic diseases.     

Performance of a novel human immunodeficiency virus (HIV) type 1 total nucleic acid-based real-time PCR assay using whole blood and dried blood spots for diagnosis of HIV in infants

The new Cobas AmpliPrep/Cobas TaqMan HIV-1 Qual test offers advanced automation for the detection of human immunodeficiency virus type 1 (HIV-1) RNA and DNA in dried blood spots (DBS) and whole blood. An analytical evaluation using an HIV-1 secondary standard yielded limits of detection of 514, 710, and 1,090 HIV RNA copies/ml for EDTA plasma, whole blood, and DBS, respectively. The precision and reproducibility of HIV-1 detection was equivalent for DBS and whole blood. Inclusivity was demonstrated for a reference panel of HIV-1 subtypes A to N. A clinical evaluation of the Cobas AmpliPrep/Cobas TaqMan HIV-1 Qual test was performed at a center for routine diagnostics in Johannesburg, South Africa, using 1,013 clinical specimens from HIV-1 exposed children. The Amplicor HIV-1 DNA test v1.5 with the MagNApure DNA isolation procedure was used as the reference method. A total of 995 valid results for whole blood with both methods yielded 691 and 303 concordant negative and positive results for the Cobas AmpliPrep/Cobas TaqMan HIV-1 Qual test, respectively. For the 800 valid DBS specimen results, 495 and 300 concordant negative and positive results were obtained, respectively. The resulting clinical specificities and sensitivities of the new test were 100% and 99.7% for whole blood and DBS, respectively. The new test was characterized by its robustness, enhanced automation, and improved sample throughput. The Cobas AmpliPrep/Cobas TaqMan HIV-1 Qual test will support early, reliable diagnosis of HIV in children in routine laboratory settings.