Levels of HIV-infected peripheral blood cells remain stable throughout the natural history of HIV-1 infection. Swiss HIV Cohort Study.

OBJECTIVE: To clarify the relationship between the number of provirus-bearing peripheral blood mononuclear cells (PBMC) and HIV-1 disease progression during the natural history of infection. DESIGN: Twenty-four HIV-1-infected subjects with known seroconversion dates and long-term follow-up were retrospectively identified using the Swiss HIV Cohort Database. PBMC specimens from this cohort were retrieved from storage for analysis. METHODS: Infected PBMC equivalents were determined by HIV-1 DNA quantitative competitive (QC)-PCR. The results were analysed with respect to HIV-1 disease stage and compared with a mathematical model of long-term HIV-1 disease progression. RESULTS: PBMC HIV-1 DNA did not correlate with major indices of disease progression, including time following primary infection, time before reaching a CD4 cell count less than 200 x 10(6)/l, and time before death. The number of PBMC harbouring HIV-1 provirus was relatively constant throughout the clinical stages of HIV-1 infection, consistent with simulated data from a mathematical model of long-term HIV-1 infection. We also showed that a biased interpretation of the QC-PCR data may arise when the values are expressed as HIV-1 DNA copies per PBMC or per CD4 cell. CONCLUSIONS: This analysis suggests that levels of provirus-bearing PBMC remain constant during the natural course of HIV-1 infection, whereas plasma virus load typically increases logarithmically during the same period. The hypothesis that plasma virus levels are directly related to the number of infected cells may deserve reconsideration.

Impaired liver function and retroviral activity are risk factors contributing to HIV-associated thrombocytopenia. Swiss HIV Cohort Study.

OBJECTIVE: To investigate the relationship between thrombopoetin (TPO) serum levels and HIV-associated thrombocytopenia. DESIGN AND METHODS: The relationship between TPO levels and severity of HIV-associated thrombocytopenia was investigated. Thirty-eight patients (19 patients with 30-96x10(9) platelets/l and 19 patients with <10x10(9) platelets/l) were matched with 38 HIV-positive non-thrombocytopenic patients (>150x10(9) platelets/l). RESULTS: HIV-positive patients with normal platelet counts had a median TPO serum level of 137 pg/ml. Patients with 30-96x10(9) platelets/l had decreased TPO levels with a median of 90 pg/ml (P = 0.016), and were more likely to have elevated serum aspartate-transferase levels (P<0.001) and hepatomegaly by palpation or ultrasound imaging (P = 0.005). The median TPO serum level of HIV-infected patients with severe thrombocytopenia was 110 pg/ml (non-significant). All patients with severe thrombocytopenia were positive for antibodies against hepatitis B virus core antigen, compared with 80% of HIV-infected persons without thrombocytopenia. Patients with severe thrombocytopenia were more likely to have high HIV replication compared to patients with normal platelet counts (P = 0.02), and reduction of plasma HIV-1 RNA levels was associated with increasing platelet counts. Severe thrombocytopenia was not associated with liver disease. CONCLUSIONS: Liver disease predisposes for low TPO serum levels and mild thrombocytopenia. High retroviral activity predisposes for severe, immune thrombocytopenic purpura-like thrombocytopenia. At least two distinct categories of severe HIV-associated thrombocytopenia exist, one responsive to antiretroviral treatment and one non-responsive to antiretroviral treatment.

Impact of TNFalpha, LTalpha, Fc gammaRII and complement receptor on HIV-1 trapping in lymphoid tissue from HIV-infected patients.

OBJECTIVES: To investigate HIV trapping mechanisms in patients with acute infection and in asymptomatic individuals prior to and during antiretroviral therapy. To determine the role of complement receptor (CR), Fc gamma receptor II (Fc gammaRII), tumour necrosis factor alpha (TNFalpha), and lymphotoxin alpha (LTalpha) expression in HIV trapping efficiency. METHODS: Lymphoid tissues from three acutely HIV-infected patients and six asymptomatic, chronically HIV-infected patients collected prior to and during antiretroviral therapy were compared with lymphoid tissues from six HIV-seronegative subjects. HIV, TNFalpha and LTalpha RNA expression was detected and quantified by fluorescence in situ hybridization. CR, Fc gammaRII and HIV p24 antigen were detected and quantified by fluorescence immunohistochemistry. RESULTS: The amount of trapped HIV did not differ significantly between patients with acute HIV infection and asymptomatic individuals, and was independent of the presence of CR or Fc gammaRII expression. However, in patients with acute infection, the amount of trapped virus was correlated inversely with the number of HIV-infected cells (P = 0.0092) and with the size of the light zone (P = 0.037). In these patients, the number of TNFalpha-expressing cells was correlated inversely with the amount of trapped virus (P = 0.014) and positively correlated with the size of the light zone in germinal centers (P = 0.041). No correlations were observed between TNFalpha or LTalpha expression and Fc gammaRII or CR expression. CONCLUSION: This report provides the first evidence that in humans TNFalpha is involved in the development of lymphoid follicles, HIV trapping, and, consequently, in early host immune responses. A model is proposed for early events in patients during acute HIV infection.

Highly active antiretroviral therapy during early HIV infection reverses T-cell activation and maturation abnormalities. Swiss HIV Cohort Study.

OBJECTIVES: To evaluate the impact of early initiation of highly active antiretroviral therapy (HAART) on disease-induced T-cell activation and maturation abnormalities during asymptomatic HIV infection. DESIGN: A prospective open-label trial of zidovudine, lamivudine and ritonavir in treatment-naive asymptomatic HIV-infected individuals with CD4 cells > or = 400 x 10(6)/l. METHODS: Peripheral blood CD4+ and CD8+ T cells derived from 15 asymptomatic HIV-infected individuals (median baseline CD4+ cells, 608 x 10(6)/l; CD8+ cells, 894 x 10(6)/l; plasma HIV RNA, 3.93 log10 copies/ml) undergoing therapy with zidovudine (300 mg twice daily), lamivudine (150 mg twice daily), and ritonavir (600 mg twice daily) were assessed for changes in expression of phenotypic markers of T-cell activation (HLA-DR and CD38) and maturation (CD45RA and CD45RO). At weeks 0, 2, 4, 8, 12, 16, 20 and 24, T-cell subsets were quantified by flow cytometry and plasma HIV viral loads determined using reverse transcription PCR. RESULTS: HAART-induced decrease in plasma HIV RNA levels coincided with a significant reduction in numbers of activated CD4+/HLA-DR+ (maximum change, -36%; P < or = 0.05), CD8+/HLA-DR+ (maximum change, -66%; P < or = 0.005) and CD8+/CD38+ (maximum change, -51%; P < or = 0.01) T cells. A concomitant significant increase in numbers of naive CD4+/CD45RA+ (maximum change, +12%; P < or = 0.005) and memory CD4+/CD45RO+ (maximum change, +6%; P < or = 0.05) T cells was also evident, which contrasted with a significant decrease in memory CD8+/CD45RO+ cells (maximum change, -42%; P < or = 0.005). CONCLUSION: The observed ability of HAART during early asymptomatic HIV infection to initiate rapid reversal of disease-induced T-cell activation and maturation abnormalities, while preserving pretherapy levels of immune function, supports the concept that therapeutic advantage is to be gained by commencing early aggressive antiretroviral therapy.

Potent antiretroviral treatment of HIV-infection results in suppression of the seminal shedding of HIV. The Swiss HIV Cohort Study.

OBJECTIVE: The amount of HIV in semen likely influences infectiousness. Antiretroviral therapy decreases HIV-RNA in semen, but data on HIV concentrations in semen in a large cohort of men with suppressed HIV-RNA in blood is unavailable. METHODS: Male patients with a treatment-induced reduction of HIV-RNA load in plasma below 400 copies/ml were asked to donate a semen and blood sample. Blood and seminal plasma were tested for the presence of HIV-RNA by the NucliSens method (detection limit 400 copies/ml). Seminal cell samples from 67 patients were further analysed for the presence of HIV-DNA using a nested DNA-polymerase chain reaction. Results of RNA and DNA testing in semen were compared with 55 HIV-positive antiretroviral therapy-naive men. RESULTS: A total of 114 patients participated in the study. Seminal plasma HIV-RNA was detectable in only two patients [1.8%, 95% confidence ratio (CI), 0-4.2%] compared with a detection frequency of 67% in untreated controls [Odds ratio (OR), 0.01; 95% CI, 0-0.03]. Detection of cell-associated HIV-DNA in semen was significantly less frequent (16 versus 38%) in patients receiving suppressive therapy compared with untreated controls (OR, 0.32; 95% CI, 0.12-0.80). CONCLUSION: In patients with treatment-induced suppression of blood viral load the likelihood of having detectable HIV in semen is very low (< 4%). In addition, seminal shedding of cell-free and cell-associated HIV is significantly lower than in an untreated population of HIV-infected asymptomatic men. On a population basis, this effect of therapy may help to reduce sexual transmission of HIV. However, individual patients may still be infected as evidenced by continued shedding of cells harbouring the HIV provirus.

Time of initiation of antiretroviral therapy: impact on HIV-1 viraemia. The Swiss HIV Cohort Study.

OBJECTIVE: The current recommendation that patients infected with HIV-1 be treated early is based on little evidence. We examined whether the early initiation of antiretroviral treatment affects residual HIV-1 viraemia. METHODS: Viraemia was measured using an assay with a detection limit of 3 HIV-1 RNA copies/ml in drug-naive patients who started antiretroviral therapy at the time of primary HIV-1 infection (PHI) (n = 10), during chronic infection without immune suppression (CD4 cell counts > or = 500/mm3; median 577) (n = 10), or after immune suppression developed (CD4 cell counts < 500/mm3; median 113) (n = 21). RESULTS: In 249 samples collected 24 to 120 weeks after treatment initiation, the mean proportion of samples with HIV-1 RNA levels of less than 3 copies/ml was 75% for PHI patients compared with 32 and 8% for immunocompetent and immunosuppressed chronically infected patients, respectively. Fifty per cent of PHI patients, but none of the chronically infected patients, had persistently fewer than 3 HIV-1 RNA copies/mL. PHI patients had lower residual HIV-1 RNA levels than chronically infected patients, and immunocompetent patients had lower residual HIV-1 RNA levels than immunosuppressed patients (all pairwise, P< 0.001). The mean residual HIV-1 RNA level was independently associated with the initiation of therapy during PHI and baseline CD4 cell counts (P < 0.001 for both associations). CONCLUSION: Viraemia levels are associated with clinical progression and predict virological treatment failure. The initiation of antiretroviral therapy at the time of PHI and while CD4 cell counts are high results in lower residual viraemia. These results support early antiretroviral therapy in HIV-1-infected patients.

Improved in situ hybridization to HIV with RNA probes derived from PCR products.

These experiments tested the hypothesis that a pool of PCR-derived RNA probes with defined length and even representation of the target sequences could produce more specific and intense in situ hybridization signals than randomly size-reduced, plasmid-derived RNA probes. In situ hybridization was performed with sense and anti-sense HIV-1 RNA probes that were derived from PCR products tailed with the T7 RNA polymerase promoter or from plasmid DNA. In situ hybridization using a pool of seven anti-sense or sense PCR-derived RNA probes (1805 nucleotides of HIV sequence, 257 nucleotides average probe length) was compared with hybridization using anti-sense or sense RNA probes made from a plasmid representing the HIV-1 env gene (3151 nucleotides of HIV-1 target). The pooled PCR-derived probes resulted in stronger in situ hybridization signals and less background than those produced with plasmid-derived RNA probes. This method for creating PCR-derived RNA probes improves the feasibility of synthesizing multiple, discrete RNA probes for studies of specific mRNA expression because it does not require the subcloning steps used to construct plasmids. PCR-derived RNA probes may provide a viable alternative to the use of plasmid-derived RNA probes for in situ hybridization.

PCR-derived ssDNA probes for fluorescent in situ hybridization to HIV-1 RNA.

We developed a simple and rapid technique to synthesize single-stranded DNA (ssDNA) probes for fluorescent in situ hybridization (ISH) to human immunodeficiency virus 1 (HIV-1) RNA. The target HIV-1 regions were amplified by the polymerase chain reaction (PCR) and were simultaneously labeled with dUTP. This product served as template for an optimized asymmetric PCR (one-primer PCR) that incorporated digoxigenin (dig)-labeled dUTP. The input DNA was subsequently digested by uracil DNA glycosylase, leaving intact, single-stranded, digoxigenin-labeled DNA probe. A cocktail of ssDNA probes representing 55% of the HIV-1 genome was hybridized to HIV-1-infected 8E5 T-cells and uninfected H9 T-cells. For comparison, parallel hybridizations were done with a plasmid-derived RNA probe mix covering 85% of the genome and a PCR-derived RNA probe mix covering 63% of the genome. All three probe types produced bright signals, but the best signal-to-noise ratios and the highest sensitivities were obtained with the ssDNA probe. In addition, the ssDNA probe syntheses generated large amounts of probe (0.5 to 1 microg ssDNA probe per synthesis) and were easier to perform than the RNA probe syntheses. These results suggest that ssDNA probes may be preferable to RNA probes for fluorescent ISH. (J Histochem Cytochem 48:285-293, 2000)

Residual HIV-RNA levels persist for up to 2.5 years in peripheral blood mononuclear cells of patients on potent antiretroviral therapy.

The long-term response of 10 asymptomatic, antiretroviral therapy-naive HIV-1-infected patients to potent combination antiretroviral therapy was characterized by monitoring levels of HIV-1 RNA in plasma, peripheral blood mononuclear cells (PBMC), and lymphoid tissue using highly sensitive HIV-1 RNA assays. Although plasma viral loads were continuously suppressed to levels below 50 HIV-1 RNA copies/ml for up to 2.5 years (60-128 weeks), HIV-1 RNA was still detectable at very low levels (1 to 49 HIV-1 RNA copies/ml) in 25% of the samples. In corresponding PBMC specimens, residual HIV-RNA was detectable in as much as 91% of samples tested (1 to 420 HIV-1 RNA copies/microg total RNA). Similarly, HIV-1 RNA levels in lymphoid tissue also remained detectable at a high frequency (86%). A highly significant correlation was demonstrated between therapy-induced change in PBMC HIV-1 RNA levels and change in plasma HIV-1 RNA levels (r2 = 0.69; p = 0.003). These findings support the concept that measurement of HIV-1 RNA in the easily accessible PBMC compartment is relevant for evaluating the potency of current and future antiretroviral therapies.

Lack of detection of human immunodeficiency virus type 1 in the saliva of infected children and adolescents.

OBJECTIVE: To determine the prevalence of human immunodeficiency virus type 1 (HIV-1) in the saliva of infected children and adolescents. METHODS: Saliva and blood samples were collected from 13 patients (age range, 1-15 years) with HIV-1 infection. Eleven were taking antiretroviral agents. The presence of HIV-1 was determined by polymerase chain reaction analysis of RNA and DNA as well as by viral culture of the saliva samples and by culture of peripheral blood mononuclear cells. RESULTS: Although HIV-1 was cultured from peripheral blood mononuclear cells of 12 patients, it was not cultured from their saliva. Only 1 of 13 saliva samples yielded positive test results for HIV-1 RNA, and none did so for HIV-1 DNA. The specimen containing HIV-1 RNA was from an untreated 10-year-old asymptomatic boy with a CD4+ lymphocyte count of 0.91 x 10(9)/L (913 cells/microL) and no infectious virus detected in plasma. CONCLUSION: The prevalence of HIV-1 in the saliva of HIV-1-infected children and adolescents is low and may not be infectious.

Human herpesvirus 6 and pneumonia.

Studies of lung tissues and bronchoalveolar lavage specimens from patients with pneumonia have led some investigators to propose that human herpesvirus 6 (HHV-6) can cause pneumonia. As the etiologic role of HHV-6 in pneumonia remains poorly defined, cases of pneumonia that are associated with pulmonary HHV-6 infection are best described as HHV-6-associated pneumonias. The clinical spectrum of HHV-6-associated pneumonia encompasses both mild and severe cases. Most cases reported so far occurred in immunosuppressed individuals following bone marrow transplantation or HIV infection. Some studies have identified coinfections with HHV-6 and other viruses in a surprising number of HHV-6-associated pneumonias. Although no systematic evaluation of treatment regimens is available at this time, coincidental administration of antiviral drugs did not generally result in clinical improvement. Controlled, prospective studies are needed to understand the clinical presentation, clinical course, etiology, diagnosis and treatment of HHV-6-associated pneumonias.

Reanalysis of unruptured intracranial aneurysm management: effect of a new international study on the threshold probabilities.

OBJECTIVE: This report updates previous clinical decision analysis for patients with unruptured intracranial aneurysm (UN-AN) based on newly published data and discusses the role of reanalysis in individual decision making. METHODS: The authors employed probabilities for the natural history of UN-AN and results of preventive surgery based on the report by the International Study of Unruptured Intracranial Aneurysms. Probabilistic sensitivity analysis with Monte Carlo simulation and traditional n-way sensitivity analyses were used to assess the uncertainty of clinical decisions. RESULTS: The baseline decision in favor of preventive surgery is reversed by new data from the international study. Probabilistic sensitivity analyses revealed several populations showing heterogeneity in terms of strategy selection. One- and two-way sensitivity analyses detected two important factors for decision making: annual rupture rate and utility for knowingly living with UN-AN. CONCLUSIONS: Annual UN-AN rupture rate and the utility for knowingly living with UN-AN are key factors when deciding on a therapeutic strategy. Also, updating published decision analyses can improve clinical decision making by integrating clinical judgment and newly available clinical data.

Human herpesvirus 6 as a possible cause of pneumonia.

Case reports and limited retrospective studies have established that human herpesvirus 6 (HHV-6) can be found in lung tissues from patients with pneumonia. Most cases of HHV-6-associated pneumonia involve immunocompromised patients, especially those who have received a bone marrow transplant or who have been infected by the human immunodeficiency virus. Additional data suggest that HHV-6 commonly reactivates in immunosuppressed individuals. The virus has been identified in a variety of organ systems, including the respiratory tract, without associated pathology. A better understanding of HHV-6 pathogenesis is needed before rational clinical intervention for HHV-6-associated pneumonias can be implemented.

Coamplified positive control detects inhibition of polymerase chain reactions.

Polymerase chain reactions (PCR) may fail to react because the substrate DNA is absent (true negative) or because of inhibition of specific amplification (false negative). The use of positive controls can increase confidence in negative PCR results by ruling out failure due to inhibition as a cause of the lack of amplification products. This report describes the construction and application of coamplified positive controls for herpes simplex virus and human herpesvirus 6 amplifications. Herpes simplex virus and human herpesvirus 6 PCR products were modified to generate control PCR products in which the original probe sequences were replaced by a Drosophila probe sequence. Implementation of the coamplified controls increased our specimen throughput in comparison with the parallel control amplifications used previously. Clinical laboratories using PCR for diagnosis of infectious diseases may find positive controls particularly helpful for increasing confidence that negative amplifications represent truly negative specimens.

Extended duration of herpes simplex virus DNA in genital lesions detected by the polymerase chain reaction.

To evaluate the utility of the polymerase chain reaction (PCR) for documenting herpes simplex virus (HSV) in persons with reactivated genital lesions viral isolation was compared with a recently developed PCR method. Three women experiencing four episodes of recurrent genital herpes were followed for 10 days per episode with daily examination and duplicate swabs of the lesions, one for HSV culture and one for PCR. HSV type 2 was cultured from three of four episodes and the mean duration of viral isolation from recurrent genital lesions was 2.6 days. PCR detected HSV DNA from lesion swabs during all four episodes, and HSV DNA was positive for an average of 6.8 days. HSV DNA was demonstrated in ulcerative lesions on 15 of 17 days versus 3 of 17 days by viral isolation (P less than .01). HSV PCR became negative when the lesions reepithelialized. These data suggest that PCR is a more sensitive measure of HSV infection than routine viral culture and that PCR detects the presence of HSV at times when culture is negative.

Conservation of HPV-16 E6/E7 ORF sequences in a cervical carcinoma.

A cervical carcinoma that contained human papillomavirus (HPV)-16 homologous DNA was analyzed. Each tumor cell genome contained a single, incomplete copy of HPV-16 DNA. The E6 and E7 open reading frames (ORFs) were completely conserved relative to other published HPV-16 sequences. Much of the non-coding region (NCR) was free of base changes, including complete conservation of several regulatory elements. Multiple mutations were identified in the remaining integrated HPV-16 DNA, which was composed of parts of the L1 and E1 ORFs. The extraordinary conservation of the E6/E7 DNA sequence, as compared with other regions of the integrated HPV-16 DNA, supports the role of E6/E7 in tumorigenesis.

Human herpesvirus 6 infections after bone marrow transplantation: clinical and virologic manifestations.

Human herpesvirus 6 (HHV-6) DNA levels in peripheral blood mononuclear cells were prospectively evaluated in 20 cytomegalovirus-seronegative allogeneic marrow transplant patients and in 10 healthy control subjects. Blood and saliva specimens obtained weekly for 3 months after transplant were evaluated by quantitative HHV-6 polymerase chain reaction. One of 20 patients experienced primary HHV-6 infection after marrow transplant (seroconversion, HHV-6 viremia, skin rash); 18 of 20 had increased peripheral blood mononuclear cell HHV-6 DNA levels consistent with asymptomatic reactivations, and 1 patient experienced a reactivation-associated skin rash. Genotyping revealed HHV-6 variant B DNA in all cases. Therapy with acyclovir or intravenous immunoglobulin was not correlated with lower HHV-6 DNA levels. Thus, asymptomatic HHV-6 reactivations appear to be common following allogeneic marrow transplantation. Among HHV-6-seronegative and viral DNA-negative patients, primary HHV-6 infection can ensue in association with self-limited clinical symptoms, including diffuse maculopapular rash.

Incomplete HIV-1 activation in latently infected U1 cells demonstrated by double in situ hybridization.

Triple combination antiretroviral therapy can reduce HIV-1 infection to a relatively small pool of latently infected cells. To eliminate this residual source of virus, new therapies designed to activate latently infected cells are currently being tested. We therefore investigated the kinetics of in vitro HIV-1 RNA induction using chronically infected U1 cells. A new two-probe fluorescence in situ hybridization (double ISH) method was devised to simultaneously assess total HIV-1 RNA (T-RNA) and unspliced HIV-1 RNA (U-RNA) expression in individual cells. Activation of the U1 cells resulted in increasing expression of T-RNA between 0 and 24 h with lagging expression of U-RNA between 6 and 30 h. Both the positive area per cell and the number of positive cells increased with time. Although activation induced 98.5% of the cells to express HIV-1 T-RNA by 24 h, 52% remained negative for U-RNA. In contrast, 100% of 8E5 cells, which constitutively express HIV-1, scored positive for U-RNA as well as T-RNA with the double ISH. This study provides, for the first time, a semiquantitative cell-by-cell analysis of HIV-1 mRNA subsets in latently infected cells. Our results establish the advantages of using double fluorescence ISH to study gene expression and demonstrate that chronically infected U1 cells remain in a partially induced state despite potent activation.