Clinical utility of genotypic resistance tests for HIV-1-infected patients with low-level virological failure.

The usefulness of genotypic resistance tests (GRT) among HIV-1 patients with low-level virological failure (LLVF) was evaluated. Up to 78% of samples with <1,000 copies/ml were sequenced successfully. For samples with 50 to 200 copies/ml, the success rate was as high as 69%. LLVF should not deter clinicians from requesting GRT.

Genotyping of B and non-B subtypes of human immunodeficiency virus type 1.

Current HIV-1 genotyping assays were developed using subtype B viruses prevalent in Western countries. It is not clear whether these assays are appropriate for use among African patients, who are likely to be infected with non-B subtypes. We evaluated the Bayer TRUGENE HIV-1 genotyping (TG) assay using prospectively collected samples from HIV-1-infected individuals who acquired infection in either sub-Saharan Africa or the West (Europe, North America, and Australia). Plasma samples from 208 individuals with an HIV-1 viral load of >1,000 copies/ml were tested using version 1 primers supplied with the TG assay. If these failed, an alternative primer set version 1.5 was used. Of the 208 individuals, the likely origin of infection was Africa (n = 104), Western (n = 87) and "Others" (i.e., all other geographic locations or origin not certain; n = 17). Among the three groups, the version 1 primers were successful in 85 (82%), 77 (89%), and 13 (76%) individuals, respectively (P = 0.1). Of the remaining 32 samples, 30 were successfully amplified by using the version 1.5 primers. HIV-1 subtypes deduced from the reverse transcriptase sequences correlated with the likely origin of infection: Africa (28A, 3B, 33C, 13D, 6G, 4J, 2K, 5CRF01_AE, and 10CRF02_AG), Western (86B and 1K), and Others (1A and 16B). The success of the version 1 primers correlated with viral load (P < 0.014) and not with HIV-1 subtypes. A protocol based on version 1 primers, followed by 1.5 primers, was successful in sequencing 99% of the samples in this cohort.

Potential risk of cross-infection during peripheral-venous access by contamination of tourniquets.

We found that a high proportion of reusable tourniquets are contaminated with blood and bacterial pathogens. Their use contravenes hospital cross-infection control protocols and we therefore recommend the use of disposable tourniquets.

Feasibility of named antenatal HIV screening in an inner city population.

The object of this study was to assess the resource use, feasibility, uptake and consumers' perspective of introducing routine named human immunodeficiency virus (HIV) testing into an inner city hospital antenatal clinic (St Thomas Hospital, part of Guy's and St Thomas Hospital Trust). Following the introduction of a new service offering routine named HIV testing at booking appointments, the length of the appointments were recorded over a three-month period and compared with appointment lengths in the previously existing service. Women being offered routine named HIV testing were asked to complete a questionnaire before and after their appointment to assess knowledge, attitudes and acceptability of HIV testing. Subjects were three-hundred-and-eighty-one pregnant women attending the antenatal clinic for their booking appointment. There was no significant difference in the length of the booking appointments following the introduction of offering routine named HIV testing. One-hundred-and-fifty-eight women (42%) consented to testing with no positive results detected. There was a significant rise in the uptake of the test over the three-month period. No other variables (demographics, perceived risk, having read the leaflet) were predictive of uptake. Anxiety scores were significantly less after the booking appointment. Routine named HIV testing was feasibly introduced into a hospital antenatal clinic with minimum resource implications. Further study is needed to monitor the ongoing uptake rate and identification of HIV-positive women before a decision can be made as to the long-term benefit of this new service. Women considered it acceptable to be offered the HIV test and were less anxious after their appointments.

Women's knowledge and attitudes, and the acceptability of voluntary antenatal HIV testing.

OBJECTIVE: To assess pregnant women's knowledge of, and attitudes towards, antenatal HIV testing, and its acceptability to them. SETTING: Antenatal clinic at Guy's Hospital, London, six community antenatal clinics and a midwifery group practice. POPULATION: Eight hundred and forty-three women attending the antenatal clinics. METHOD: The women received a leaflet explaining HIV testing, and completed a questionnaire before and after their booking appointment. This included an assessment of their knowledge of, and attitudes towards HIV testing, and its acceptability. RESULTS: Seven hundred and eighty-nine women (94%) completed questionnaires. Fifty-one percent (n = 405) were Caucasian, 25% (n = 195) African, 11% (n = 86) West Indian and 13% (n = 100) were from other ethnic groups. Fifty-eight percent received the HIV information leaflet, of whom 86% had read it. Knowledge relating to HIV was good, the median knowledge score being 6 out of a possible 8, but it was less in non-Caucasian women and those with lower educational qualifications. Knowledge was not related to uptake of testing. Thirty-five percent of women accepted the offer of an HIV test, rates being higher in hospital clinics (41%) than in the midwifery group practice (10%) and the community clinics (30%). Women more likely to accept the offer of an HIV test were non-Caucasian (P = 0.0443), those who had thought about the HIV test before this pregnancy (P = 0.0298) and those seeing one particular midwife (P = 0.0003). Most women (67%) thought that all pregnant women should be offered the HIV test and then make their own decision. Overall, 64% women did not change their original pre-discussion decision on testing for HIV. Thirty-six percent of women changed their decision from 'yes' to 'no' or 'don't know' after seeing the midwife. Women attending the community clinics (P = 0.003) and those who had been tested before (P = 0.0451) were more likely to change their decision. CONCLUSION: This study, in a multiethnic population, has shown that knowledge regarding HIV is good but does not increase the uptake of testing. Women prefer to be offered the HIV test and make their own choice regarding whether to accept it.

Assisted conception in HIV discordant couples: evaluation of semen processing techniques in reducing HIV viral load.

Artificial insemination with motile spermatozoa prepared from HIV-infected men using standard procedures has been employed with many HIV-discordant couples. We have demonstrated that processing semen from HIV positive men can reduce HIV levels, measured as HIV1 RNA copies/ml using nucleic acid based sequence amplification (NASBA), to undetectable levels (less than 400 copies/ml) but not in all samples. We believe that all processed samples should be tested prior to insemination.

Voluntary, named testing for HIV in a community based antenatal clinic: a pilot study.

Despite the increasing advantages of identifying HIV infection in pregnant women, only some 12% of HIV positive women attending antenatal clinics in London have been identified by named testing. As virtually all antenatal care will be community based within the next two to three years, we assessed the problems of introducing named HIV testing during pregnancy into the primary care setting. Planning the service took a considerable time and required the production of educational material for both staff and pregnant women and some reorganisation of procedures. Over a one year period an uptake of 44% was noted. Several problems were encountered including an average of 21 minutes needed to give information on AIDS and HIV, an adverse effect on the midwife-mother relationship, and anxiety (affecting both women and midwives). Possible solutions to this difficult problem are discussed.

Is HIV testing in antenatal clinics worthwhile? Can we afford it?

Since 0.2-0.4% of pregnant women attending antenatal clinics (ANC) at St Thomas's Hospital are HIV positive, and as the Department of Health (DH) have recommended that universal voluntary HIV testing be made available to women attending ANC in areas of "known or suspected higher prevalence of HIV infection", we examined the implications of the DH initiative in an Inner London Teaching Hospital as well as in a General Practice involved in shared care. The cost of the programme (148,300 pounds to 193,900 pounds), 80% of which relates to the need to obtain informed consent, was approximately 2.7-3.5 times that calculated by the DH. The DH based much of their costing on additional time for counselling rather than calculating the additional staff required. We estimated that 25% of women will require specialized counselling since 17% are of African ethnicity and others are injecting drug users or 'worried well'. Various means of reducing costs were considered but, until such time as explicit, informed consent is no longer considered necessary, the above resources will be required. Unless the DH continues to provide central direction to Providing Agencies to give priority to these recommendations and, where necessary, provides additional funding, we fear that this important public health initiative will be unsuccessful.

Isolation of a new foamy retrovirus from orangutans.

We have isolated a new foamy virus from blood samples taken from two apparently healthy orangutans (Pongo pygmaeus). The older orangutan has since died with encephalopathy after a brief acute illness, while the younger one, his grandson, remains well. These animals and 12 other orangutans had specific antibodies to foamy virus as measured by immunofluorescence. The new foamy virus and the antisera showed strong and specific neutralization, with only weak cross-reaction with other simian foamy virus strains. Southern blotting with gag and env probes of human foamy virus and PCR amplification showed that the new foamy virus, designated SFV-11, is related to, yet distinct from, previously characterized strains from humans, chimpanzees, and monkeys.

Mapping of linear B cell epitopes on capsid proteins of bovine papillomavirus: identification of three external type-restricted epitopes.

Immunodominant, conserved and type-restricted external epitopes of bovine papillomavirus (BPV) major (L1) capsid protein have been identified using BPV particles and synthetic peptides. Antisera to disrupted BPV-1 recognized BPV-1 and BPV-2 particles in immune electron microscopy (IEM) studies and inhibited BPV-2-induced focus formation of NIH/3T3 cells. Thus BPV-1/BPV-2 cross-reactive epitopes occur on the surface of virions. The L1 protein appeared to be immunodominant as the antisera reacted with three dominant BPV-1/BPV-2 conserved B cell epitopes (amino acids 111 to 125, 131 to 145 and 191 to 205) in Pepscan assays of BPV-1 L1, whereas no common epitopes and less frequent antibody binding to peptides were detected in Pepscans of the L2 protein of BPV-1. Four discrete variable regions were identified in the sequences of L1 proteins of BPV-1 and BPV-2. Antisera against synthetic peptides corresponding to three of the four variable regions (amino acids 42 to 56, 435 to 449 and 485 to 499) of BPV-2 L1 caused clumping of BPV-2, but not of BPV-1, particles as examined by IEM, and antisera to one peptide (amino acids 485 to 499) inhibited BPV-2-induced focus formation of NIH/3T3 cells. These data suggest that these regions are type-specific BPV-2 L1 epitopes and that they occur on the virion surface. Although conformation-dependent epitopes remain to be identified on papillomaviruses, the linear epitopes identified in this study may be worthy of further study as constituents of experimental prophylactic vaccines.

Electron microscopic detection of human parvovirus (B19) in a patient with HIV infection.

We report the findings on an HIV-positive patient found to be infected with human parvovirus B19. In a comparative study of sequential sera, direct electron microscopy (EM) had the same sensitivity as DNA hydridization for the detection of the virus. EM did not require specific reagents and also yielded information on the state of the virus; i.e., whether random or complexed, and the type of antibody involved. The presence of parvovirus complicated the judgement as to drug treatment. This case also highlighted the importance of diagnosing the presence of opportunistic viral infections that have no, or low-grade, pathogenic activity.

Recovery of antigenically reactive HIV-2 cores.

Negative staining studies of human immunodeficiency virus (HIV) have been hampered by the fragile nature of the particles. Although detergent treatment is capable of releasing cores from HIV-2 particles, these are unstable and do not retain morphological integrity. Addition of glutaraldehyde will stabilise these structures but, if used at too high a concentration, will destroy their antigenicity. This study shows that if both detergent and glutaraldehyde are used in correct proportions, antigenically reactive cores can be recovered from HIV-2 cell cultures. More specifically we show that a mixture of 0.1% Nonidet P40 and 0.1% glutaraldehyde produces preparations of HIV-2 cores that are suitable for immune electron microscopy. These cores reacted positively, that is, formed immune complexes, with both human HIV-2 antisera and a mouse monoclonal antibody that, although directed against p24 (HIV-1), reacts also with p25 (HIV-2).

Further studies of HIV morphology by negative staining.

Thin-section studies of HIV-1- and HIV-2-infected cells were used to establish peak virus productivity and distribution of virus on and around infected cells. Maximum virus yields occurred 7 days after passage; cells at that stage were used as a source of virus for negative staining. Various methods of separating virus and cells were assessed: results showed that gentle homogenization in a Tenbroek-type homogenizer yielded considerably more virus than other techniques. Virus obtained in this way mainly appeared in the form of large clumps. Because of the large numbers of virus particles obtained it was possible to visualize what is probably the immature form of the virus. The inner component of this particle is spherical and, as is discussed, is a transient form proceeding to the now well established, mature, cone-shaped virus core.

The morphology of human immunodeficiency virus (HIV) by negative staining.

We have examined preparations of human immunodeficiency virus 1 (HIV-1) and HIV-2 by negative staining electron microscopy. HIV-2 cultures contained large numbers of 130-200 nm particles containing a 130-nm-long by 30-70 nm-wide core. This core is probably of conical or pear-shaped morphology. Some particles exhibited a short fringe that could be seen to comprise a regular arrangement of repeating subunits when visualised end on. Identical particles were found in HIV-1 cultures but in much lower numbers. Attempts to carry out negative staining immune EM were unsuccessful. Also detected in both HIV-1 and HIV-2 cultures were small (70-80 nm) fringed viruslike particles. The possible significance of these particles is discussed.

Development of a lymphocyte transformation assay for rotavirus in whole blood and breast milk.

A multivariate assay was developed for detecting lymphoproliferative responses to rotavirus in whole blood and breast milk. Detection of lymphocyte transformation in samples from healthy uninfected adults required an assay incorporating a large number of antigen and cell concentrations and several incubation periods before pulsing. In contrast, lymphoproliferation to rotavirus was observed over a wider range of these variables in blood collected from a kidney recipient nine days post-rotavirus infection. Lymphoproliferation to rotavirus was detected in 32.5% of breast milk samples tested. The magnitude of lymphoproliferation to rotavirus did not correlate with rotavirus-specific IgG ELISA antibody titres in sera or with rotavirus-specific IgA ELISA antibody titres in breast milk.