Serum IgD and IgE concentrations in immunodeficiency diseases.

Concentrations of IgD and IgE were measured in sera from 165 patients with well-defined immunodeficiency in an effort to find information possibly relevant to the roles of antibodies of these classes in host defense. Values for both immunoglobulins were generally quite low in patients who had marked deficiencies of all three major immunoglobulins, although occasional normal or high normal values for IgD were seen in hypogammaglobulinemic patients. Group mean IgD concentrations were also depressed in patients with Wiskott-Aldrich syndrome and in those with selective IgA deficiency; IgE concentrations were depressed in patients with X-linked immunodeficiency with hyper-IgM and in those with ataxia telangiectasia. IgD and IgE were both significantly elevated in patients with extreme hyperimmunoglobulinemia E and undue susceptibility to infection and in a patient with the Nezelof syndrome; none of these patients had histories suggestive of atopy. In addition, the mean IgE concentration was significantly elevated in patients with selective IgA deficiency, many of whom were atopic, and in those with the Wiskott-Aldrich syndrome. The highest IgD concentration (163 mg/100 ml) was found in serum from a boy with variable immunodeficiency who had a lifelong history of severe recurrent pharyngeal infections, primarily streptococcal in etiology. Recurrent staphylococcal infection was a feature common to many but not all patients with elevated serum IgE concentration. These data may prove useful in the future delineation of biologic roles for antibodies in these two immunoglobulin classes.

Genotypic analysis of the protease and reverse transcriptase of HIV type 1 subtype C isolates from antiretroviral drug-naive adults in Malawi.

The protease (PR) and reverse transcriptase (RT) regions of HIV-1 isolates from 21 antiretroviral (ARV)-naive Malawian adults were sequenced and analyzed to determine the prevalence of drug resistance-associated mutations in this population. Phylogenetic analysis confirmed that all isolates grouped with HIV-1 subtype C, the predominant subtype in Malawi. No major mutations associated with resistance to PR inhibitors (PIs), nucleoside RT inhibitors (NRTIs), or nonnucleoside RT inhibitors (NNRTIs) were found. In contrast, accessory mutations were found in the protease region at positions 10, 20, 36, 63, 77, and 93, and in the RT region at positions 118, 211, and 214. Further studies will be needed to determine the clinical impact of these polymorphisms on viral susceptibility to existing antiretroviral drugs.

Quantification of HIV in semen: correlation with antiviral treatment and immune status.

OBJECTIVE: This study examined the concentration of HIV in semen and the effects of biological factors on HIV excretion. METHODS: Semen samples from 101 men at different stages of the disease were evaluated by quantitative HIV culture and HIV RNA detection. Blood plasma samples were available from 56 patients. The effects of CD4 and CD8 count, blood plasma RNA levels, treatment status and clinical staging on the shedding of HIV were evaluated. RESULTS: HIV RNA levels in semen correlated with quantitative HIV culture of seminal cells and a strong association of positive seminal cell culture with high RNA levels was observed. CD4 count and antiviral treatment were inversely correlated with the concentration of HIV in semen. Blood plasma HIV RNA values were correlated with HIV RNA levels in semen, although some patients had highly discrepant results. CONCLUSIONS: The strong correlation between seminal cell culture and concentration of HIV RNA in seminal plasma suggested that HIV detected in seminal plasma was released by productively infected cells in the male genital tract. The study showed that the concentration of HIV in semen, which was likely to be correlated with HIV infectivity, was a function of the immune status of the HIV-infected individual. The results suggested that antiviral therapy may have reduced the concentration of HIV in semen.

Effect of antiviral treatment on the shedding of HIV-1 in semen.

OBJECTIVE: The potential role of antiretroviral treatment on the infectiousness of HIV-1-infected men was examined by studying the effect of antiviral treatment on the shedding of HIV-1 in semen. METHODS: Forty-four patients enrolled in various treatment protocols were asked to donate a semen sample before they began a new antiviral treatment and at a follow-up visit after 6 to 15 weeks of treatment. Since most patients were on blinded protocols, patients were stratified by response of blood viral load. The effect of each patient's treatment was classified as good (n = 24), fair (n = 8) and marginal (n = 13) by measurement of the HIV RNA reduction in blood plasma (> 1.0 log10; 0.5-1.0 log10 and < 0.5 log10 HIV RNA copies/ml reduction, respectively). The effect of treatment on shedding of HIV-1 in semen was documented by the reduction of HIV RNA concentration in seminal plasma and by quantitative HIV-1 seminal cell culture. RESULTS: Overall, antiviral treatment resulted in a significant fall in the viral load in semen (RNA and culture) that paralleled the reduction of viral load in blood. More pronounced reductions of HIV RNA in semen were observed as the effectiveness of treatment on blood HIV RNA levels increased (median drop from baseline 0, 0.3 log10 and 0.8 log10 RNA copies/ml in patients with marginal, fair and good treatment effect, respectively). Thirteen patients lost detectable HIV RNA in blood on treatment and all of these had undetectable levels of HIV-1 in semen by culture and RNA analysis at follow-up. In 19 of the 31 patients (62%) who still had HIV RNA in their blood during treatment, semen HIV levels were below detection in semen at follow-up. CONCLUSIONS: Treatment-induced changes of HIV RNA concentration in blood are generally associated with a corresponding change in seminal HIV RNA: If confirmed in larger studies, potent antiretroviral therapy might reduce the spread of HIV-1.

Resistance of HIV-1 to antiretroviral agents in blood and seminal plasma: implications for transmission.

OBJECTIVES: To evaluate blood and genital secretions from HIV-infected men for HIV-1 resistant to antiretroviral agents. DESIGN: A longitudinal study of 11 men with HIV infection and persistent detectable HIV RNA levels in blood and semen on antiretroviral therapy. METHODS: HIV-1 from the blood and seminal plasma, obtained before the initiation of a new therapeutic regimen and on therapy, were evaluated by population-based sequencing of reverse transcriptase (RT) and protease RNA for the development of resistance to antiretroviral therapy. The genetic relatedness of sequences over time was compared. RESULTS: RT genotypic resistance markers were present in seminal plasma at baseline in three out of six individuals with previous RT inhibitor experience. Eight out of 10 men, from whom the viral sequence was available on new therapy, demonstrated the evolution of new resistance mutations in the blood or seminal plasma, or both. The evolution of resistance mutations in blood and semen were frequently discordant, although over time similar patterns were seen. In two individuals, protease inhibitor resistance mutations evolved in the blood but not in the major variant in seminal plasma. Comparisons of the viral sequences between blood and seminal plasma from six men revealed two patterns. Three men showed a clustering of sequences from blood and semen. Three had sequences that appeared to evolve separately in the two compartments. CONCLUSIONS: HIV-1 variants with genotypic resistance markers are present in the male genital tract and evolve over time on incompletely suppressive antiretroviral therapy. The absence of genotypic changes consistent with protease inhibitor resistance in the semen, despite their presence in blood plasma, suggests the possibility of limited penetration of these agents into the male genital tract. Sexual transmission of resistant variants may have a negative impact on treatment outcome in newly infected individuals and on the spread of the diseases within a population. Therapeutic strategies that fully suppress HIV-1 in the genital tract should be a public health priority.

Detection and biologic characterization of infectious HIV-1 in semen of seropositive men.

OBJECTIVE: Factors that influence the infectivity of an individual and the impact of antiviral treatment on infectivity are not well defined. This study investigated the value of a sensitive method for detecting infectious HIV in semen for use as a marker for infectivity. DESIGN: A cross-sectional study of infectious HIV in the semen of 33 HIV-positive men. METHODS: A sensitive method for detecting infectious HIV in semen was used. The correlation of culture in semen with clinical and laboratory data was investigated. Biological phenotypes of isolates from blood and semen were tested using an MT-2 assay. RESULTS: HIV cultures from seminal cells were positive in 18 patients (55%) and in one patient from seminal plasma. Higher recovery rates of HIV from semen correlated with a low CD4 count (80% in patients with a CD4 count > 100 x 10(6)/l versus 33% in patients with a CD4 count < 100 x 10(6) cells; P < 0.025) and symptomatic disease (78 versus 27% in asymptomatic patients; P < 0.01). Recovery of HIV from semen was independent of presence or absence of plasma viremia and the biological phenotype of blood isolates. Ten patients with syncytium-inducing (SI) isolates in their blood had positive semen cultures for HIV. Seven of the 10 patients had SI isolates recovered from their semen, whereas three had non-SI isolates only. CONCLUSION: Data from partner studies show higher rates of HIV transmission for patients with low CD4 counts and symptomatic disease. The compatibility of epidemiologic data with our finding that significantly more HIV is recovered in semen from patients with advanced disease, suggests that HIV culture of semen samples may provide a useful surrogate marker to measure infectivity in clinical studies. Further studies are needed to define the inoculum required to transmit HIV and to study the impact of sexually transmitted diseases and HIV-1 phenotype on semen infectivity.

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.

Plasma HIV burden in Malawian children co-infected with rotavirus.

Fifty-eight HIV-infected children with acute rotavirus diarrhea were tested for plasma HIV RNA. There was no difference between acute and convalescent mean viral loads, and little change in CD4 cell counts. Compared with the 16 children who died within 4 weeks, 31 survivors had slightly lower viral loads at presentation and significantly higher CD4 cell counts. Low CD4 cell counts, but not HIV-1-RNA concentrations, were predictive of Death. Local, enteric rotavirus infection did not appear to affect blood HIV viral load or CD4 cell counts in this small group of children.

Viral burden in genital secretions determines male-to-female sexual transmission of HIV-1: a probabilistic empiric model.

OBJECTIVE: To develop a model to predict transmission of HIV-1 from men to women. DESIGN: HIV-1 in seminal plasma, and endocervical CCR5 receptors were correlated with epidemiological studies of HIV-1 transmission to develop a probabilistic model. SETTINGS: Semen samples were collected from patient subjects in Seattle Washington, Chapel Hill, North Carolina, and St. Gallen, Switzerland. Endocervical biopsy specimens were obtained from women in Chicago, Illinois. PARTICIPANTS: Eighty-six men (not receiving antiretroviral therapy) in whom CD4 cell count and semen volume were available, and 24 women in whom the number of endocervical CCR5 receptors were determined. MAIN OUTCOME MEASURES: Prediction of transmission of HIV-1 from men to women per episode of vaginal intercourse based on the absolute burden of HIV (volume x HIV RNA copies/ml seminal plasma). RESULTS: The model suggests efficient heterosexual transmission of HIV-1 when semen viral burden is high. When semen contains 100 000 copies of non-syncytium-inducing (NSI) HIV RNA the probability of HIV-1 transmission is 1 per 100 episodes of intercourse; conversely, with 1000 copies NSI HIV RNA in semen, transmission probability is 3 per 10 000 episodes of intercourse. CONCLUSIONS: This model links biological and epidemiological data related to heterosexual HIV-1 transmission. The model can be used to estimate transmission of HIV from men with high semen viral burden from inflammation, or reduced burden after antiretroviral therapy. The results offer a biological explanation for the magnitude of the HIV epidemic in places where earlier studies have shown men have high semen viral burden, such as in sub-Saharan Africa. The model can be used to develop and test HIV-1 prevention strategies.

HIV in body fluids during primary HIV infection: implications for pathogenesis, treatment and public health.

OBJECTIVE: To describe initial viral dissemination to peripheral tissues and infectious body fluids during human primary HIV infection. DESIGN: Observational cohort study. METHODS: Blood plasma, cerebrospinal fluid (CSF), seminal plasma, cervicovaginal lavage fluid and/or saliva were sampled from 17 individuals with primary HIV infection (range of time from symptoms onset to sampling, 8--70 days) and one individual with early infection (168 days). Subjects' HIV-1 RNA levels in each fluid were compared with levels from antiretroviral-naive controls with established HIV infection. For study subjects, correlations were assessed between HIV-1 RNA levels and time from symptoms onset. Responses to antiretroviral therapy with didanosine + stavudine + nevirapine +/- hydroxyurea were assessed in each compartment. RESULTS: HIV-1 RNA levels were highest closest to symptoms onset in blood plasma (18 patients) and saliva (11 patients). CSF HIV-1 RNA levels (five patients) appeared lower closer to symptoms onset, although they were higher overall in primary versus established infection. Shedding into seminal plasma (eight patients) and cervicovaginal fluid (two patients) was established at levels observed in chronic infection within 3--5 weeks of symptoms onset. High-level seminal plasma shedding was associated with coinfection with other sexually transmitted pathogens. Virus replication was suppressed in all compartments by antiretroviral therapy. CONCLUSIONS: Peak level HIV replication is established in blood, oropharyngeal tissues and genital tract, but potentially not in CSF, by the time patients are commonly diagnosed with primary HIV infection. Antiretroviral therapy is unlikely to limit initial virus spread to most tissue compartments, but may control genital tract shedding and central nervous system expansion in primary infection.

The effect of Plasmodium falciparum malaria on HIV-1 RNA blood plasma concentration.

OBJECTIVES: This study was undertaken to determine the relative effect of malaria infection on HIV concentration in blood plasma, and prospectively to monitor viral concentrations after antimalarial therapy. DESIGN: A prospective, double cohort study was designed to compare the blood HIV-1 RNA concentrations of HIV-positive individuals with and without acute malaria illness. Subjects were followed for 4 weeks after successful malaria therapy, or for 4 weeks from enrollment (controls). METHODS: Malawian adults with symptomatic Plasmodium falciparum parasitemia (malaria group) and asymptomatic, aparasitemic blood donors (control group) were tested for HIV-1 antibodies to identify appropriate study groups. The malaria group received antimalarial chemotherapy only and were followed with sequential blood films. In both groups, blood plasma HIV-1 RNA viral concentrations were determined at enrollment and again at 1, 2 and 4 weeks. RESULTS: Forty-seven malaria patients and 42 blood donors were enrolled. At enrollment blood plasma HIV-1 RNA concentrations were approximately sevenfold higher in patients with malaria than in blood donors (medians 15.1 x 10(4) and 2.24 x 10(4) copies/ml, respectively, P = 0.0001). No significant changes in median HIV-1 concentrations occurred in the 21 blood donors followed to week 4 (P = 0.68). In the 27 subjects successfully treated for malaria who were followed to week 4, a reduction in plasma HIV-1 RNA was observed from a median of 19.1 x 10(4) RNA copies/ml at enrollment, to 12.0 x 10(4) copies/ml at week 4, (P = 0.02). Plasma HIV-1 concentrations remained higher in malaria patients than controls (median 12.0 x 10(4) compared with 4.17 x 10(4) copies/ml, P = 0.086). CONCLUSIONS: HIV-1 blood viral burden is higher in patients with P. falciparum malaria than in controls and this viral burden can, in some patients, be partly reduced with antimalarial therapy.

Infectious immune complexes in HIV-1-infected patients.

Using polyethylene glycol (PEG) precipitation we have found that most HIV-1 seropositive patients have IgG containing-circulating immune complexes (CIC). In addition these CIC sometimes contain IgA, IgM, C3, and/or HIV p24 antigen. Previous work has demonstrated that patients who have plasma viremia, CD4 cell counts less than 170/mm3, or who are symptomatic are more apt to have HIV that is precipitable with PEG. In this study we report that the infectious HIV found in the plasma of patients with plasma viremia could only be found in the 2% PEG precipitates, i.e., PEG supernatants never contained infectious HIV, although they often contained noninfectious p24 antigen. These results suggested that at least some of the infectious HIV circulating in the plasma of infected patients is in the form of immune complexes. To support this idea we also demonstrated that infectious HIV could be precipitated with antiserum raised to either immunogloblins or complement components.

Factors associated with changes in HIV shedding in semen.

The efficiency and duration of transmissibilty of HIV seems to be highly variable and dependent on a number of factors related to both the donor and the recipient as well as characteristics intrinsic to the virus itself. Some of the factors likely to be important in sexual transmission of HIV include stage of disease, antiretroviral therapy, and concomitant systemic or mucosal infections, including sexually transmitted diseases. This paper describes recent work from our group in each of these areas.

Quality control of HIV cultures in a clinical retrovirology laboratory.

The culture of primary HIV isolates requires fresh phytohemagglutinin (PHA) stimulated peripheral blood mononuclear cells (PBMC) from seronegative donors. The variation inherent in donor cells, obtained from many different individuals, might affect the likelihood of virus isolation. We developed a quality control (QC) procedure which could determine quantitatively the susceptibility of random donor cells to HIV infection. The QC reagents consisted of cells and supernatants infected with a syncytium-inducing clinical isolate of HIV. Six 5-fold dilutions of QC cells (starting at 1000 cells) and supernatant (starting at a 1:200 dilution) were cultured in parallel with 1 x 10(6) 2-3-day-old PHA stimulated donor cells. After 7 or 14 days the resulting culture supernatant was tested for syncytia formation with MT-2 cells. A total of 127 sequential donors were tested over an 11 month period. All but one donor PBMC preparation was capable of being infected by the QC reagents (and this particular donor was permissive for several other primary isolates). The standard variation observed among all cultures was about one 5-fold dilution. The coefficient of variation ranged from 10.7 to 17.3%. These results suggest that the mononuclear cells from most, if not all, individuals are permissive for HIV to approximately the same level. Other quality control measures used in the laboratory are described.

Sensitive method for the detection of infectious HIV in semen of seropositive individuals.

The aim of the study was to develop a sensitive culture method for the detection of HIV in semen. Antimicrobials were evaluated to suppress bacterial and fungal contamination of HIV cultures. Toxicity of seminal plasma was reduced using a short incubation of seminal fluid with culture cells. The detection rate was determined by adding known amounts of diluted primary virus isolates to uninfected cells. The sensitivity of the method was determined in a cohort of 33 HIV seropositive men. The use of a virus transport medium with high doses of antibiotics reduced contamination of HIV cultures. Toxicity of seminal plasma on culture cells was limited when seminal fluid was incubated with culture cells for only 90 min. Detection levels for cell-associated virus and cell-free virus from semen were 12 infected cells per ejaculate and 100 infectious units per milliliter of seminal fluid, respectively. Cell-associated HIV could be recovered from 18 semen samples of 33 HIV positive men (55%). Recovery of cell-free virus from seminal fluid was infrequent. It is concluded that the method is highly sensitive for the detection of HIV from semen. However, the recovery rate of infectious cell-free virus in seminal fluid from clinical samples is infrequent.

Quantitation of human immunodeficiency virus type 1 RNA in cell free seminal plasma: comparison of NASBA with Amplicor reverse transcription-PCR amplification and correlation with quantitative culture.

Human immunodeficiency virus type 1 (HIV-1) is transmitted by infected males in semen. However, the inoculum required for infection is unknown. The ability to collect such information will rely on the availability of reliable quantitative assays of HIV-1 in semen. We examined the comparative performance of NASBA and Amplicor Monitor RT-PCR in quantifying HIV-1 RNA in cell free seminal plasma from seropositive men and correlated the results obtained with viral titres measured by a seminal cell quantitative microculture (QMC) assay. Of samples analysed, 68% and 56% by both NASBA and RT-PCR contained measurable HIV-1 RNA, respectively. Amplification inhibition frequently affected RT-PCR but not NASBA. Excluding samples with complete RT-PCR inhibition, there was 90% qualitative concordance and a strong positive correlation (r = 0.86) of RNA levels measured by the two methods. Comparison of the concentration of HIV-1 RNA in seminal plasma samples, as measured by NASBA, with QMC viral titres indicated that RNA levels probably reflect the infectiousness of whole semen. NASBA is a reliable technique for quantitating HIV-1 RNA in seminal plasma and should become a valuable tool in the study of factors that influence the sexual transmission of HIV.

Hyper-excretion of human immunodeficiency virus type 1 RNA in saliva.

Anatomical compartments (e.g., the reproductive tract) are reservoirs of human immunodeficiency virus type-1 (HIV-1) and potential sites of residual infection in patients receiving anti-retroviral therapy (ART). Viral hyper-excretion relative to blood is a hallmark of reservoirs. To determine whether hyper-excretion can occur in the oral cavity, we compared viral loads in blood plasma and saliva of 67 adults. Salivary viral hyperexcretion was defined as a four-fold or higher viral load in saliva than in plasma. HIV-1 RNA was detected in 79% of plasma samples, in 44% of unfiltered saliva samples, in 16% of filtered saliva samples, and in 59% of saliva-derived cell pellets. Compared with non-hyper-excretors (n = 62), hyper-excretors (n = 5) had elevated levels of viral RNA in unfiltered saliva and saliva-derived cells, HIV-associated periodontal disease, gingival inflammation, and no combination ART. Morphological characterization of cell pellets identified lymphocytes as a likely HIV-1 source. These collective findings are consistent with an oral HIV-1 reservoir in selected individuals.

Variation in mitogen-induced ovine lymphocyte blastogenesis: adherent cells or 2-mercaptoethanol restore randomly depressed responses.

Peripheral blood leucocytes (PBL) isolated 23 times over a 6-week period from four normal sheep showed considerable variation in serially tested responses to predetermined optimal concentrations of concanavalin A (Con A), phytohaemagglutinin (PHA), and pokeweed mitogen (PWM). Con A responses, in particular, varied widely and were often randomly depressed (21 of 91 times compared to 15 of 91 times for PHA or PWM). The addition of as few as 1% adherent cells (AC) to depressed cultures fully restored the PBL proliferative response to normal levels. Addition of greater numbers of AC (5 or 10%) had little further enhancing effect on depressed cultures. The addition of 1, 5, or 10% AC to cultures that were responding at normal levels increased responses only slightly. Autologous or allogeneic AC were equally effective. Addition of 2-mercaptoethanol (2-ME) to depressed cultures only partially restored the blastogenic response to Con A and had little effect on normal cultures.

Molecular Diagnosis of HIV-1 by PCR.

Most individuals can be diagnosed as being infected with HIV-1 with an enzyme immunoassay (EIA), which detects antibodies to the virus. Repeatedly positive results must be confirmed, usually by Western blot. These assays have been improved since their introduction in 1985. When used together, the EIA and Western blot are now extremely reliable, with sensitivities of 99.5% and specificities of 99.8% for the diagnosis of HIV-1 (1). These tests are fairly rapid, reproducible across laboratories, well quality-controlled, relatively inexpensive, and form the basis of the protection of the blood supply.

Collection and Processing of Seminal Plasma for the Quantitation of HIV-1 RNA by NASBA and RT-PCR.

Semen is the major vehicle for the sexual transmission of HIV-1. The ability to isolate infectious HIV from the semen and to quantitate viral burden in the form of cell-free or cell-associated HIV-1 RNA in semen are important for epidemiologic and public health aspects of the epidemic. Earlier studies used viral culture to detect HIV in semen. Cell associated culturable virus recovery rates ranged from 8 to 55% (1-8). Much lower recovery rates (3-15%) were reported by these investigators for cell-free seminal plasma. In general, subjects with lower CD4 counts, higher seminal plasma viral load (>3.5-4 log(10)), and an AIDS diagnosis were more apt to have positive seminal cell HIV cultures.