Persistence of human immunodeficiency virus type 1 subtype B DNA in dried-blood samples on FTA filter paper

The stability of human immunodeficiency virus type 1 (HIV-1) DNA in whole blood collected on filter paper (FTA Card) was evaluated. After >4 years of storage at room temperature in the dark our qualitative assay detected virus at a rate similar to that of our initial test (58 of 60, 97%; P = 0.16), suggesting long-term HIV-1 DNA stability.     

Neutralization kinetics of sensitive and resistant subtype B primary human immunodeficiency virus type 1 isolates

The aim of the study was to determine if sensitive and resistant human immunodeficiency virus type 1 (HIV-1) subtype B primary isolates have different neutralization kinetics. Neutralization assays were undertaken where either the time allowed for virus to react with antibodies or the subsequent period of this mixture's exposure to target cells were varied. The relative neutralization sensitivity/resistance is a reproducible property of the isolates. In a minority of combinations, the titre falls exponentially for as long as the free virions are exposed to antibody. In the remainder, neutralization kinetics shows deviations which may be attributed to events occurring after the virus-antibody mixture is added to the target cells: significant neutralization with minimal exposure of the free virions to antibody; a plot where reduction in virus titre is parallel to the duration of the incubation phase of the assay. Neutralization rate constants are similar for primary HIV-1 SF33, HIV-1 SF162, and HIV-1 89.6, reaching 5 x 10(5)-1 x 10(6)/M sec for the monoclonal antibody IgG1 b12. However, although increased antibody levels produced greater reductions in virus titre the rate of neutralization was not proportional to the antibody concentration. Neutralization of either the free virion or cell-associated virus does not correlate with the resistance/sensitivity of primary subtype B isolates. The target cells play an active role, so that in designing neutralization assays with primary isolates of HIV-1, events following the virus-antibody complex binding to the cell surface have to be taken into consideration.     

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.     

Simple, sensitive, and specific detection of human immunodeficiency virus type 1 in clinical specimens by polymerase chain reaction with nested primers.

A simple, sensitive, and specific polymerase chain reaction (PCR) protocol for the detection of human immunodeficiency virus type 1 (HIV-1) is described. We have improved all three PCR steps: sample preparation, DNA amplification, and detection of the amplified product. Some of the improvements have been described previously, but they have never been combined into a complete PCR protocol. Peripheral blood mononuclear cells were lysed directly in a buffer containing sodium dodecyl sulfate, Triton X-100, and proteinase K. This crude cell lysate was amplified in a two-step PCR, first with outer primers and then with inner primers nested within the first primers. The PCR product was visualized by agarose gel electrophoresis and ethidium bromide staining. Thus, we avoided conventional DNA extraction as well as hybridization for the detection of the PCR product. The samples were analyzed with four sets of nested primers (JA4 through JA7, JA9 through JA12, JA13 through JA16, and JA17 through JA20) designed to amplify HIV-1 gag, env gp120, env gp41, and pol sequences, respectively. We were able to amplify HIV-1 sequences in all samples from 90 HIV-1-seropositive individuals with mostly mild symptoms. Of these individuals, 24 were negative in HIV-1 isolation and 9 were selected because they were infected by African and Haitian HIV-1 strains. Eighty-five (94%) individuals were positive with at least three of four primer sets. Samples from 26 healthy blood donors, as well as cells infected in vitro with human immunodeficiency virus type 2 and human T-cell leukemia virus type I, were negative in PCR, thus demonstrating the specificity of the amplification.     

Stability of human immunodeficiency virus type 1 proviral DNA in whole-blood samples

The Roche Amplicor HIV-1 Test requires that whole blood be processed within four days of collection. However, this requirement may be too limiting for use in international settings. Thus, we demonstrate that blood may be processed up to 10 days after collection if maintained under ambient conditions (2 to 25 degrees C).     

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.     

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.     

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     

Use of dried blood spot specimens in the detection of human immunodeficiency virus type 1 by the polymerase chain reaction.

Dried blood spots (DBSs) constitute a potentially valuable source of material for human immunodeficiency virus (HIV) serologic and molecular testing. To facilitate molecular testing, we have adapted the polymerase chain reaction (PCR) to the detection of HIV proviral DNA in DBS samples. The method is highly reproducible, with 75 microliters of whole dried blood providing sufficient DNA for duplicate testing with three primer sets. By using DBS PCR, 66 of 69 (95.6%) seropositive at-risk individuals tested positive by at least two primer sets and 85 of 85 (100%) low-risk seronegative blood donors tested negative by all three sets of primers. The frequency of HIV DNA detection in seronegative at-risk individuals was low, with only 1 of 58 (1.7%) individuals testing positive. These results show that in a clinical environment, HIV PCR analysis of DBS specimens is specific and sensitive. The method is cost effective and presents a useful alternative to the isolation of HIV from seropositive babies with an undefined infection status.     

Evaluation of the ultrasensitive human immunodeficiency virus type 1 (HIV-1) p24 antigen assay performed on dried blood spots for diagnosis of HIV-1 infection in infants

The diagnostic accuracy of the modified p24 antigen assay performed on pediatric dried blood spots was evaluated. Samples analyzed within 6 weeks of collection yielded no false-positive results (specificity, 100%) and few false-negative results (sensitivity, 96.5% to 98.3%). Laboratory services with limited resources should assess this option for routine infant diagnosis.     

Laboratory methods for early detection of human immunodeficiency virus type 1 in newborns and infants.

Cumulative data on serological testing of newborns and infants have shown that (i) maternal and newborn anti-HIV-1 IgG titers are high at delivery, which may explain the persistence of antibody in the infants of seropositive mothers; (ii) in some situations, serial HIV-1 antibody testing may identify infected infants; and (iii) detection of anti-HIV-1 IgA or IgM is specific for infection but the sensitivity of this assay may be compromised in certain situations, such as when infected infants are hypogammaglobulinemic or when the rise and fall of HIV-1-specific IgM synthesis following acute infection has been completed before delivery of the infant. Cumulative data on PCR, viral culture, and tests for antigen in newborns and infants have shown that (i) among all age groups, viral culture is probably the most specific test available for detection of HIV-1, as PCR and the p24 antigen test may (though rarely) give false-positive results; (ii) the sensitivity of these tests increases in the order of antigen, culture, and PCR, with relatively insensitive results in the first 3 months of life for all of these tests; (iii) the sensitivity of all of these tests improves and approximates 90 to 100% when infants over 6 months of age are tested; and (iv) data regarding the sensitivity, specificity, and usefulness of these virological assays in infants under 3 months of age are very scant and inconclusive.     

Automated multiplex assay system for simultaneous detection of hepatitis B virus DNA, hepatitis C virus RNA, and human immunodeficiency virus type 1 RNA

We have developed an automated multiplex system for simultaneously screening hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-1) in blood donations. The assay, designated AMPLINAT MPX HBV/HCV/HIV-1 Test (AMPLINAT MPX), consists of virus extraction and target sequence-specific probe capture on specimen preparation workstation GT-X (Roche Diagnostics K.K., Tokyo, Japan) and amplification and detection by TaqMan PCR on the ABI PRISM 7700 Analyzer (Perkin-Elmer Applied Biosystems, Foster City, Calif.). An internal control (IC) is incorporated in the assay to monitor the extraction, target amplification, and detection processes. The assay yields qualitative results without discrimination of the three targets. Detection limits (95% confidence interval) are 22 to 60 copies/ml for HBV, 61 to 112 IU/ml for HCV, and 33 to 66 copies/ml for HIV-1, using a specimen input volume of 0.2 ml. The AMPLINAT MPX assay detects a broad range of genotypes or subtypes for all three viruses and has a specificity of 99.6% for all three viruses with seronegative specimens. In an evaluation of seroconversion panels, the AMPLINAT MPX assay detects HBV infection an average of 24 days before the detection of HBsAg by enzyme immunoassay. HCV RNA was detected an average of 31 days before HCV antibody. HIV-1 RNA was detected an average of 14 days before HIV-1 antibody and an average of 9 days before p24 antigen. The Japanese Red Cross has been evaluating the AMPLINAT MPX system since October 1999. The clinical performance indicates that the AMPLINAT MPX system is robust, sensitive, and reproducible, with a high percentage of valid assay runs (96.8%), a low false-positive rate (0.34%), and a low IC failure rate (0.24%).     

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.     

Plasma RNA viral load in human immunodeficiency virus type 2 subtype A and subtype B infections

Human immunodeficiency virus type 2 (HIV-2) is much less pathogenic than HIV-1, and HIV-2 infection is associated with plasma viral loads significantly lower than those found in HIV-1 infection. We have developed a real-time quantitative PCR method for measuring the HIV-2 RNA load that covers the range of genetic diversity of HIV-2 isolates and that detects extremely low viral loads. Samples from 49 patients were studied. Proviral DNA was first detected and quantified. The strains that were detected were then genotyped: 21 patients were infected with HIV-2 subtype A and 15 patients were infected with HIV-2 subtype B; 1 patient was infected with a highly divergent strain. Env PCR failed for the remaining 12 patients, so subtypes could not be determined. For viral RNA quantification, a stock of HIV-2 strain NIHZ, which was counted by electron microscopy, was used as the standard. Several primer sets targeting the highly conserved gag region were evaluated. Various primer combinations failed to amplify subtype B strains. With the final primer pair selected, which detected both subtype A and subtype B strains, the sensitivity of the assay was 100% at a viral load of 250 copies/ml and 66% at a viral load of 125 copies/ml. We found a correlation between the CD4(+)-cell count, the clinical stage, and the plasma HIV-2 RNA level. The median plasma HIV-2 RNA value for the 33 asymptomatic patients was 2.14 log(10), whereas it was 3.1 log(10) for the 16 patients with AIDS (P < 0.01). Proviral DNA was detectable in 18 symptom-free patients with high CD4(+)-cell counts, in whom viral RNA was undetectable.     

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.     

Early infant human immunodeficiency virus type 1 detection suitable for resource-limited settings with multiple circulating subtypes by use of nested three-monoplex DNA PCR and dried blood spots

The early detection of human immunodeficiency virus type 1 (HIV-1) infection in infants is complicated by the persistence of maternal antibodies and by diverse HIV-1 subtypes. We developed a nested, three-monoplex HIV-1 DNA PCR (N3M-PCR) assay to detect diverse HIV-1 subtypes in infants born to infected mothers. We optimized the test for use with dried blood spot (DBS) samples for ease of storage and transport from rural China to central laboratories. Six pairs of primers were designed that targeted env, gag, and pol genes, and the test was run in three reactions with an analytical sensitivity of 10 copies DNA per reaction to cover nine HIV-1 subtypes, A, B, C, D, F, G, CRF01-AE, CRF08-BC, and CRF07-BC. The assay performance was evaluated on 347 DBS specimens from 151 exposed infants in four diverse provinces of China in which multiple subtypes were circulating. The results of this test were compared to those of HIV antibody enzyme immunoassay and Western blotting confirmation for the infants at ≥18 months of age or to convincing clinical and epidemiologic data for deceased infants. The sensitivity of the N3M-PCR assay was 30.0% (3/10) for infants at 48 h after birth, 91.7% (11/12) at 1 to 2 months of age, and 93.7% (15/16) at 3 to 6 months of age. The specificity was 100% (94/94) at all three time points. The PCR reproducibility in the three DNA regions was 100% for samples at 48 h after birth, 96.7% at 1 to 2 months, and 100% at 3 to 6 months of age. The HIV-1 DNA N3M-PCR assay on DBSs offers a simple and affordable approach for early infant HIV-1 diagnosis in regions with diverse HIV-1 circulating subtypes.