Dried-plasma transport using a novel matrix and collection system for human immunodeficiency virus and hepatitis C virus virologic testing.

A novel method for the collection and transportation of dried-blood-plasma samples, SampleTanker (ST), was developed and compared to standard shipping protocols for frozen-plasma specimens containing human immunodeficiency virus type 1 (HIV-1) and/or hepatitis C virus (HCV). Matched frozen and dried 1-ml EDTA-containing plasma samples were collected and analyzed by several molecular-based virologic assays. After addition of 1.175 ml of reconstitution buffer, 1.035 ml of dried plasma was recovered. Mean intra-assay variances were 0.05, 0.05, and 0.06 log(10) copies/ml for the Versant, Amplicor, and NucliSens QT HIV-1 load assays, respectively (P, not significant). However, mean HIV-1 viral load was consistently reduced in dried samples by 0.32 to 0.51 log(10) copies/ml, depending on assay type (P < 0.05). Infectious HIV-1 was not recovered from dried ST plasma. There was no significant difference in HIV-1 viral load results obtained using ST after 8 weeks of storage at ambient temperature. Compared to frozen plasma, HIV-1 genotypic results were >99% concordant at the nucleotide and amino acid levels, as well as for resistance-associated mutations. We further demonstrated successful detection of multiple analytes, including HIV-1 viral load, HIV-1 antiretroviral resistance genotype, and HCV genotype, from a single ST unit. Dried plasma collected with ST yielded comparable results to frozen samples for multiple-analyte clinical testing. As such, ST could be a useful alternative for virologic tests and clinical trials worldwide by significantly diminishing transportation cost and the sample volume restrictions associated with dried-blood-spot technology.

Laboratory safety considerations.

In the laboratory it is important that potentially pathogenic agents be controlled to protect the laboratory worker from infection and the experiment from contamination. The operation of a safe laboratory depends on many factors: the training and judgement of laboratory personnel; the implementation of protocols, the selection and use of equipment and reagents; and the location and design of the laboratory. These should be integrated in order to provide maximum safety for the personnel without impeding operation of the laboratory.

Biologic properties of human herpesvirus 7 strain SB.

The growth characteristics of human herpesvirus 7 strain SB (HHV-7 (SB)) were studied in human umbilical cord blood lymphocyte (CBL) cultures. The virus has approximately a 4-day growth cycle, as measured by immunofluorescence analysis, quantitation of the relative viral DNA concentration, and examination of infected cells by electron microscopy on consecutive days post-infection. By systematically varying the culture media components, improved culturing conditions were established. Activated lymphocytes were required for virus growth. HHV-7(SB) grew best in phytohemagglutinin-stimulated CBL cultured in media containing 0.01 mg/ml hydrocortisone. Addition of recombinant human interleukin 2 (IL-2) at concentrations exceeding 1-10 U/ml inhibited virus growth in most CBL cultures. Addition of exogenous IL-2 to the culture media had no effect on viral DNA production. However, the percentage of virus antigen-positive cells was highest when 0.1-1 U/ml was added to the media. Differences in the ability of individual CBL cultures to replicate HHV-7(SB) was not explained by differing CD4+ cell concentrations. However, individual cultures varied in the level of endogenous IL-2 production, which may contribute to the virus growth variability in CBL. HHV-7(SB) grew in the CD4-positive T-cell line SupT1, but not in a variety of other lymphocyte, fibroblast, or epithelial cell lines. Nine compounds were tested for antiviral activity against HHV-7 in vitro. Phosphonoformic acid inhibited virus growth with a 50% effective concentration of 4.8 microM. Ganciclovir (200 microM) and phosphonoacetic acid (100 microM) inhibited more than 90% of virus production. None of the compounds were cytotoxic at concentrations which inhibited the virus. A generalized increase in host cell protein synthesis was also observed in virus-infected cells similar to that seen in CBL infected with human herpesvirus 6.

Lack of correlation between human herpesvirus-6 infection and the course of human immunodeficiency virus infection.

Human herpesvirus-6 (HHV-6) and human immunodeficiency virus (HIV) are both tropic for CD4+ lymphocytes. To determine whether HHV-6 infection affects the susceptibility to or the course of HIV infection, HHV-6 titers were measured by an anticomplement immunofluorescence assay in serum of three groups of homosexual or bisexual men: (1) those with AIDS (n = 78), (2) those with HIV-associated lymphadenopathy (LAS; n = 81), and (3) those who were HIV-seronegative (n = 55). Early and late serum samples were available for 45 men with LAS (median interval 49 months). Men with early LAS did not differ from HIV-seronegative men in either the percentage that were HHV-6-seropositive or in the distribution of titers. There was a significantly lower percentage of seropositives in AIDS patients than in the other two groups (P less than .01). LAS patients who progressed to AIDS did not differ in percentage seropositivity or distribution of titers from nonprogressors. HHV-6 titers tended to decrease over time. HHV-6 titers late in LAS were similar to those in AIDS patients. These findings suggest that it is unlikely that previous exposure to HHV-6 either predisposes to or affects the course of HIV infection.

Comparative study with in situ hybridization and immunocytochemistry in detection of HIV-1 in formalin-fixed paraffin-embedded cell cultures.

The sensitivities of three immunohistological techniques were compared in this study for detecting human immunodeficiency virus (HIV-1) in infected cultured human lymphocytes that had been formalin-fixed and paraffin-embedded. The techniques included in situ hybridization (ISH) with HIV-1 cDNA; immunocytochemistry with HIV-1 p24 monoclonal antibody (ICC-m); and immunocytochemistry with HIV-1 polyclonal antibody from a patient with acquired immunodeficiency syndrome (AIDS) (ICC-p). Procedures were optimized for enzyme digestion and for antibody reaction conditions. HIV-1--infected cells and noninfected control cells were tested. Noninfected controls were uniformally negative by all three methods. Infected cells had the highest positivity rate by the ISH method (p less than or equal to 0.0001), and the ICC-p method was more positive than the ICC-m (p less than or equal to 0.0001). Both the ICC-p and the ICC-m techniques were more positive with the cocultivated cell cultures than the ISH, which was more sensitive with the infected continuous cell line (P less than or equal to 0.0001). The ICC-p method had a lower standard deviation on positive results than either the ICC-m or ISH method. The variability observed with these test procedures, reagents, and specimens suggests that these are important technological parameters in detecting p24, with implications for detecting other HIV-1 markers in infected tissues.

Demonstration of human immunodeficiency virus by colorimetric in situ hybridization: a rapid technique for formalin-fixed paraffin-embedded material.

A colorimetric method of in situ hybridization has been developed for the rapid detection of human immunodeficiency virus (HIV) in formalin-fixed paraffin-embedded material. Following optimization of digestion conditions, biotin-labeled DNA probes are detected with an alkaline phosphatase conjugate. The method is verified using fixed paraffin-embedded cell blocks of HIV-infected and uninfected lymphocyte cell cultures. Hybridization specifically detects both viral RNA and proviral DNA. Formalin fixation for intervals up to 21 d did not significantly hamper the signal under the appropriate digestion conditions; however, Trump's fixation for even 12 h greatly reduced the intensity of the hybridization. This technique for in situ hybridization is amenable to automation, provides results within 6 h, and results in good morphologic preservation. A key feature of the technique is the use of human placental DNA as an endogenous positive control to optimize the empirically determined conditions for protein digestion.

Condoms as physical and chemical barriers against human immunodeficiency virus.

In an in vitro model, 20 condoms containing 0.9 mL of 6.6% (vol/vol) nonoxynol 9 and ten condoms without nonoxynol 9 were tested as physical and chemical barriers against human immunodeficiency virus (HIV). Each condom was mounted on a hollow dildo and placed in a glass cylinder. The HIV inoculum and HIV-free medium were placed on opposite sites of the condom. Intercourse was simulated by pumping the dildo up and down in the cylinder before and after deliberate rupture of the condom. Samples for HIV culture were taken from outside and inside the condom, before and after rupture. After rupture of nonoxynol 9-containing condoms, an outside nonoxynol 9 concentration of 0.25% was reached. No condom without nonoxynol 9 leaked HIV before rupture, but after rupture HIV could be detected in medium outside of seven of ten condoms tested. In none of 20 nonoxynol 9-containing condoms could HIV be detected in outside medium after rupture. Thus, undamaged condoms provide an effective physical barrier against HIV, and nonoxynol 9 may provide an effective chemical barrier as well.

Isolation of human immune deficiency virus from African AIDS patients and from persons without AIDS or IgG antibody to human immune deficiency virus.

We previously reported a high incidence of acquired immune deficiency syndrome (AIDS) in Kinshasa, Zaire, as well as a high frequency of antibody to human immunodeficiency virus (HIV), which includes HTLV-III and LAV viruses, in persons without AIDS. In this report we assessed the frequency of HIV virus infection in persons with and without clinical AIDS and the association of virus isolation to presence of antibody. We isolated HIV from 27 (77%) of 35 patients with AIDS, and 5 of 9 patients with AIDS-related complex (ARC). Virus was also isolated from plasma and cerebrospinal fluid of patients in the study. The presence of antibody was a reliable marker for virus infection in African patients with AIDS. HIV was isolated from 5 of 27 control patients without AIDS, 3 of whom had normal T helper to T suppressor ratios and normal numbers of T helper cells. Two of these patients had no detectable antibody to HIV by ELISA or Western blot methods. In a population, such as the general heterosexual population of Kinshasa, with frequent infection by HIV and with few clearly definable risk groups, screening for antibodies to HIV may not be sufficient to identify some virus infected persons.

PIP: This study represented the 1st attempt to isolate human immunodeficiency virus (HIV) from African acquired immunodeficiency syndrome (AIDS) patients and controls. HIV was isolated from 27 (77%) of 35 Zairians with AIDS and from 5 (55%) of 9 patients with AIDS-related complex (ARC). In addition, 5 (19%) of 27 controls admitted to Zaire's Mama Yemo Hospital for causes unrelated to AIDS were found to be positive for antibodies to HIV. Next, an effort was made to isolate the virus from 42 AIDS or ARC patients on whom data were already available on the results of an enzyme-linked immunosorbent assay (ELISA). HIV was isolated from 30 (81%) of 37 patients with positive ELISA tests and from none of the 5 patients with a negative assay. Among controls, antibodies were found in a higher proportion of patients with abnormal helper: suppressor ratios or a low absolute T helper cell count. On the other hand, these abnormalities were not found in 3 of the 5 control patients from whom HIV was isolated, including 2 without HIV antibody. This suggests that neither of these criterion are good indicators of virus infection. The isolation of HIV infection from 5 hospital controls with no clinical signs of infection suggests that either the rate of asymptomatic HIV virus infection is high in Zaire or that common tropical diseases such as malaria or tuberculosis may be associated with HIV infection. The frequency of HIV isolation from AIDS and ARC patients in this study is higher than that in earlier reports from non-Africans, but is comparable to current statistics from the US.

Micromethod for assaying reverse transcriptase of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus.

A micromethod for assaying the reverse transcriptase enzyme of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus in cocultures of clinical specimens for viral isolation was developed and compared with the macromethod in use. Ultracentrifuged, pelleted, and solubilized viral culture supernatants were transferred into either tubes (macromethod) or microtiter plates (micromethod) and incubated with tritiated enzyme substrate. Trichloroacetic acid-precipitated DNA was collected on individual filter papers with a Millipore filtration manifold (macromethod) or on filter sheets using a semiautomated cell harvester (micromethod). Filters were then placed in scintillation fluid and counted on a beta scintillation counter. Results of the micromethod significantly correlated to those of the macromethod, with a linear relationship between the two. The cutoffs for positivity based on the mean + 2 standard deviations for a set of known negative specimens (n = 19) was 4,973 cpm for the micromethod compared with 5,336 for the macromethod. The intrarun and interrun variations were comparable for both methods. There was a 67% increase in the maximal daily number of specimens which could be run (100 versus 60) as well as a reduction in reagent use. In summary, the micromethod utilizing a semiautomated cell harvester is comparable to the existing macromethod in accuracy and is an improvement due to savings in time and reagents.

Antibody against the human immunodeficiency virus in commercial intravenous gammaglobulin preparations.

In a 6-month study, antibody levels against the human immunodeficiency virus (HIV) were determined in intravenous gammaglobulin preparations and 45 serum samples from 20 patients on gammaglobulin therapy. All 10 lots of a reduced and alkylated preparation and 4 of 8 lots of a pH4/pepsin-treated preparation were seropositive by enzyme-linked immunosorbent assay (ELISA). By Western blot analysis, 8 of 10 lots of the reduced and alkylated preparation and 3 of 8 lots of the pH4/pepsin-treated preparation were positive. Before gammaglobulin infusion, 2 of 45 preinfusion samples were seropositive by ELISA but seronegative by Western blot. After infusion, 15 of 45 samples were seropositive by ELISA, and 8 had antibody against p24 by Western blot. Seropositivity persisted for less than 1 month. Cultures of HIV-positive intravenous gammaglobulin lots were negative for reverse transcriptase activity or viral antigen expression. These results suggest that current methods of preparation either exclude or inactivate HIV.

T-cytotoxic/suppressor cell phenotypes in a group of asymptomatic homosexual men with and without exposure to HTLV-III/LAV.

The number of lymphocytes bearing the Leu 2+ or T8+ (suppressor/cytotoxic) phenotype is elevated in asymptomatic homosexual men. By two-color immunofluorescence using paired monoclonal antibodies (alpha-Leu 2 and alpha-Leu 15, alpha-Leu 2 and alpha-Leu 7, alpha-Leu 7 and alpha-Leu 11), we enumerated phenotypic subpopulations that are associated with cytotoxic, suppressor, or natural killer function. Both cytotoxic (Leu 2+15-) and suppressor (bright Leu 2+15+) cell populations are elevated in homosexual men. Homosexual men who have been exposed to human T-lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV) have higher numbers of Leu 2+15- and Leu 2+7- cells than homosexual men who have not been exposed. Phenotypic subpopulations (dim Leu 2+ Leu 15+ and Leu 7-11+) that are associated with the most potent natural killer activity (against K562 target cells) were not found to be elevated in homosexual men.

The safety of the hepatitis B vaccine. Inactivation of the AIDS virus during routine vaccine manufacture.

In the United States, one hepatitis B vaccine (Heptavax-B) has been licensed for the prevention of hepatitis B virus infections. Even though this vaccine has been shown to be highly effective and well tolerated in controlled trials and has been recommended for use in those at risk for acquiring infection by hepatitis B virus, many individuals have been reluctant to be immunized for fear of contracting acquired immunodeficiency syndrome (AIDS). In this study, we demonstrate that each of the three inactivation steps used in the manufacture of Heptavax-B independently will inactivate the infectivity of high-titered preparations of the AIDS virus; recipients of the hepatitis B vaccine do not develop antibodies to the AIDS virus; the hepatitis B vaccine does not contain detectable levels of nucleic acids related to the AIDS virus. These observations clearly demonstrate that vaccination with the currently available hepatitis B vaccine poses no demonstrable risk for acquiring AIDS.

Laboratory and epidemiologic evaluation of an enzyme immunoassay for antibodies to HTLV-III.

The enzyme immunoassays (EIAs) for antibody to human T-cell lymphotropic virus type III (HTLV-III) were rapidly adopted for screening donated blood and plasma. To evaluate the significance of a positive EIA reaction, test performance was examined in a blood bank screening program. Specimens were tested by EIA, Western blot assay, and HTLV-III/lymphadenopathy-associated virus (LAV) culture. The EIA was positive in 0.25% of 67 190 blood donations. Specimens were categorized and 57.3% had low (weak) reactivity, 12.7% had moderate reactivity, and 30.0% had high reactivity. Highly reactive specimens were strongly associated with a positive Western blot or culture (86.7%) in contrast to moderately and weakly reactive specimens (1.9%). Twenty-five of 29 donors interviewed with a highly reactive EIA had risk factors for HTLV-III/LAV infection. Risk factors were not identified for 74 of 75 interviewed donors with specimens of lower reactivity. The minimum calculated specificity was 99.82%. The use of the HTLV-III EIA has virtually eliminated the use of blood and plasma from HTLV-III/LAV infected donors.

HTLV-III/LAV infection in hemodialysis patients.

Twenty-five (4.8%) of 520 hemodialysis patients were seropositive for antibody to human T-cell lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV) by enzyme immunoassay. Four had high reactivity on enzyme immunoassay and positive results of Western blot tests, and one of the four had a positive culture. The remaining 21 seropositive patients had low reactivity on enzyme immunoassay, negative results of Western blot tests, and negative cultures. All had received blood transfusions and 19 had antibodies to antigens associated with the H9 cell line used to propagate HTLV-III for serological tests. We found that HTLV-III/LAV was not transmitted in the dialysis centers. Frequent blood transfusion places dialysis patients at risk for HTLV-III/LAV infection, but may more commonly lead to false-positive results of enzyme immunoassay tests.

The natural history of infection with the lymphadenopathy-associated virus/human T-lymphotropic virus type III.

Over half of the persons infected with the lymphadenopathy-associated virus/human T-lymphotropic virus type III (LAV/HTLV-III), the retrovirus that causes the acquired immunodeficiency syndrome (AIDS), become persistently infected with the virus. These "carriers" serve as the major reservoir of infection for others. Virus from their vascular and lymphatic spaces infects others through direct blood or mucous membrane exposure. After months to years, a high proportion of those infected will develop clinical manifestations of infection. For infected homosexual men, approximately 25% have developed AIDS-related conditions, mainly lymphadenopathy, and approximately 10% have developed AIDS. Because of the large number of infected persons in the United States, increasing rates of disease can be expected.

Transfusion-associated acquired immunodeficiency syndrome in the United States.

By Aug 15, 1985, one hundred ninety-four cases of possible transfusion-associated acquired immunodeficiency syndrome (AIDS) had been reported to the Centers for Disease Control. Cases received their transfusions in 30 states. Infants account for 10% of the cases, suggesting an increased susceptibility to developing AIDS. Investigations one to six years after the transfusions have identified high-risk donors to 47 cases. Of 47 high-risk donors tested, 40 had a reactive serology for human T-cell lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV) antibody, including five with no risk for AIDS by history. The HTLV-III/LAV was isolated from 23 of 26 seroreactive high-risk donors, most of whom remained asymptomatic. Blood components that transmitted HTLV-III/LAV included red cells, platelets, plasma, and whole blood. The time from transfusion to diagnosis of AIDS ranged from four to 84 months. The risk of developing AIDS after a blood transfusion has been low and will be lowered further by using both self-deferral and antibody screening.