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.

Problems in the interpretation of HIV-1 viral load assays using commercial reagents.

During routine monitoring of human immunodeficiency virus (HIV) viral load, two problems arose. First, a number of patients, the majority being African, were found to have low viral loads by the Chiron branched-chain DNA assay in conjunction with low CD4(+) cell numbers. In order to determine whether this was due to failure of the branched-chain DNA assay to detect non-B subtypes of HIV, selected samples were subtyped and HIV RNA quantified by branched-chain DNA, NASBA, and the Roche Monitor RT-PCR assay. Twenty-eight (97%) of 29 Africans were infected with a non-B subtype of HIV and 15 (93.7%) of 16 non-Africans with subtype B. Twenty-three samples had a low viral load by branched-chain DNA, which was confirmed by the NASBA and RT-PCR assays. All three assays detected B and non-B subtypes with similar efficiency; NASBA failed to detect HIV RNA in a small number of non-B samples. Discrepancies between viral load and CD4(+) cell numbers did not appear therefore to be related to subtype. Second, while quantification of HIV RNA was being conducted using version 2 of the branched-chain DNA assay (lower detection limit 500 HIV RNA copies/ml) the manufacturers had developed a more sensitive assay and a comparative evaluation was therefore conducted. In approximately 30% of samples the viral load was up to 10 times higher with the more sensitive assay. These experiences emphasise the importance of close collaboration between the clinic and the laboratory.

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.

A comparative study of higher primate foamy viruses, including a new virus from a gorilla.

Few foamy (spuma) retroviruses have been investigated in molecular detail, despite their previous isolation from several mamalian species, including ten neutralization serotypes from various primates. Here, we have studied a new gorilla foamy virus (SFV-Gg) and investigated its functional and phylogenetic relationship to the human (HFV) and other primate foamy viruses, including that recently described in orangutans (SFV-11). Nucleotide sequencing of PCR products obtained from the R/U5 region of the LTR, gag, and pol genes revealed a close relationship between HFV and three chimpanzee isolates (SFV-6, SFV-7, and SFV-cpz). The SFV-Gg, SFV-11, rhesus macaque (SFV-1), and African green monkey (SFV-3) isolates were more divergent. To explore functional relationships, primate foamy virus transactivation of HFV LTR driven beta-galactosidase expression in a newly constructed cell line, BHLL, was investigated. HFV, SFV-6, and SFV-7 potently transactivated HFV LTR driven lacZ gene expression, SFV-Gg induced expression approximately 10-fold less efficiently, and SFV types 1, 2, 3, and 11 did not significantly transactivate the HFV LTR. It was, thus, possible to assay serum neutralizing activity in SFV-infected primates against HFV, SFV-6, and SFV-7 by reduction of beta-galactosidase activity following infection of the indicator cell line. Sera from infected chimpanzees and gorillas neutralized, to varying degrees, each of these three viruses, whereas orangutan sera did not. Our results, based on DNA sequences and functional assays, support the conclusion that HFV is closely related to foamy viruses of chimpanzee origin.

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.