Enzyme linked immunosorbent assay (ELISA) for the detection of serum antibodies to the inner core lipopolysaccharide of Neisseria meningitidis group B.

We have developed a solid-phase ELISA to study the human immune response to inner core lipopolysaccharide (LPS) of Neisseria meningitidis (Nm) using structurally defined glycolipids from a genetically defined mutant (galE) of a serogroup B Nm strain. Previous studies had demonstrated that a galE (inner core) LPS epitope is conserved in approximately 70% Nm strains and was accessible to antibody in fully encapsulated wild-type Nm strains. A murine monoclonal antibody, MAb B5, raised to a galE mutant of serogroup B Nm strain, immunotype L3 (B.15.P1.7,16) was used to determine the specificity of the inner core LPS ELISA by inhibition studies using purified galE LPS and human sera. The intra-assay coefficient of variation (CV) was 5-6% and inter-assay CV was 19-22%. Using this ELISA, significant differences in the geometric mean titres (GMTs) of naturally occurring serum antibodies (specific to inner core LPS) between healthy adults (18-65 years, N=54) and healthy infants (3-4 months, N=144) of both IgG and IgM classes were found (P<0.0001). GMTs were expressed in galE arbitrary units (AU/ml) (95% confidence intervals): IgG antibodies in adults 5.7 (5. 0,6.9) and in infants 1.1 (1.0,1.3); IgM antibodies in adults 7.7 (5. 7,10.4), and in infants 0.85 (0.7,1.1). In age-matched children aged 26-113 months a difference (P=0.04) in specific IgG was found in healthy infants and infants in the acute phase of invasive Nm disease (GMT (95%CI) in AU/ml: in healthy infants 7.7 (5.3,11.0), in acute phase infants 4.2 (2.5,7.2). However, there was no difference in specific IgM (P=0.98) between these groups healthy infants 4.7 (3. 1,7.0), acute phase 4.6 (2.9, 7.4). In eleven children (5-181 months) there were differences in the GMTs of specific IgG and IgM (P=0.02, P=0.008 respectively) between paired acute and convalescent sera (GMT) (95%CI) in AU/ml: IgG acute 1.95 (0.98, 3.8), convalescent 5.2 (2.2,12.4); IgM acute 1.78 (1.05,3.0), convalescent 4.38 (2.6,7.3). We conclude that ELISA is a specific, sensitive and reproducible method for the detection of antibodies to inner core LPS of Nm and that an epitope defined by MAb B5 can be immunogenic in infants and adults. These findings are relevant to the potential candidacy of inner core LPS as a vaccine.

Random mandatory drugs testing of prisoners: a biassed means of gathering information.

BACKGROUND: Our objective was to develop and test a methodology for inferring the percentage of prisoners currently using opiates from the percentage of prisoners testing positive for opiates in random mandatory drugs testing (rMDT). METHODS: The study used results from Willing Anonymous Salivary HIV (WASH) studies (1994-6) in six adult Scottish prisons, and surveys (1994-5 and 1997) in 14 prisons in England and Wales. For Scottish prisons, the percentage of prisoners currently using opiates was determined by assuming, with varying empirical support, that: current users of opiates in prison were 1.5 times as many as current inside-injectors; and current inside-injectors were 0.75 times as many as ever injectors in prison. We also assumed that current inside-users' frequency of use of opiates (by any route) was equal to the frequency of inside-injecting by current inside-injectors in Aberdeen and Lowmoss Prisons in 1996, namely six times in 4 weeks. We assumed that some scheduling of heroin-use prior to weekends takes place, so that only 50% of current inside-users of opiates would test positive for opiates in rMDT: these assumptions allow us to arrive at WASH-based expectations for the total percentage of prisoners testing positive for opiates in rMDT. For England and Wales, a multiplier of 118/68 was applied which was derived from prisoners' interviews, to convert the results from ever inside-injectors, as determined by WASH studies, to the percentage of current inside users of opiates. We made the same assumptions on frequency of inside-use of opiates as in dealing with the Scottish results. RESULTS: We expected 202.7 opiate positive results in April to September 1997 in rMDTs at six adult prisons in Scotland, 226 were observed. We expected 227.0 at a set of 13 adult prisons and one other in England and Wales; 211 were observed. CONCLUSIONS: Further testing of the methodology for prisons in England and Wales will be possible when 1997 WASH data are released. So far, the methodology has performed well. From it, we infer that 24% of inmates at the six adult prisons in Scotland were current inside-users of opiates, compared to 11% at the 14 adult prisons where survey data were available in England and Wales. The corresponding April to September 1997 percentage of opiate positives in rMDT were: 13% (results from the six Scottish prisons) and 5.4% (results from 14 prisons in England and Wales), a two-fold under-estimate of % current users of opiates in prison (24% and 11%). Planning of drug rehabilitation places for prisoners should thus be based on twice the percentage of prisoners testing opiate positive in rMDT. This correction factor of two should be kept under review.

Method used to identify previously undiagnosed infections in the HIV outbreak at Glenochil prison.

Four years after the occurrence of an outbreak of hepatitis B and HIV infection among injecting drug user inmates at Her Majesty's Prison Glenochil in Scotland, a study design was developed to complete the epidemiological account of the HIV outbreak. Our aim was to identify potential cases of (1) HIV transmission not diagnosed during the original outbreak investigation and (2) the source(s) of the outbreak. Scotland's HIV positive case register was searched for matches to a soundexed list of 636 Glenochil inmates imprisoned during January-June 1993. Eight HIV infections that may have been acquired in Glenochil and four possible sources of the outbreak were identified. The second stage of follow-up molecular epidemiological techniques used on stored sera samples from identified individuals is described in the companion paper. Without breach of medical or prisoner confidentiality, indirect and anonymous follow-up has proved possible for the Glenochil inmates.

Conservation and accessibility of an inner core lipopolysaccharide epitope of Neisseria meningitidis.

We investigated the conservation and antibody accessibility of inner core epitopes of Neisseria meningitidis lipopolysaccharide (LPS) because of their potential as vaccine candidates. An immunoglobulin G3 murine monoclonal antibody (MAb), designated MAb B5, was obtained by immunizing mice with a galE mutant of N. meningitidis H44/76 (B. 15.P1.7,16 immunotype L3). We have shown that MAb B5 can bind to the core LPS of wild-type encapsulated MC58 (B.15.P1.7,16 immunotype L3) organisms in vitro and ex vivo. An inner core structure recognized by MAb B5 is conserved and accessible in 26 of 34 (76%) of group B and 78 of 112 (70%) of groups A, C, W, X, Y, and Z strains. N. meningitidis strains which possess this epitope are immunotypes in which phosphoethanolamine (PEtn) is linked to the 3-position of the beta-chain heptose (HepII) of the inner core. In contrast, N. meningitidis strains lacking reactivity with MAb B5 have an alternative core structure in which PEtn is linked to an exocyclic position (i.e., position 6 or 7) of HepII (immunotypes L2, L4, and L6) or is absent (immunotype L5). We conclude that MAb B5 defines one or more of the major inner core glycoforms of N. meningitidis LPS. These findings support the possibility that immunogens capable of eliciting functional antibodies specific to inner core structures could be the basis of a vaccine against invasive infections caused by N. meningitidis.

How many drug rehabilitation places are needed in prisons to reduce the risk of bloodborne virus infection?

Transmission of HIV and hepatitis B virus infection has been recognised in prisons, and injecting drug use is a major route of infection. Combined results of two pilot health care surveys showed that 47% of prisoners with a history of injecting drug use wanted help to give up class A drugs but only 11% of non-injecting drug users expressed a similar wish. It would therefore seem appropriate for prisons to estimate the number of inmates with a history of injecting drug use and provide drug rehabilitation places for half that number (47% rounded up). Data from three prisons in England and Scotland for which the numbers of drug rehabilitation places were known showed that they provided less than quarter of the minimum requirement based on this formula. The proportion of inmates with a history of injecting or of non-injecting drug use who want help to give up class A drugs requires further investigation in order to refine the needs formula.

Prisoners' views about the drugs problem in prisons, and the new Prison Service drug strategy.

Three hundred and seventy-five out of 575 prisoners (222/299 drug users and 153/267 non-users) who responded to a self-completion health care questionnaire at two prisons in 1997 commented on drugs in prisons. One hundred and forty-eight out of 176 responses expressed negative opinions about mandatory drugs testing (MDT), and 107 said that MDT promoted switching to or increased use of heroin/hard drugs'. Sixty-two prisoners suggested that more help/counselling was needed for drug users, 52 segregation of drug users/drug-free wings, and 50 more security on visits/in corridors after medication. The new Prison Service drug strategy has revised random MDT. It targets those who supply drugs, and supports those who want to stop using drugs, and accords with prisoners' views about the heroin problem in prisons.

Prevalence of hepatitis C in prisons: WASH-C surveillance linked to self-reported risk behaviours.

We used cross-sectional willing anonymous salivary hepatitis C (WASH-C) surveillance linked to self-completed risk-factor questionnaires to estimate the prevalence of salivary hepatitis C antibodies (HepCAbS) in five Scottish prisons from 1994 to 1996. Of 2121 available inmates, 1864 (88%) participated and 1532/1864 (82%) stored samples were suitable for testing. Overall 311/1532 (20.3%, prevalence 95% CI 18.3-22.3%) were HepCAbS-positive: 265/536 (49%, 95% CI 45-54%) injector-inmates but only 27/899 (3%, 95% CI 2-4%) non-injector-inmates. Among injectors, HepCAbS positivity was only slightly higher (p = 0.03) in those who had injected inside prison (53%, 162/305) than in those who had not (44%, 98/224). Those who began injecting in 1992-96 were much less likely to be HepCAbS-positive than those who started pre-1992 (31%, 35/114 vs. 55%, 230/422; p < 0.001). Even with injectors who began in 1992-96 but had never injected inside prison, the prevalence of hepatitis C carriage was 17/63 (95% CI 16-38%). The prevalence and potential transmissibility of hepatitis C in injector-inmates are both high. Promoting 'off injecting' before 'off drugs' (both inside and outside prison), methadone prescription during short incarcerations, alternatives to prison, and support of HepCAbS-positive inmates in becoming eligible for treatment, all warrant urgent consideration.

X-Linked agammaglobulinemia patients are not infected with Epstein-Barr virus: implications for the biology of the virus.

Epstein-Barr virus (EBV) infects both B lymphocytes and squamous epithelial cells in vitro, but the cell type(s) required to establish primary and persistent infection in vivo has not been definitively elucidated. The aim of this study was to investigate a group of individuals who lack mature B lymphocytes due to the rare heritable disorder X-linked agammaglobulinemia in order to determine the role of the B cell in the infection process. The results show that none of these individuals harbored EBV in their blood or throat washings. Furthermore, no EBV-specific memory cytotoxic T lymphocytes were found, suggesting that they had not undergone infection in the past. In contrast, 50% of individuals were found to carry human herpesvirus 6, showing that they are infectible by another lymphotropic herpesvirus. These results add weight to the theory that B lymphocytes, and not oropharyngeal epithelial cells, may be required for primary infection with EBV.

Study size and documentation to detect injection-related hepatitis C in prison.

We used existing data on hepatitis C prevalence, injection-related hepatitis C transmission and needle use in prisons and new data on infectiousness, to estimate the size of study required to detect injection-related hepatitis C in UK prisons. A pilot study of 500 prisoners followed for 10 weeks would have a 65% chance of detecting a hepatitis C seroconversion, conservatively assuming one injection per prisoner per week, and a 3% transmission rate per injection, but uncertainty might persist as to whether transmission had occurred during a short incarceration or before it. If the actual transmission rate was 10%, as recently documented, then such a study would have more adequate statistical power. A definitive study of 3000 prisoners for 10 weeks would expect to detect about six seroconversions, even with conservative estimates of injection frequency and transmission rate. Adequate design and power of these studies is important because of the complacency that could result from false-negative findings. We suggest six risk-factor themes that studies should document.

Harm reduction measures and injecting inside prison versus mandatory drugs testing: results of a cross sectional anonymous questionnaire survey. The European Commission Network on HIV Infection and Hepatitis in Prison.

OBJECTIVES: (a) To determine both the frequency of injecting inside prison and use of sterilising tablets to clean needles in the previous four weeks; (b) to assess the efficiency of random mandatory drugs testing at detecting prisoners who inject heroin inside prison; (c) to determine the percentage of prisoners who had been offered vaccination against hepatitis B. DESIGN: Cross sectional willing anonymous salivary HIV surveillance linked to a self completion risk factor questionnaire. SETTING: Lowmoss prison, Glasgow, and Aberdeen prison on 11 and 30 October 1996. SUBJECTS: 293 (94%) of all 312 inmates at Lowmoss and 146 (93%) of all 157 at Aberdeen, resulting in 286 and 143 valid questionnaires. MAIN OUTCOME MEASURES: Frequency of injecting inside prison in the previous four weeks by injector inmates who had been in prison for at least four weeks. RESULTS: 116 (41%) Lowmoss and 53 (37%) Aberdeen prisoners had a history of injecting drug use but only 4% of inmates (17/395; 95% confidence interval 2% to 6%) had ever been offered vaccination against hepatitis B. 42 Lowmoss prisoners (estimated 207 injections and 258 uses of sterilising tablets) and 31 Aberdeen prisoners (229 injections, 221 uses) had injected inside prison in the previous four weeks. The prisons together held 112 injector inmates who had been in prison for more than four weeks, of whom 57 (51%; 42% to 60%) had injected in prison in the past four weeks; their estimated mean number of injections was 6.0 (SD 5.7). Prisoners injecting heroin six times in four weeks will test positive in random mandatory drugs testing on at most 18 days out of 28. CONCLUSIONS: Sterilising tablets and hepatitis B vaccination should be offered to all prisoners. Random mandatory drugs testing seriously underestimates injector inmates' harm reduction needs.

Anonymous HIV surveillance with risk-factor elicitation: at Perth (for men) and Cornton Vale (for women) prisons in Scotland.

434 male and 145 female prisoners were available to participate in cross-sectional, voluntary anonymous HIV surveillance (using saliva samples) with linked self-completion questionnaire at HMP (Her Majesty's Prison) Perth on 17 May and at HMP Cornton Vale on 18 May 1995. Three hundred and four men (70%) and 136 women (94%) completed a risk-factor questionnaire and 304 and 135 samples were received for HIV antibody testing. Two hundred and eighty-two and 132 questionnaires passed logical checks. Six saliva samples from Perth (all injectors) out of 304 and none from Cornton Vale out of 134 tested were HIV antibody positive. Four were presumptively from known HIV-infected male inmates; the other 2 were local men, under 26 years, who began injecting in 1989-91, and both reported having had a recent HIV test. Overall HIV prevalence was estimated at 2% compared to a known prevalence of 1.4% (6/434), giving a 1.5 ratio of overall: disclosed HIV prevalence at HMP Perth. HIV prevalence was estimated at 7% (6/82) for injector-participants and 14% (5/35) for local injector participants. At Cornton Vale, where both known HIV-infected inmates abstained, overall and disclosed HIV prevalence, were equal at 1.4%. At Perth Prison, 29% of prisoners had injected drugs (82/278); 85% of injector-inmates reported having injected inside (some prison and 31% (25/80) had started to inject while inside, 7 during their present sentence. Of all 21 injector-inmates who first injected after 1991, 10 had started to inject inside, including one of 69 male inmates who had never been inside before. The corresponding figures for Cornton Vale, where 46% of inmates were injectors (58/132), were that 57% of injector-inmates had injected inside (32/56) but only one woman, for whom this was not her first sentence, had started to inject inside. Twenty-eight per cent of male prisoners (78/277) and 57% of male injector-inmates (47/82) had had a personal HIV test since January 1993, as had 35% of female prisoners (43/124) and 57% of female injector-inmates (30/53). A much higher proportion of Glasgow's female prisoners (64%: 38/60) were injectors than of women prisoners from the Edinburgh, Dundee and Fife area (21%: 5/26) or from elsewhere (34%: 15/45). Rape was reported by 23% of women (30/130). Women who had been raped had a more polarized distribution of male sexual partners (none to 2 plus) in the year before sentencing than other women and were more likely to report anal sex (11/30 vs 11/100, P < 0.001). Prostitution had been engaged in by 19% of female injector-inmates (11/57) and was acknowledged by one other woman. However, only 5% of women (6/130) reported ever having been treated for an STD.

Immunodeficiency in non-tuberculous mycobacterial disease.

T-cell immunity was investigated in eight patients with non-tuberculous mycobacterial disease, to see whether impaired immune function might be the explanation for their infection. Cellular immune function was evaluated in vitro by measuring the proliferation of peripheral blood mononuclear cells in response to both non-specific mitogens (phytohaemagglutinin and pokeweed mitogen) and specific recall antigens (streptokinase-streptodornase and purified protein derivative from Mycobacterium tuberculosis), and in vivo, by measuring the skin test response to a panel of recall antigens. Functionally relevant T-lymphocyte sub-populations (CD4, CD8, activated CD3 and gamma/delta T-cells) were enumerated by two-colour flow cytometry. The results were compared with those for a group of patients with pulmonary tuberculosis, with groups of controls matched for age and smoking habit, and with a patient group receiving steroid treatment. The patients with non-tuberculous mycobacterial disease had poor or absent skin test responses; in vitro, their response to recall antigens was depressed, although their response to mitogens was normal. The patients had significantly raised levels of CD8 lymphocytes and activated T-cells, but lacked any circulating gamma/delta T-cells. There were also differences between the various control groups. In conclusion, this study demonstrates a deficiency in the cellular immune system of these patients, which is most readily detectable by skin testing, or by measuring lymphocyte proliferative responses to recall antigens. However, the study also shows changes in cellular immune responses in controls matched for age and smoking and in patients on steroid treatment, and underscores the need for matched controls. Further work needs to be done to ascertain whether the cellular immune deficiency is a cause of, or is caused by, the mycobacterial infections, and also to investigate the pathological significance of the alterations in T-cell sub-populations.

Cost implications of random mandatory drugs tests in prisons.

BACKGROUND: Compulsory urine testing of prisoners for drugs, a control initiative, was introduced in eight prisons in England and Wales early in 1995. Despite no evidence of effectiveness, testing was extended to all prisons in England and Wales by March 1996. We consider the cost of testing. METHODS: We combined the costs of refusals, confirmatory tests, punishment of confirmed positives for cannabis or for class A drugs to estimate the average costs of random compulsory drugs testing. These costs were then compared to: i) the healthcare budget for a prison; and ii) the cost of putting in place a credible prisons' drugs reduction programme. We then used Scottish data on incarceration and regional prevalence of injecting drug users to estimate the extent of the injecting drug use problem that prisons face. FINDINGS: Costs per 28 days of the random mandatory drugs testing control initiative in an establishment for 500 inmates where refusal rate is a) 10% or b) nil; and 35% of urine samples test positive, one tenth of them for class A drugs were estimated at between a) 22,800 UK pounds and b) 16,000 UK pounds per 28 days [a) $US35,100 and b) $US24,600]. This cost was equivalent to twice the cost of running a credible drugs reduction and rehabilitation programme, and around half the total healthcare expenditure for a prison of 500 which averaged 41,114 UK pounds per 28 days [$US64,860]. Major cost-generating events were the punishment of refusals--over one third of cost a)--and testing positive for cannabis--over 50% of cost a). In Scotland, around 5% of injecting drug users (IDUs) are incarcerated at any time: 5% of Lothian's drugs care, treatment and prevention costs and 2.5% of its HIV/AIDS prevention budget in 1993-94 amounted to 101,300 UK pounds per annum--or 7770 UK pounds per 28 days ($US11,970)--and about 35% of monthly MDT costs. INTERPRETATION: We suggest that 5% of current resources for drugs prevention and treatment and for IDU-targetted HIV/AIDS prevention should be directed towards the prisons because in the prisons, where 5% of the clients are at any time, injectors have less access to harm reduction measures than on the outside.

Human cytomegalovirus infection is not increased in common variable immunodeficiency.

It has been postulated that human cytomegalovirus (HCMV) infection may have a role in the pathogenesis of common variable immunodeficiency (CVID). Many patients have a lymphocyte phenotype similar to that seen in HCMV infection, HCMV mononucleosis may precipitate hypogammaglobulinaemia, and a previous small study of common variable immunodeficient patients reported a high rate of active HCMV infection. This study investigated the presence and activity of HCMV infection in 102 CVID patients. Buffy coats were examined for the presence of HCMV IE and glycoprotein B genes using highly sensitive nested PCR. 30 blood donors of known HCMV serologic status were used as controls. There was no significant difference in HCMV positivity by PCR between patients and controls. Enrichment for mononuclear cells prior to PCR had no effect on sensitivity. Twenty-five patients were also examined for HCMV antigenaemia by staining buffy coat cytospins with monoclonal antibodies directed against the HCMV pp65 lower matrix protein, a technique widely used for diagnosis of active HCMV disease. Only one patient was positive (and also positive by PCR). Whilst these results do not exclude prior infection contributing to antibody deficiency in a small proportion of CVID patients, this study refutes the previously reported increase in active HCMV infection in CVID.