HIV DNA PCR Research Articles

治療後ウエスタンブロット法にて抗HIV 抗体が陰性化し 持続しているHIV-1 感染症の1 例

Western blot (WB) is the most widely accepted confirmatory assay for detecting antibodies to the human immunodeficiency virus 1 (HIV-1). We report the case of an HIV-1 patient whose WB was negative for over two years. A 41-year-old Japanese man with Pneumocystis pneumonia (PCP) and pulmonary tuberculosis referred in March 2005 was found to have positive HIV-1 ELISA and HIV RNA PCR, but HIV-1 WB with only two bands, at gp160 and p18, and no WB HIV-2 band. The CD4 count was 37/microL, and total immunoglobulin, IgG, IgM, and IgG subclasses were normal. The man was treated for PCP and pulmonary tuberculosis, then underwent antiretroviral therapy. He had taken short-terms steroids to treat a drug allergy and immune reconstitution syndrome. Six months later, his serological ELISA tests for HIV-1 and HIV DNA PCR were negative and WB showed no positive band. The CD4 count recovered gradually, and exceeded 350/microL two years later, but WB remained negative. Lymphoproliferative assays and interferon y expression against HIV-pl7, p24, and p41 were studied and compared to those of other HIV-1 infected patients. Our patient showed no response to p17 or p24 and only a weak response to p41. Other patients showed a response to HIV-antigens, but patients with antiretroviral therapy or with histories of steroid use responded more weakly than those with neither. These findings show that HIV-specific lymphocytes decline with antiretroviral therapy and steroid treatment within early HIV infection. It is therefore important to interpret negative serological tests carefully in patients such as ours.

Role of the Laboratory in Ensuring Global Access to ARV Treatment for HIV-Infected Children: Consensus Statement on the Performance of Laboratory Assays for Early Infant Diagnosis

A two day meeting hosted by the World Health Organization (WHO) and the U.S. Centers for Disease Control and Prevention (CDC) was held in May 2006 in Entebbe, Uganda to review the laboratory performance of virologic molecular methods, particularly the Roche Amplicor DNA PCR version 1.5 assay, in the diagnosis of HIV-1 infection in infants. The meeting was attended by approximately 60 participants from 17 countries. Data on the performance and limitations of the HIV-1 DNA PCR assay from 9 African countries with high-burdens of HIV/AIDS were shared with respect to different settings and HIV- subtypes. A consensus statement on the use of the assay for early infant diagnosis was developed and areas of needed operational research were identified. In addition, consensus was reached on the usefulness of dried blood spot (DBS) specimens in childhood as a means for ensuring greater accessibility to serologic and virologic HIV testing for the paediatric population.

Prevention of mother-to-child transmission: program changes and the effect on uptake of the HIVNET 012 regimen in Malawi

To evaluate uptake of HIV testing in a prevention of mother-to-child transmission program (PMTCT) in Lilongwe, Malawi from April 2002 until December 2006. Retrospective analysis of monthly reports from the beginning of the program. Four antenatal clinics in Lilongwe, Malawi. Pregnant women attending urban antenatal clinics in Lilongwe were invited to participate in a PMTCT program. Women were given information and education on antenatal care and PMTCT in groups of 8 to 12. Written informed consent for HIV testing was obtained privately. Women returned for the test result 1-2 weeks later. Mothers and infants were given the HIVNET 012 regimen. Rapid HIV testing and 'opt-out' testing were instituted in July 2003 and April 2005, respectively. Infants were tested using HIV DNA PCR and, if HIV positive, a CD4 cell percentage was obtained and the infants were referred for further medical evaluation and treatment. The program reached 20 000 pregnant women in the first 12 months. Acceptance of HIV testing increased from 45% to 73% (P < 0.001) when rapid, same day testing was instituted. When opt-out testing was instituted, 99% of the mothers agreed to testing. Of the infants tested, 15.5% were HIV positive. Rapid HIV testing using the opt-out method increased acceptance of HIV testing in the PMTCT program to 99% in urban Lilongwe, Malawi.

Development and challenge of HIV/AIDS testing laboratory network and quality assurance system in China

This paper describes the development and challenge of HIV/AIDS testing laboratory network and quality assurance system in China. At present, the HIV/AIDS testing laboratories includes three classes, the National AIDS Reference Laboratory, HIV/AIDS confirmatory laboratories and HIV/AIDS screening laboratories. All of them are accredited by the health authorities, and each class of laboratories take charge of their function strictly according to the “National Management of HIV/AIDS Detection (2006)”. A complete quality assurance and quality control system for HIV/AIDS testing has been developed, which includes technical training, strict laboratory monitoring and approval, examination or proficiency testing on HIV/AIDS detection, and quality evaluation and supervision of HIV/AIDS diagnostic kits. Besides conduct the routine anti-HIV antibody test, more and more laboratories began to conduct other tests, such as CD4+T lymphocyte cell counting, HIV viral load, HIV DNA PCR, genotyping, drug resistance, and HIV-1 recent infection test. The primary challenges faced by the HIV/AIDS testing laboratory network are in the areas of laboratory management and quality control. For example, the provincial PT program is inefficient, the internal quality control is conducted perfunctorily, personnel training can not met the needs of the workplace, which need to be improved.

Low risk of contamination with automated and manual excision of dried blood spots for HIV DNA PCR testing in the routine laboratory

In low resource settings the inability to diagnose HIV in infants early presents a major obstacle to providing care for HIV-infected children. Dried blood spot samples offer a solution to the scarcity of skills available for venesecting young infants but pose challenges to laboratories because their processing requirements are distinct from that of the liquid blood samples widely used for viral detection assays. Different methods of excising 287 paired HIV-positive and HIV-negative dried blood spot samples ( n = 574) for testing by the Roche Amplicor™ HIV-1 DNA assay version 1.5 were assessed. A manual punch in conjunction with three different cleaning protocols ( n = 372) and an automated punch (BSD 1000 GenePunch) using a single cleaning protocol ( n = 202) was assessed for the risk of cross contamination between samples. A single false positive result obtained using the automated punch may have been attributable to contamination during the excision step of the assay. Excision of dried blood spot samples is associated with a very low risk of cross contamination regardless of the instrument or cleaning intervention used. The process of excising dried blood spot samples should not compromise the results of HIV viral detection assays performed on dried blood spots in routine laboratories which is encouraging for increasing access to an accurate diagnosis of HIV in infants.

A Reverse Transcriptase Assay for Early Diagnosis of Infant HIV Infection in Resource-limited Settings

Early diagnosis of pediatric HIV infection is confounded by persistence of maternal antibodies until 18 months, necessitating the use of expensive assays such as HIV-1 DNA PCR, an untenable option in resource-limited settings. This is the first report of a low-cost, commercial, reverse transcriptase (RT) assay for the diagnosis of HIV-1 infection in infants. RT assays were performed on 42 samples from 30 HIV-exposed Kenyan infants under 15 months of age. When correlated with serologic testing conducted after 18 months, the sensitivity, specificity, positive and negative predictive values of the RT assay were 92%, 93%, 87% and 96%. A low-cost assay for infant HIV diagnosis is urgently needed, and these results merit further evaluation.

Diagnosis and monitoring of HIV infection

The latest statistics indicate that the number of people infected with human immunodeficiency virus type 1 (HIV-1) worldwide in 40.3 million, 25.8 million of whom live in sub-Saharan Africa. In 2004, 29.5% of South African women attending antenatal clinics were infected. The virus infects people of all ages and social classes. A diagnosis of HIV has serious physical, emotional and social implications for the patient. HIV-infected patients are susceptible to numerous opportunistic and other infections, as well as to non-infectious diseases such as tumours. They eventually require lifelong treatment with potentially toxic medication. It in therefore essential that a timeous and correct diagnosis be made.An understanding of the tests available for the diagnosis and monitoring of HIV in essential for all clinicians working in South Africa. Tests available for the diagnosis of HIV in patients older than 18 months include HIV-specific antibody assays, fourth-generation combination antibody-antigen assays and Western Blot. The diagnosis of HIV infection in infants younger than 18 months requires detection of the virus itself by means of p24 antigen detection or HIV DNA PCR. The CD4+ T lymphocyte count and HIV viral load are used for monitoring disease progression and response to therapy.

Increased severe anemia in HIV-1-exposed and HIV-1-positive infants and children during acute malaria

Since the primary hematological complication in both pediatric HIV-1 and malaria is anemia, co-infection with these pathogens may promote life-threatening severe malarial anemia (SMA). The primary objective of the study was to determine if HIV-1 exposure [HIV-1(exp)] and/or HIV-1 infection [HIV-1(+)] increased the prevalence of SMA in children with acute malaria. The effect of HIV-1 exposure and HIV-1 infection on the prevalence of SMA (hemoglobin < 6.0 g/dl), parasitemia (parasites/microl), and high-density parasitemia (HDP, >or= 10 000 parasites/mul) was investigated in children <or= 2 years of age presenting at hospital with acute Plasmodium falciparum malaria in a rural holoendemic malaria transmission area of western Kenya. Upon enrollment, a complete hematological and clinical evaluation was performed on all children. Malaria parasitemia was determined and children with acute P. falciparum malaria were evaluated for HIV-1 exposure and infection by two rapid serological antibody tests and HIV-1 DNA PCR, respectively. Relative to HIV-1(-) group (n = 194), the HIV-1(exp) (n = 100) and HIV-1(+) (n = 23) groups had lower hemoglobin concentrations (P < 0.001 and P < 0.001, respectively), while parasitemia and HDP were equivalent between the three groups. Multivariate analyses demonstrated that the risk of SMA was elevated in HIV-1(exp) children (odds ratio, 2.17; 95% confidence interval, 1.25-3.78; P < 0.01) and HIV-1(+) children (odds ratio, 8.71; 95% confidence interval, 3.37-22.51; P < 0.0001). The multivariate model further revealed that HIV-1 exposure or infection were not significantly associated with HDP. Results presented here demonstrate that both HIV-1 exposure and HIV-1 infection are associated with increased prevalence of SMA during acute P. falciparum infection, independent of parasite density.

Prospective Clinical Trials Designed to Assess the Use of Hormonal Contraceptives and Risk of HIV Acquisition

Prospective Clinical Trials Designed to Assess the Use of Hormonal Contraceptives and Risk of HIV Acquisition

Quantitation of human immunodeficiency virus type 1 in breast milk.

The distribution and stability of human immunodeficiency virus type 1 (HIV-1) in breast milk (BM) components remain largely unknown. Inhibitory effects, if any, of BM on HIV RNA and DNA PCR amplification are poorly understood. We have addressed these issues by using virus-spiked BM samples from HIV-negative women. BM samples from HIV-negative women were spiked with HIV-1 virions or cells containing a single integrated copy of HIV DNA (8E5/LAV). After incubation under different experimental conditions, viral RNA was detected by the Roche Amplicor UltraSensitive assay in whole-milk, skim milk, and lipid fractions. We found excellent correlation between HIV-1 input copy and recovery in whole milk (r = 0.965, P < 0.0001), skim milk (r = 0.972, P < 0.0001), and the lipid fraction (r = 0.905, P < 0.001). PCR inhibition was observed in less than 10% of the spiked samples. Similar levels of inhibition were noted in BM samples collected from HIV-infected women. HIV proviral DNA was detected in BM samples using real-time PCR (linear correlation between the threshold cycle versus log DNA copy number, >0.982). The effects of incubation duration and temperature and repeated freeze-thaw cycles on HIV RNA recovery were analyzed. HIV RNA levels were remarkably stable in whole milk after three freeze-thaw cycles and for up to 30 h at room temperature. Our findings improve the understanding of the dynamics of HIV detection in BM and the conditions for BM sample collection, storage, and processing.

Subtype-specific problems with qualitative Amplicor HIV-1 DNA PCR test.

commercial HIV-1 qualitative DNA PCR tests have the potential to detect virus in patients in whom antibody tests may be ineffective, such as patients with primary HIV infection and infants born to HIV seropositive mothers. However, the genetic diversity of HIV-1 raises concern about the ability of the PCR tests to detect all current subtypes. to asses the sensitivity of the Amplicor HIV-1 test on 126 whole-blood samples representing seven different subtypes and to investigate the sensitivity when the standard assay was modified by including the primer pair SK145 and SKCC1B. of the 126 HIV-1 infected persons, 113 were tested positive and 13 were DNA PCR negative. On the basis of these results, the standard Amplicor HIV-1 test had a sensitivity of 90% in our cohort. In addition, 9% of the positive samples showed a low reactivity but above the cut-off of the assay. The standard assay yielded sensitivities of 100% for subtype B (n=16), D (n=9) and G (n=1), but only 83% for subtype A (n=41), 98% for subtype C (n=43), 79% for subtype E (n=14) and 0% for subtype F (n=2). All samples with low reactivity were non-B subtype. Eight of the DNA PCR negative samples, four subtype A, one C and three E were amplified with the modified Amplicor HIV-1 test with addition of SK145/SKCC1B primers. Using this modified protocol, six samples out of eight became positive. However, two samples (one A and one C) remained DNA PCR negative. this study confirms that the Amplicor HIV-1 test does not detect all subtypes with equivalent sensitivity and 10% of the samples, tested negative. Thus, it is preferable to add the SK145/SKCC1B primers to the standard test, where infection with non-B subtype is suspected.

Interventions to prevent vertical transmission of HIV-1: effect on viral detection rate in early infant samples.

To determine whether mode of delivery or the use of maternal or neonatal antiretroviral prophylaxis influence the age when HIV-1 can first be detected in infected infants, particularly the probability of detection at birth. In a collaboration between four multicentre studies, data on 422 HIV-1 infected infants who were assessed by HIV-1 DNA PCR or cell culture before 14 days of age were analysed. Weibull mixture models were used to estimate the cumulative proportion of infants with detectable levels of HIV-1 according to use of maternal/neonatal antiretroviral therapy (mainly zidovudine monotherapy) and mode of delivery. HIV-1 was detected in 162 infants (38%) when they were first tested, at a median age of 2 days. At birth, it was estimated that 36% [95% confidence interval (CI), 31-41%] of infants have levels of virus that can be detected by DNA PCR or cell culture. This percentage was not associated with either mode of delivery (35% for vaginal delivery versus 40% for cesarean section delivery; P = 0.4) or the use of maternal or neonatal antiretroviral prophylaxis. Among infants with undetectable levels of HIV-1 at birth, the median time to viral detectability was estimated to be 14.8 days (95% CI, 12.9-16.8 days). This time was increased by 15% (95% CI, -11 to 48%; P = 0.3) among infants who were exposed to antiretroviral therapy postnatally compared with infants who were not exposed. No effect was observed for mode of delivery. The outcome of an early virological test for HIV-1 is thought to be related directly to the timing of transmission and cesarean section delivery primarily reduces the risk of intrapartum transmission. The absence of an association between mode of delivery and viral detectability at birth was therefore unexpected. There was no evidence that foetal or neonatal exposure to prophylactic zidovudine delays substantially the diagnosis of infection, although this cannot be inferred for combination antiretroviral therapy.

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.

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. 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. 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. 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. 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. 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.

DIAGNOSIS OF HIV INFECTION IN CHILDREN

Many advances have been made in the area of HIV diagnostics. Commercially available virologic assays are sensitive and specific for the early detection of HIV in perinatal infection. The timing of the transmission of HIV from mother to child (in utero, at the time of birth, or postnatally by breast-feeding) is a critical consideration in the appropriate diagnosis of infants. Several algorithms can be used to define early infection and the potential timing of acquisition of infection that combine different assays and timing of specimens. The use of virologic assays, including HIV DNA PCR and HIV RNA detection methods and culture, can define and rule out infection in infants less than 18 months of age. Serologic diagnostic methods, including HIV ELISA, immunofluorescence, and western blot assays, can be used to diagnose infants more than 18 months of age, when transplacental antibody has disappeared in uninfected HIV-exposed infants. The challenge of the early and accurate diagnosis of perinatally HIV-exposed infants is the use of new assays to detect different HIV subtype infections that are prevalent in developing countries. Rapid, simple, and inexpensive serologic and virologic assays are being developed for worldwide use.

Infant feeding and HIV-1 transmission

Anna Coutsoudis and colleagues present a potentially important piece of the puzzle concerning the transmission of HIV-1 through breast-feeding in the first few months of life. Breast-feeding poses a substantial risk of acquiring HIV-1 infection for the baby of an HIV-1-infected mother. Samples from 549 infants were tested by HIV-DNA PCR on day 1 and at ages 1 month and 3 months. Results revealed that the percentages of infection were similar for the three groups between 1 month and 3 months, and that the real increase in rate of infection occurred after the first month of life. This suggests that there may be a protective early effect of exclusive breast-feeding and that hence a breast-feeding HIV-1-infected mother should avoid giving any other foods for at least the first 3 months of life. Results in this study highlight the need for re-evaluation of the role of breast-feeding in the transmission of HIV-1.

The relationship between maternal-infant antibody levels and vertical transmission of HIV-1 infection.

This study assesses the predictive value of the ratio of HIV-1 antibodies in the newborn at birth to that in the mother for perinatally transmitted infection confirmed subsequently by age 18 months. The ratio of HIV-1 (EIA) antibody levels in the baby at birth to that in the seropositive mother after the first trimester (sequenstration index SI) was available in 114 of a perinatal cohort of 137 infants. We related this ratio to the HIV infection status of the children by 18 months, HIV-1 DNA PCR and HIV-specific IgA antibody detection at birth, between 3 and 6 months, and morbidity and mortality. Thirty-five of the 137 (26 per cent) children were diagnosed as infected by 18 months. The mean (SD) HIV SI was 1.57 (0.88) in 29 infected and 0.83 (0.42) in 85 uninfected infants (P < 0.0001). Sensitivity and specificity of a threshold SI of 1.27 (mean +/- 2 SD of uninfected group) for the prediction of perinatal HIV-1 infection were 41 and 98 per cent, respectively. The reason for the higher SI in the infected babies is the combination of lower antibody titres in the transmitting mothers with raised levels in the infected babies. A similar analysis of antibody ratios showed no statistical differences for measles and tetanus (P > 0.1) between HIV infected and uninfected groups. There was a tendency to increased morbidity (Pearson's correlation coefficient r = 0.31) and more severe disease in those with higher HIV-1 SI. Three of 17 (18 per cent) peripheral blood samples from infected children at birth were PCR positive; all had SI's above the threshold. Overall sensitivity and specificity of PCR were 85 per cent each. Eleven of the 29 infected children were HIV-1 specific IgA positive at birth; six (64 per cent) of these had an SI > 1.27. This simple SI of HIV-1 EIA antibodies at birth is comparable to elaborate techniques in its power to predict perinatally acquired infection. It may be a cheap, reliable and rapid screening test for vertically transmitted HIV-1 infection.

P24 antigen detection, viral isolation, DNA-PCR and in vitro antibody production for the diagnosis of HIV-1 latent infection in heterosexual women at high risk for HIV-1 infection.

The report of the existence of at-risk seronegative subjects, latently infected with HIV-1 and producing "in vitro" HIV-1 specific antibodies, prompted the authors to evaluate extensively twenty-five heterosexual HIV-1 seronegative women at high risk for HIV-1 infection. The capability of peripheral blood mononuclear cells from such subjects to produce "in vitro" HIV-1 specific antibodies after pokeweed-mitogen stimulation, was studied. Silent HIV-1 infection was investigated by HIV-1 DNA PCR, viral isolation and serum p24 Ag detection at entry and after 6 and 12 months. Three seroconversions took place within 12 months, but no HIV-1 infections were found in the absence of detectable serum anti HIV-1 antibodies, even in subjects who apparently produced such antibodies in vitro. The antibodies produced in vitro by the seronegative women studied appeared of narrow specificity, reacting mainly with gp 160/120 envelope glycoproteins. A strong concordance was found between the serological status and the other markers for HIV-1 infection, suggesting that the phenomenon of HIV-1 "latent infection" is a very rare event, if it occurs at all. Seronegative women sexually exposed to the virus may produce in vitro anti HIV-1 antibodies of narrow specificity in the absence of other signs of infection and this phenomenon might be related to an anamnestic response to the virus.

Quality assurance and use of PCR in clinical trials.

Qualitative and quantitative HIV-1 DNA and RNA PCR assays are proving to be useful in the diagnosis of HIV-1 infection in infants and in assessing the in vivo antiviral activity of new therapies and regimens in clinical trials. The use of these standardized commercial assays in conjunction with an external quality assurance program has ensured that results from different laboratories are comparable. In addition, real-time proficiency monitoring has the potential to detect problems immediately before patient data are compromised.

Quality assurance for HIV-1 DNAPCR

Quality assurance for HIV-1 DNAPCR