An evaluation of health protection practices for the investigation and management of Cryptosporidium in England and Wales.

Background: Cryptosporidium is a major cause of gastroenteritis (cryptosporidiosis). Case and outbreak report rates vary geographically, which may in part reflect public health practice. Methods: To examine the public health management of cryptosporidiosis, an online questionnaire was administered to the 28 Health Protection Teams (HPTs) in England and Wales in 2014. Practices for investigation and management of cases and outbreaks were compared. Results: Practice varied among the 24 (86%) respondents in terms of who undertook actions (HPT or Local Authority) to investigate and manage cryptosporidiosis. HPTs without exceedance monitoring detected fewer outbreaks (1/5, 20%) than those with it (13/19, 68%) (P = 0.12), and those that always administered a risk-factor questionnaire detected more outbreaks (12/19, 63%) than those who did this only sometimes (2/5, 40%) (P = 0.62). Significantly more HPTs with a system to detect common exposures reported at least one outbreak (14/19, 74%) compared to HPTs with no system (0/5) (P = 0.01). Conclusions: Applying exceedance monitoring, using a standardized questionnaire taking into account the incubation period for Cryptosporidium, and having a structured system to detect common exposures increased outbreak detection. Information about all cases should be shared between local public health authorities, and current guidance used for the prevention of spread.

A randomised placebo-controlled trial to explore the effect of suppressive therapy with acyclovir on genital shedding of HIV-1 and herpes simplex virus type 2 among Zimbabwean sex workers.

OBJECTIVES: To determine the effect of daily acyclovir on genital shedding of HIV-1 and herpes simplex virus type 2 (HSV-2) in a randomised placebo-controlled trial among rural Zimbabwean sex workers. METHODS: 214 women were recruited and tested for HIV-1 and HSV-2 antibodies, HIV plasma viral load, CD4 lymphocyte count and genital swabs for qualitative detection of HIV-1 and HSV-2 genital shedding. Women were randomly assigned to acyclovir 400 mg twice a day for 12 weeks or matching placebo and were followed weekly to detect HIV-1 or HSV-2 genital shedding. Shedding analyses were only undertaken on 125 women co-infected with HSV-2 and HIV-1. Data were analysed using logistic regression, with random effects modelling used to account for repeated measurements on the same women. RESULTS: All women were randomly assigned to acyclovir or placebo; 125 of whom were co-infected with HIV-1 and HSV-2. 69 women were randomly assigned to acyclovir and 56 to placebo. Although twice daily acyclovir reduced rates of HSV-2 genital shedding, (adjusted odds ratio (AOR) 0.24; 95% CI 0.12 to 0.48; less than p<0.001), it had no effect on the proportion of visits at which HIV-1 shedding was detected (AOR 1.08; 95% CI 0.48 to 2.42; p = 0.9). Adherence varied between participants but even when adherence was high (as determined by pill count and extent of HSV-2 suppression) HIV-1 shedding was not reduced. CONCLUSION: Among these HIV-1 and HSV-2-seropositive women, suppressive acyclovir therapy had no effect on the rate of HIV genital shedding despite a reduction in genital HSV-2. Treatment adherence and its measurement clearly affect the interpretation of these results.

Decreased adenosine receptor binding in dystonic brains of the dt(sz) mutant.

In patients with paroxysmal non-kinesigenic dyskinesias, episodes of dystonia can be provoked by stress and also by methylxanthines (e.g. caffeine), which inhibit adenosine A(1)/A(2A) receptors. In the dt(sz) mutant hamster, a model of this movement disorder, adenosine A(1) receptor antagonists were previously found to worsen dystonia, while adenosine A(1) and A(2A) receptor agonists exerted pronounced beneficial effects. Therefore, in the present study, adenosine receptor A(1) and A(2A) binding was determined by autoradiographic analyses in dt(sz) hamsters under basal conditions, i.e. in the absence of a dystonic attack, and in a group of mutant hamsters which exhibited severe stress-induced dystonic attacks prior to kill. In comparison with non-dystonic control hamsters, [(3)H]DPCPX (8-cyclopentyl-1,3-dipropylxanthine) binding to adenosine A(1) receptors and [(3)H]CGS 21680 (2p-(2carboxyethylphen-ethylamino-5'-N-ethlycarboxamindoadenosine) binding to adenosine A(2A) receptors were significantly lower throughout the brain of dystonic animals. Under normal resting conditions, mutant hamsters showed significant decreases in adenosine A(1) (-12 to-42%) and in A(2A) (-19 to-34%) receptor binding compared with controls. Stressful stimulation increased adenosine A(1) and A(2A) receptor binding in almost all brain regions in both control and dystonic hamsters. The stress-induced increase was more marked in mutant hamsters, leading to a disappearance of differences in most regions compared with stimulated controls, except the striatum. In view of previous findings of striking beneficial effects of adenosine A(1) and A(2A) receptor agonists and of striatal dysfunctions in the dt(sz) mutant, the reduced adenosine receptor binding may be an important factor in the pathogenesis of paroxysmal dystonia.

National surveillance of HIV-1 subtypes for England and Wales: design, methods, and initial findings.

The HIV-1 infections detected in an ongoing national unlinked anonymous HIV surveillance program were subtyped and analyzed according to demographic and risk characteristics. Of the 893 anti--HIV-1--positive specimens allocated to an exposure group, 70% could be subtyped. Almost 25% of infections subtyped were non-B, mostly subtypes A, C, and D. Non-B infections were rare in homosexual and bisexual men and in drug injectors. Forty percent of infections in heterosexual men attending genitourinary medicine clinics were non-B subtypes; of these, 25% were subtype A infections and 59% were subtype C infections. For female clinic attendees, 61% were non-B subtype infections, of which 48% were subtype A infections and 42% were subtype C infections. Of the clinic attendees born in the United Kingdom and Europe, 14% of the men and 35% of the women were infected with non-B subtypes. In contrast, 78% of infections in antenatal patients were non-B subtypes, of which 61% were subtype A and 29% were subtype C. Extrapolation to the estimated 29,700 prevalent adult infections in the United Kingdom indicates that approximately 8,100 (27%) such infections are non-B subtypes and that these are found almost entirely in heterosexuals. The findings suggest spread of infection of non-B subtypes to heterosexuals born in the United Kingdom from individuals infected in regions of high prevalence.

Recombinant strains of HIV type 1 in the United Kingdom.

Twenty-five recombinant (mosaic) HIV-1 genomes were detected among 151 samples comprising 118 non-B subtype sequences and 33 samples containing subtype B sequences. Seven of the 25 mosaic patterns were similar to characterized circulating recombinant forms (two A/E, four A/G, and one D/F) and one was a MAL-like A/D recombinant. Eighteen of the recombinants had evidence of subtype A sequences in at least one region of their genome. One sample was found to contain a novel recombinant form (pol F, env K). Two samples could not be characterized unambiguously as recombinant forms and a further one appeared to be a complex C/J/D/A genomic form. The majority of the mosaic genomes were recombinants between gag, pol, or env, whereas the C/J/D/A mosaic had cross-over breakpoints within pol. These findings suggest that almost 20% of non-B subtype isolates of HIV-1 circulating in the United Kingdom have mosaic genomes. This shows the diverse origin of HIV-1 strains circulating in the United Kingdom and may have implications for antiretroviral drug resistance.

Viral genome characterisation by the heteroduplex mobility and heteroduplex tracking assays.

The heteroduplex mobility assay (HMA) is a means of comparing two PCR amplicons or, in the variation known as the heteroduplex tracking assay (HTA), a means of estimating the quasispecies diversity of a viral genome. Heteroduplex assays have many applications including subtyping viral genomes, screening for low frequency variants in a population, scanning the relative genetic diversity across a genome and screening for recombinant clones. They can be used to detect dual infections, superinfections, contaminated blood products and laboratory contaminations. PCR amplicons of about 65% sequence similarity or greater will form heteroduplexes under appropriate conditions, and phylogenetic trees can be drawn from heteroduplex mobility data. While homoduplexes indicate more than 98% similarity between two DNA sequences, heteroduplexes indicate at least seven mismatches in a 500-bp amplicon, or a three-base pair gap in 1000-bp. Minority variants comprising 1% to 5% of the genome population can be detected and quantified by HTA. Thus far, heteroduplex assays have been described for HIV and other lentiviruses, hepatitis C and G viruses, Norwalk-like viruses, influenza, measles and poliovirus. They could be applied to a wide range of other viral species.

A gag gene heteroduplex mobility assay for subtyping HIV-1.

A heteroduplex mobility assay (HMA) using 753 and 446 base pair (bp) amplicons of the p17/p24 region of the gag gene of HIV-1 has been developed and validated with reference clones and clinical samples representative of subtypes A, B, C, D, E, G, and H. There was complete concordance between the gag HMA assigned subtype and the subtype known from gag or env sequence data or env HMA. The heteroduplexes from both amplicons can be clearly resolved on either MetaPhor XR agarose or MDE polyacrylamide gels. The MetaPhor XR gel system was the more convenient and is the preferred choice for routine HMA subtyping. This gag HMA provides a rapid, simple and inexpensive method for subtyping HIV-1 based on a genomic region other than the commonly used env gene target. The incorporation of gag HMA into subtype determination algorithms should allow the detection of gag/env recombinant strains of HIV-1.

Post-transfusion HIV infection despite donor screening: a report of three cases.

In August 1996, a blood donation was collected which subsequently infected three patients post-transfusion with HIV 1. The donation itself was originally screened as negative for anti-HIV 1/2 using a sensitive EIA method, but subsequently was shown to contain p24 Ag and HIV RNA by an amplification technique. The proposed introduction of nucleic acid testing of all blood donations in the UK for hepatitis C, hepatitis B and HIV may further reduce the remote risk of further episodes of post-transfusion infection. The infection in the index recipient was detected on routine pre-transplant virological screening but proved difficult to confirm, at a time when she had recently received myeloablative treatment for a haematological malignancy which impaired the immune response. There is a need for continued vigilance in such patients to exclude post-transfusion infection, at a time when natural immunological responses have been impaired by their disease or treatment.

Performance of the Amplicor human immunodeficiency virus type 1 PCR and analysis of specimens with false-negative results.

Over a 4-year period, the Roche Amplicor kit was used in a United Kingdom reference laboratory for the detection or confirmation of human immunodeficiency virus (HIV) type 1 infection, particularly in infants born to HIV-infected mothers. Of 408 specimens from adults and older children tested, the 122 seronegative specimens were all Amplicor negative. Of the 286 seropositive specimens, 268 were Amplicor positive. On the basis of these results, the Amplicor assay has a specificity of 100% and a sensitivity of 93.7%. In addition, for 247 specimens from infants and young children, serological results may not have been diagnostic because of placental transfer of maternal antibodies. Forty-eight were Amplicor positive, and of the 199 Amplicor-negative specimens, 19 were assumed to be false negative on the basis of clinical data, serological markers (including p24 antigen), and/or results for previous or follow-up specimens. This represents a sensitivity of 75% for the Amplicor test for specimens from patients under 2 years of age. Of these 37 false-negative specimens plus 2 specimens from other laboratories, 31 could be characterized by amplifying extracted material from them by an in-house nested gag PCR spanning the Amplicor target region. The amplicons were sequenced and found to represent subtypes A (35.5%), B (22.6%), C (22.6%), D (16.1%), and G (3.2%). False-negative results by the Amplicor assay may be ascribed to low-target copy number, the physical behavior of one primer (SK462), and sequence variation in the target region of the other primer (SK431).

Analysis and genotyping of PCR products of the Amplicor HIV-1 kit.

The Roche Amplicor PCR kit was used to detect HIV-1 DNA in UK patients of known serostatus. Four false-negative and/or equivocal results were obtained from patients who were known to be anti HIV seropositive (Tosswill et al., 1994). Cells from the blood of these patients were shown to contain HIV DNA after extraction, concentration and amplification by nested PCR using primers flanking those in the kit. To determine whether DNA sequence divergence was the cause of these discrepancies, the gag region targeted by the primers in the kit was sequenced for specimens giving positive, equivocal and false-negative results. No greater degree of sequence divergence was found within the primer and probe target regions among the equivocals and false-negatives than among the positive control specimens. The few misleading results were probably attributable to low copy numbers of proviral DNA in these specimens. Sequences obtained from the target and flanking regions of the kit were sufficient to allow the genotype of the virus to be determined.

Genomic diversity of small round structured viruses in the United Kingdom.

Fifty-two faecal specimens collected in the United Kingdom between 1986 and 1992, which contained small round structured virus (SRSV) particles, were tested by reverse transcriptase polymerase chain reaction assays using two primer pairs derived from sequences of Snow Mountain Agent and Norwalk virus. There was poor correlation between results obtained with each primer pair. Twenty specimens (38%) gave positive bands with SM51/31 primers and 18 (34%) were positive with SM52/32 primers, with a total of 30 specimens (57.7%) giving amplification products of the expected size with one or both primer pairs. Genomic variation was investigated by sequencing a 266 bp region of the RNA polymerase gene from nine strains which had been antigenically typed by solid phase immune electron microscopy (SPIEM). RNA sequence identities ranged from 53 to 99%. Three genomic groups were suggested by phylogenic analysis, the first of which contained Norwalk virus, Southampton virus, and strains typed by SPIEM as SRSV UK2. The second contained Snow Mountain agent and strains typed as either SRSV UK3 or UK4. The third contained strains typed as SRSV UK1 and strains untypeable by SPIEM. Some correlation was demonstrated when antigen typing by SPIEM and phylogenic grouping based on sequence data were compared.