Detection of human immunodeficiency virus-1 by polymerase chain reaction and virus cultivation

Peripheral blood of 57 patients with antibodies to human immunodeficiency virus 1 (HIV-1) and of five HIV-1 seronegative subjects at risk for HIV-1 infection were analysed by polymerase chain reaction (PCR) and virus isolation. The virus was recovered from peripheral blood cells in 89% and from plasma in 75% of the HIV-1 seropositive cases. In contrast, proviral HIV-1 DNA was detected in all HIV-1 seropositive patients by dot blot hybridization of the amplified fragments. The intensities of the dot blot reactions were less pronounced in asymptomatic HIV-1 seropositive individuals than in patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC), suggesting an increase in proviral DNA with advancing disease. Three of five seronegative patients with signs or symptoms suggesting HIV-1 infection, but none of the controls, were positive for HIV-1 DNA by one or two primer pairs. These results show a high sensitivity of the PCR for detecting HIV-1 DNA in patients of all stages of HIV-1 infection. Proviral DNA can also be detected in some individuals without detectable antibodies to the virus. The virus load in peripheral blood, as determined by virus cultivation and PCR, seems to increase with progression of the infection.     

Comparison of human immunodeficiency virus type 1 RNA sequence heterogeneity in cerebrospinal fluid and plasma

The source of human immunodeficiency virus type 1 (HIV-1) RNA in cerebrospinal fluid (CSF) during HIV-1 infection is uncertain. The sequence heterogeneity of HIV-1 RNA in simultaneous CSF and plasma samples was characterized for five patients at the baseline and during the first week of antiretroviral therapy by two commercial genotyping methodologies. In individual subjects, the sequences in CSF samples differed significantly from those in plasma. In contrast, the viral sequences in CSF at the baseline did not differ from the sequences in CSF during treatment. Similarly, viral sequences in plasma did not vary over this interval. This study provides evidence that HIV-1 RNA in CSF and plasma arise from distinct compartments.     

Human immunodeficiency virus type 1 is present in the cerebrospinal fluid of a majority of infected individuals.

Cerebrospinal fluid (CSF) specimens from 63 patients with different severities of human immunodeficiency virus (HIV-1) infection, including asymptomatic virus carriers, were examined for the presence of HIV-1 by using polymerase chain reaction (PCR) and virus isolation. Polyadenylated RNA, presumably associated with virus particles, was extracted and reverse transcribed, and the pol region was amplified in a nested PCR. Virus could be detected in 90% of the CSF specimens examined by PCR, and data on isolation of virus from CSF were in agreement with these figures. In fact, when several CSF specimens from the same individual were studied, HIV-1 could be isolated from 80% of the patients. The presence of the viral RNA in CSF was independent of the clinical stage of infection and of neurological symptoms. These results show that the spread of HIV-1 to the brain represents an early event during infection and occurs in the majority of asymptomatic individuals.     

Analysis of ENV V3 sequences from HIV-1-infected brain indicates restrained virus expression throughout the disease

The isolation of human immunodeficiency virus type 1 (HIV-1) from the cerebrospinal fluid (CSF) of asymptomatic virus carriers suggests that the viral infection spreading to the brain occurs early during infection. The aim of the present study was to investigate whether HIV-1 infection of the brain parenchyma also occurs during the early phase of infection. We also wished to compare the degree of replication of the virus in the brain at different clinical stages associated with HIV-1 infection. With the use of polymerase chain reaction (PCR), the viral genomes present in seven of eight brain specimens obtained from two asymptomatic HIV-1 carriers and six AIDS patients were amplified. Thereafter, the number of viral copies present in each brain specimen was quantified, the third variable region (V3) of the gp120 glycoprotein was sequenced and these results compared with the histopathological findings in the tissue. The HIV-1 DNA genome was amplified from seven of the eight brain tissues, including the specimens obtained from the two asymptomatic carriers. An increased number of viral copies in the brain was found in association with histopathological findings of HIV-1 encephalitis. The analysis of the V3 sequences, however, revealed the presence of a homogeneous virus population in the brain at every clinical stage of the disease. These results suggest that, although entry of the virus in the parenchyma may occur early during infection, HIV-1 replication in the brain is constrained until the terminal phase of AIDS encephalitis. © 1996 Wiley-Liss, Inc.     

Higher HIV-1 RNA cutoff level required in cerebrospinal fluid than in blood to predict positive HIV-1 isolation

HIV-1 can be isolated from the vast majority of blood samples taken from HIV-1-seropositive patients not treated with antiretroviral drugs. Isolation rates from cerebrospinal fluid (CSF) samples are considerably lower, ranging between 20-70%. The objective of this study was to determine the cutoff levels for HIV-1 RNA that would yield a positive predictive value > or =90% for positive virus isolation from CSF and blood. Quantitative HIV-1 RNA PCR (Amplicor HIV monitor, version 1.0, Roche Diagnostic Systems) and virus isolation were used to examine 303 CSF samples and 278 paired blood samples from 157 HIV-1-seropositive patients. Patients on antiretroviral treatment provided 140 of the CSF samples and 131 of the blood samples. CSF samples that were positive by culture numbered 137 of 303 (45%), as compared with 216 of 278 (78%) blood samples. In the case of samples taken from patients with antiretroviral treatment, 28% were positive by culture from CSF and 63% from blood. As expected, mean HIV-1 RNA levels were higher in CSF and blood samples positive by culture than in samples negative by culture. A cutoff level of >5,000 HIV-1 RNA copies/ml was required to yield a positive predictive value for positive virus isolation from CSF samples of > or =90%, whereas the cutoff level for blood samples was just above the detection limit of the assay (>200 HIV-1 copies/ml).     

Evaluation of the Abbott Real-Time HIV-1 quantitative assay with dried blood spot specimens

The Abbott Real-Time HIV-1 assay was evaluated for its performance in quantification of human immunodeficiency virus type 1 (HIV-1) RNA in dried blood spot (DBS) samples. In total, 169 blood samples with detectable plasma HIV-1 RNA were used to extract RNA from paired DBS and liquid plasma samples, using the automated Abbott m Sample Preparation System (m2000sp). HIV-1 RNA was then quantitated by the m2000rt RealTime analyser. RNA samples suitable for real-time PCR were obtained from all but one (99.4%) of the DBS samples and HIV-1 RNA was detected in 163/168 (97.0%) samples. The correlation between HIV-1 RNA values measured in paired DBS and plasma samples was very high ( r  = 0.882), with 78.5% and 99.4% of cases differing by <0.5 and 1.0 log, respectively. Retesting of DBS replicates following 6 months of storage at 2–8°C showed no loss of HIV-1 RNA in a subset of 89 samples. The feasibility of DBS testing coupled with automated sample processing, and the use of a latest-generation FDA-approved real-time PCR-based system, represents an encouraging first step for viral load measurement in reference centres in developing countries where access to antiretroviral therapy is expanding.     

Antibody specificity for human immunodeficiency virus type 1 in serum and cerebrospinal fluid from patients with AIDS and AIDS-related complex

Since little information has been reported about the specificity of antibodies to human immunodeficiency virus type 1 (HIV-1) found in the cerebrospinal fluid (CSF), 21 CSF and serum specimens were examined from 19 patients with clinical AIDS, AIDS-related complex, or asymptomatic HIV-1 infection. The predominant specificity of antibodies using Western blot analysis in both serum (100 %) and CSF (100 %) was directed towardenv gene products. The next most common antibody specificities were to thepol gene products (serum 95 %; CSF 62 %). Less commonly found was antibody to thegag-encoded proteins (serum 71 %; CSF 38 %). The level of antibody to HIV-1 in CSF could not be predicted from the level found in serum. Also, the spectrum of antibodies seen did not correlate with disease stage or with the quantity of antibody present. The serum/CSF pairs were also examined for the presence of HIV-1 antigen by commercial enzyme immunoassay. HIV-1 antigen was present in eight of 19 (43 %) of the serum samples and five of 20 (25 %) of the CSF samples tested.     

Biological characterization of paired human immunodeficiency virus type 1 isolates from blood and cerebrospinal fluid

Virus has been isolated from the blood and cerebrospinal fluid (CSF) of eight subjects with varying severity of human immunodeficiency virus type 1 (HIV-1) infection and from the frontal lobe of one patient with AIDS. The five patients with AIDS-related complex (ARC) and AIDS also showed neurological/psychiatric complications. With the exception of one isolate from the CSF of an asymptomatic carrier, all isolates replicated in peripheral blood mononuclear cells and monocytes after cell-free transmission. Isolates obtained from the blood of patients in late stages of HIV infection replicated in 3 (of 4) cases in H9 cells, whereas none of the blood isolates from patients in the early stages did so. The capacity of CSF isolates to replicate in H9 cells was low (only 2 of 12). Paired virus isolates from blood and CSF of the same patient could be distinguished by their replicative capacity in different cell lines, type of cytopathic effect, and protein profile as tested by radioimmunoprecipitation. The results indicate that variant viruses with distinct biological characteristics may be isolated from the blood and CSF of the same patient.     

Detection of human immunodeficiency virus type 1 (HIV-1) in heel prick blood on filter paper from children born to HIV-1-seropositive mothers.

The human immunodeficiency virus type 1 (HIV-1) DNA PCR results of 94 dried blood spot (DBS) samples on filter paper and corresponding venous blood in EDTA obtained from infants born to HIV-1-seropositive mothers were compared. In addition, the results of HIV-1 DNA PCR on DBS and the HIV-1 RNA PCR from plasma of 70 paired samples were compared. A 100% specificity and a 95% sensitivity for HIV-1 DNA PCR on DBS compared with results for venous blood were observed for the 94 paired samples. The results of the DBS HIV-1 DNA PCR and HIV-1 RNA PCR of 70 corresponding plasma samples correlated perfectly (100%). The DBS HIV-1 DNA PCR method proved reliable for HIV-1 detection.     

Postmortem recovery of human immunodeficiency virus type 1 from plasma and mononuclear cells. Implications for occupational exposure.

OBJECTIVE--To determine the ability to recover human immunodeficiency virus type 1 (HIV-1) from the plasma and mononuclear cell (MNC) fractions of postmortem blood samples from patients with the acquired immunodeficiency syndrome. DESIGN--Blood was randomly cultured post mortem from 41 patients with the acquired immunodeficiency syndrome. Plasma and MNC cultures were performed as well as serum antigen assays. Evaluation parameters included MNC recovery, MNC viability, time of sample collection after death, time of inoculation of coculture following sample acquisition, and storage conditions of the body (ie, refrigeration vs nonrefrigeration). SETTING--Blood samples were obtained from patients with the acquired immunodeficiency syndrome being prepared for burial at metropolitan area funeral homes. PATIENTS--Postmortem samples were obtained from 41 patients with the acquired immunodeficiency syndrome. MAIN OUTCOME MEASURE--Virus recovery from either cells or plasma. RESULTS--Human immunodeficiency virus type 1 was recovered from 21 (51%) of 41 patients at 0.5 to 21.25 hours postmortem. Recovery of HIV-1 from plasma and/or MNC fractions was variable with 6 (15%) of 41 plasma+/MNC+, 12 (29%) of 41 plasma-/MNC+, three (7%) of 41 plasma+/MNC-, and 20 (49%) of 41 plasma-/MNC-. Plasma p24 levels (> 30 pg/mL) were detectable in 14 (48%) of 37 samples tested. Of those culture-positive patients, seven (33%) of 21 were refrigerated compared with the culture-negative group in which 10 (50%) of 20 were refrigerated. Time from death until specimen acquisition was the only factor significantly associated with recovery of HIV-1. CONCLUSION--These results should be useful for health care workers and others exposed to postmortem blood from HIV-infected individuals and should lead to changes in the processing practices of morticians and/or pathologists for HIV-1-infected cadavers.     

Cerebrospinal fluid interleukin-6 activity in HIV infection and inflammatory and noninflammatory diseases of the nervous system

Interleukin-6 (IL-6) activity was measured in the cerebrospinal fluid (CSF) of patients at different stages of human immunodeficiency (HIV) virus infection and of patients with multiple sclerosis (MS) or other inflammatory (OID) and noninflammatory neurological diseases (OND). In the advanced stages of HIV infection and in OID, IL-6 was detected more frequently (80 and 75% of the cases) and at higher concentrations than in the early stages of HIV infection. MS and OND (44, 48, and 44% of cases). Analysis of CSF and paired sera indicated that IL-6 production can be compartmentalized to either of the fluids. Evidence that altered blood-brain barrier functions can, at least in part, influence the CSF IL-6 levels was found in OID patients. No association was evident between intrathecal immunoglobulin synthesis and CSF IL-6 levels. Interleukin-1 (IL-1) levels were detectable in a minority of the samples from neurological patients; one OID patient had high levels of both CSF IL-1 and IL-6.     

Comparative evaluation of NASBA HIV-1 RNA QT, AMPLICOR-HIV monitor, and QUANTIPLEX HIV RNA assay, three methods for quantification of human immunodeficiency virus type 1 RNA in plasma.

Three commercial assays for quantifying plasma human immunodeficiency virus type 1 (HIV-1) RNA were evaluated. The assays differed in their sample volumes, the means of preparing samples, and methods of amplification and detection. Plasma samples were obtained from 36 HIV-1-infected patients representing all stages of HIV-1 infection and were analyzed as coded specimens. Measurement of HIV-1 RNA baseline levels revealed no significant difference in sensitivity between the three assays. The assays were also applied to the quantitation of HIV-1 RNA levels in the plasma of patients who were changing their antiretroviral therapy. The changes measured in HIV-1 RNA levels in plasma in response to therapy were comparable by the three assays. No close correlation was found between the amount of HIV-1 RNA and the CD4 T-cell count; HIV-1 RNA assays were more sensitive than p24 antigen assays as an indicator of plasma viremia. Overall, the study demonstrates that all three quantitative assays for HIV-1 RNA can be used to measure the HIV-1 RNA copy number representing the HIV-1 viremia status in patients with HIV-1 infection. Since this copy number is likely to be useful in monitoring the effectiveness of antiviral therapy, these quantitative assays for HIV-1 RNA are ready to be built into clinical trials.     

Evaluation of an atypical HIV type 1 antibody. Serologic pattern leading to detection of HIV type 2 infection in North America

The variability and discordance of human immunodeficiency virus type 1 (HIV-1) antibody enzyme immunoassay determinations on serial specimens derived, to our knowledge, from the first documented case of HIV-2 infection in North America are described. The initial specimen was weakly reactive, but two subsequent serum specimens were both nonreactive by enzyme immunoassay. All specimens were indeterminate for HIV-1 antibody by HIV-1 Western blot analysis. Serum HIV-2 antibody was demonstrated by enzyme immunoassay using whole virus lysate, HIV-2-specific synthetic peptide assays, and HIV-2 Western blot analysis. Human immunodeficiency virus type 2 genomic sequences were demonstrated in peripheral blood mononuclear cells using gene amplification technology. Human immunodeficiency virus type 2, isolated from peripheral blood lymphocytes, had typical morphologic features of lenti-virus by electron microscopy. Western blot analysis and other specific assays should be considered in individuals with clinical evidence suggesting HIV infection who are nonreactive for HIV-1 antibody by enzyme immunoassay or who have atypical reactivity patterns.     

Application of Dried Blood Spot Specimens for Serologic Subtyping of Human Immunodeficiency Virus Type 1 in Thailand

Dried blood spot (DBS) specimens were assessed as an alternative to plasma for human immunodeficiency virus type 1 (HIV-1) serotyping by V3 loop peptide enzyme immunoassay. Nested PCR capable of distinguishing HIV-1 subtypes B and E was used as the reference standard. Ninety-two percent of DBS samples were typeable as either HIV-1 subtype B or E. Serotype results with DBS and plasma were identical for 254 of 257 specimens. A simple DBS collection method provides a convenient alternative for conducting HIV-1 serotype surveillance while retaining sensitivity and specificity.     

Transmission of retroviruses by transfusion of screened blood in patients undergoing cardiac surgery

We determined the rates of seroconversion to human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus Type I (HTLV-I) in a cohort of patients receiving transfusions of blood components screened for antibody to HIV-1. Preoperative and postoperative serum samples were collected from 4163 adults undergoing cardiac surgery who received 36,282 transfusions of blood components. The postoperative samples from all patients were tested for serologic evidence of HIV-1 infection, and those that were positive were compared with the corresponding preoperative samples. One case of HIV-1 transmission by transfusion of screened blood components was identified; two preexisting HIV-1 infections were found. Samples from 2749 patients were tested similarly for serologic evidence of HTLV-I infection; these patients received 20,963 units of blood components. Five new cases and two preexisting cases of HTLV-I infection were detected. The observed risk of HIV-1 transmission by transfusion was 0.003 percent per unit; the risk of HTLV-I transmission was 0.024 percent per unit. We conclude that there is a very small risk of HTLV-I infection from transfused blood products that have been screened for antibodies to HIV-1, but that it is nearly 10-fold higher than the risk of HIV-1 infection.     

Urinary detection of toscana virus nucleic acids in neuroinvasive infections

BackgroundToscana virus (TOSV) is a sandfly-borne pathogen causing febrile diseases and neuroinvasive infections in humans. Definitive diagnosis of TOSV infections frequently requires the detection of viral RNA in cerebrospinal fluid (CSF) or in circulation, which can be achieved prior to seroconversion.ObjectivesTo evaluate TOSV excretion in urine and impact of urine as a diagnostic specimen.Study designA total of 82 plasma, CSF and urine samples were collected from 24 individuals with a preliminary diagnosis of atypical viral encephalitis, where frequent bacterial fungal and viral causes were ruled out. Phlebovirus and WNV nucleic acids were investigated via real-time and nested polymerase chain reaction (PCR) assays. Commercial immunofluorescence assays were employed for viral IgM detection. Amplicons were characterized via cloning and sequencing.ResultsPhlebovirus PCR yielded positive results in 7 out of 14 samples that comprise 4 plasma and 3 urine specimens from 3 individuals. Amplicons were characterized as TOSV genotype A. Investigation of the follow-up samples suggested that virus shedding in urine coincides or follows viremia. Despite conserved sequences observed in paired or sequential plasma-urine specimens, L693S substitution in the viral polymerase was characterized in a urine sample.ConclusionsThese preliminary findings indicate that urine can be employed as a additional clinical sample for TOSV RNA detection in suspected cases, especially in individuals where specimens for viral diagnostics during the early stages of the infection are not available.     

HIV-1 seroprevalence in an inner-city public hospital.

In a hospital-based seroprevalence survey for human immunodeficiency virus type 1 (HIV-1) infection, a stratified sampling method based on age and gender was used to collect 5429 blood samples at an inner-city hospital. Sentinel Hospital Surveillance System (SHSS) criteria developed by the Centers for Disease Control and Prevention were used to classify patient diagnoses into two categories by the likelihood of being associated with HIV-1 infection. The two categories were those with high likelihood of association with HIV-1 (SHSS-ineligible) and those with low likelihood of association with HIV-1 infection (SHSS-eligible). Of the 5429 blood samples, 4262 were SHSS-eligible and 1167 were SHSS-ineligible. After personal identifies were removed, specimens were tested by ELISA and confirmed by Western blot analysis. The overall prevalence rate of HIV-1 infection was 0.98%. The seroprevalence rate was almost 2.6 times higher in high-association patients compared with low-association patients (1.89% versus 0.73%, P < .001). Results from this study indicate a high unsuspected HIV-1 seroprevalence rate in a subpopulation (SHSS-eligible) considered to have diagnoses with low likelihood of association with HIV-1 infection. These patients may better approximate HIV-1 seroprevalence in the general population of the area served by the hospital than would a sample of all patients. Monitoring HIV-1 seroprevalence in the SHSS-eligible group will be a useful measure for community serosurveillance for HIV-1 infection.