A sensitive immunoassay for flunitrazepam and metabolites.

The objective of this study was to develop an immunoassay that would be capable of detecting flunitrazepam and/or cross-reacting metabolites in urine and comparing the results with those obtained by gas chromatography-mass spectrometry. Doses of Rohypnol varying between 0.5 and 4 mg were given to volunteers, and urine was collected for up to two weeks postingestion. These samples were analyzed by an ELISA that was developed using an antibody raised to flunitrazepam and a drug-enzyme conjugate prepared by attaching 7-aminoflunitrazepam to horseradish peroxidase. Significant levels of flunitrazepam and/or cross-reacting metabolites were detected in urine for up to one week after ingestion. The immunoassay is selective with only diazepam cross-reacting at a level of 1000 microg/L.

Cost-effective analysis of hepatitis A prevention in Ireland.

OBJECTIVE: The purpose of this study was to determine the most cost-effective prevention strategy against hepatitis A virus (HAV) infection for healthcare workers and the general population at risk in Ireland. METHODS: Four prevention strategies were compared: active immunization with Havrix Monodose (1440E.U); screening for anti-HAV antibody and then vaccinating; passive immunization; screening for anti-HAV antibody and then passive immunization. The cost-effective ratio was calculated for each prevention strategy. Threshold analysis, sensitivity analysis, and model extension to include indirect cost from work days lost and secondary attack rates through horizontal transmission were also derived. RESULTS: The medical costs were lowest and the infection rate highest when no preventive action was taken. Vaccination was most cost effective when the prevalence of immunity was 45% or less, reducing the infection rate by 98% when compared to nonprevention. Screening before vaccination was most cost effective when the prevalence of immunity was greater than 45%. Passive immunization and screening before passive immunization were not comparable to the other strategies in cost effectiveness. Sensitivity analysis showed that the cost-effective ratio for vaccination was dependent on vaccine price, incidence of HAV, and prevalence of immunity in the target group. Extending the model to include indirect costs further increased the cost effectiveness of vaccination. CONCLUSION: The best cost-effective strategy relates to target group immunity. Where HAV immunity is 45% or less, vaccination is the strategy of choice and when immunity is greater than 45%, then screening followed by vaccination should be used. This study can be used to provide a framework within which choices can be made to achieve better health for less cost.

A comparison of five commercial immunoassays for the detection of flunitrazepam and other benzodiazepines in urine.

Five commercially available immunoassay test kits (SYVA EMIT(R) d.a.u(TM), SYVA EMIT(R) II assay, Abbott FPIA, Cozart Auto-Lyte(R) and Roche Abuscreen(R) Online(TM), all used for the benzodiazepine group of drugs) were evaluated for their ability to detect flunitrazepam, its major urinary metabolite, 7-aminoflunitrazepam, and several other benzodiazepines at serial dilutions (final concentration 25-1000 ng/ml) in drug-free urine and in urines following oral administration of flunitrazepam (1-3 mg). For comparison, gas chromatography/mass spectrometry was used to measure urinary levels of 7-aminoflunitrazepam. Levels of drug detected in the study were compared with the cross-reactivities presented by the manufacturers for each individual kit. One to three mg doses of flunitrazepam were taken by volunteers and levels excreted in urine analysed over several hours. A positive response was obtained in several samples from volunteers who had taken 2 mg or 3 mg doses, but not a 1 mg dose. Thirty-five clinical samples from the individuals suspected of benzodiazepine abuse were also examined. The results were not consistent among the kits evaluated.We conclude that the test kits evaluated in this study do not detect flunitrazepam reliably, due primarily to their poor sensitivities.

Hepatitis A in urban Ireland.

AIMS: To determine the prevalence of immunity to hepatitis A virus (HAV) infection in urban Ireland and to categorize the region into low, intermediate or high HAV endemicity, and to analyse the significance of certain commonly associated risk factors. METHODS: Two hundred and thirty three volunteers were recruited from 6 general practices in Dublin, Ireland. There were 44 volunteers in the 10 to 19 yr age group, 40 in the 20 to 29, 42 in the 30 to 39, 43 in the 40 to 49 and 64 in the over 50 age groups. Each participant completed a detailed questionnaire and was tested for anti-HAV total antibody (primarily IgG) using a competitive ELISA assay. Urban Ireland was classified into the appropriate area of HAV endemicity according to the prevalence of immunity by age group. Risk factor differences were analysed for significance using the chi square test and Fisher's exact test. RESULTS: One hundred and fifty seven (67 per cent) volunteers were immune, of whom 20 (45 per cent) were in the 10 to 19 yr age group, 17 (43 per cent) in the 20 to 29, 30 (71 per cent) in the 30 to 39, 34 (79 per cent) in the 40 to 49 and 59 (92 per cent) in the over 50 age groups. Fifty-five per cent of the individuals studied below the age of 20 yr were non-immune. The immune rates over the age of 30 were significantly greater (p < 0.01) than those in the 10 to 29 age groups. Socioeconomic pattern in the total and 10 to 19 yr age group was a significant (p < 0.0002, p < 0.004 respectively) risk factor for infection. CONCLUSION: This study concludes that urban Ireland is an area of low HAV endemicity with age and socioeconomic status as the significant influences on seropositivity. This survey provides an insight into the changing epidemiology of HAV infection in Ireland and serves as a guide for immunisation of at risk population groups.

Transmission of a homogenous hepatitis B virus population of A1896-containing strains leading to mild resolving acute hepatitis and seroconversion to hepatitis B e antigen antibodies in an adult.

The pre-core variant, A1896, which switches off hepatitis B e antigen (HBeAg) production, is common in hepatitis B e antigen antibody (anti-HBe)-positive chronic hepatitis patients. It has been observed in occasional case reports of acute hepatitis. However, transmission in the absence of HBeAg-producing strains, leading to acute nonfulminant hepatitis and clearance in adults, has not been reported. Here, we show that this event can occur, further confirming that A1896 strains are "wild-type" and can lead to all the same outcomes as G1896 strains. This is in keeping with phylogenetic evidence that A1896 is transmitted independently on a large scale in the population and explains anti-HBe- positive persons who have not had an HBeAg-positive phase documented.

The reliability of saliva as a sample for the detection of hepatitis A immunoglobulins under various sampling conditions.

BACKGROUND: Saliva is increasingly being investigated as an alternative to serum for diagnostic and epidemiological testing even though antibody levels are substantially lower in buccal cavity fluids. However, there has been little study on whether buccal cavity activity and/or the timing of saliva sampling affects the diagnostic outcome, particularly in seropositive subjects. The absence of influence by these factors may be critical to the use of saliva for pre-vaccination screening for example. OBJECTIVES: The effects of eating, brushing of teeth and circadian rhythm on the measureable salivary immune status of 42 healthy individuals known to be serum and saliva anti-HAV positive were examined. STUDY DESIGN: A total of 141 saliva samples obtained from the 42 healthy subjects, before and after meals, before and after brushing of teeth and at various timepoints throughout the day, were assayed for total anti-HAV using an in-house saliva based enzyme-immunoassay, previously shown to have a 100% correlation in terms of sensitivity and specificity with a serum based assay. RESULTS: The results indicated that total anti-HAV titres varied according to the time of day and that eating had no significant effect on the total anti-HAV titre, but brushing of teeth did. Titres never varied to the extent that a result was falsely negative at any timepoint. CONCLUSION: These results confirm the usefulness of saliva as a diagnostic sample for the detection of hepatitis A antibody, regardless of sampling times, eating or tooth-brushing.

Two year old hepatitis A vaccine is as good as new.

This was a randomized, controlled, double-blind study assessing the reactogenicity and immunogenicity of newly produced vs 2 year old hepatitis A vaccine. Overall 215 non-immune volunteers, 18-39 years old were divided into four groups and administered vaccine at months 0, 1 and 6. Three groups each received a different vaccine lot which had been stored at 4 degrees C for 2 years, and one group received recently produced vaccine as control. The mean local and general adverse reaction rates were 59.1% and 17.4%, respectively, and all vaccinees had seroconverted by month 2. There were no significant differences in geometric mean anti-hepatitis A virus (HAV) antibody titres between the four groups. In conclusion 2 year old HAV vaccine is safe and equally immunogenic as newly produced vaccine.

Hepatitis B in the non-residential mentally handicapped population.

This study describes seroprevalence and risk factors for hepatitis B in seven centres caring for non-residential mentally handicapped individuals. Overall, 11% were hepatitis B marker seropositive and 4% had the hepatitis B surface antigen (HBsAg). Male sex and increasing age were associated with seropositive status, and Down's syndrome was associated with the presence of HBsAg. Immediate family members of those with hepatitis B markers were screened and 22% had evidence of hepatitis B markers. Forty-one family members were identified when the mentally handicapped individual was HBsAg positive and of these 13 (32%) were seropositive. This study demonstrates that hepatitis B is a problem for the non-residential mentally handicapped population and confirms the risk of infection to their immediate families.

Capture immunoassay for the diagnosis of bovine mastitis using a monoclonal antibody to polymorphonuclear granulocytes.

A direct capture enzyme-linked immunosorbent assay (ELISA) was developed to measure elevated polymorphonuclear granulocyte (PMN) antigens using horseradish peroxidase (EC 1.11.1.7) conjugated rabbit polyclonal anti-PMN antisera and a monoclonal antibody specific for PMN cells. Optical densities obtained in the ELISA were used to predict the cell counts of milk samples. Predicted counts were not significantly different from actual somatic cell counts (SCC). In a total of 156 bovine milk samples the correlation coefficient between somatic cell counting, taking greater than 500,000 cells/ml as being indicative of mastitis, and the assay was 0.94, yielding an assay sensitivity of 95.2% and a specificity of 97.3%. In further trials the ELISA could detect elevated PMN antigens in milk with SCC as low as 100,000 cells/ml. The results indicate that the monoclonal antibody based direct ELISA has excellent potential in the detection and determination of bovine mastitis.

Evaluation of commercial enzyme immunoassays for detection of hepatitis delta antigen and anti-hepatitis delta virus (HDV) and immunoglobulin M anti-HDV antibodies.

Panels of hepatitis B virus surface antigen-positive sera from drug abusers were used to evaluate 14 commercial enzyme immunoassays from six companies for detecting hepatitis delta virus (HDV) markers. For detecting hepatitis delta virus antigen (HDAg), the Wellcome, Pasteur and Noctech assays had 100% sensitivity for all 42 HDAg-positive serum specimens that were confirmed in-house; the Organon reagents gave 59.5% sensitivity without detergent and 64.3% sensitivity with detergent, but there were 14 discrepant results with and without detergent. The Sorin assay detected HDAg in only 10 of the positive samples (23.8% sensitivity). For the detection of antibody to HDV (anti-HDV) all six commercial enzyme immunoassays were reactive with all 36 anti-HDV-positive specimens that were confirmed in-house. There were no false-positive results with the Wellcome, Noctech, or Sorin assay, but one specimen was false positive by the Organon assay. One HDAg-positive specimen gave a false anti-HDV-positive result in the Abbott assay and an equivocal result in the Pasteur assay (97.8% specificity). For the detection of immunoglobulin M anti-HDV, the Wellcome, Noctech, and Sorin assays agreed for the 38 positives confirmed in-house, except for one false negative with the Sorin test. We conclude that there has been a substantial improvement over previously evaluated assays in sensitivity and specificity of commercial assays for anti-HDV detection, and the sensitivities of immunoglobulin M anti-HDV assays are also comparable. However, there are still major differences in sensitivity among some assays for HDAg detection.

Progressive multifocal leukoencephalopathy-remission with cytarabine.

A 51-year-old woman who was in complete remission from non-Hodgkin's lymphoma, developed a rapidly progressive dementia. Progressive multifocal leukoencephalopathy (PML) was diagnosed on the basis of a rising antibody titre to JC polyomavirus in cerebro-spinal fluid and serum and the presence of diffuse white matter changes on magnetic resonance imaging. She was treated initially with intravenous cytarabine and showed minimal improvement. Rapid improvement occurred when intrathecal cytarabine was added and the patient is in complete remission from both lymphoma and PML 20 months later.

Acquired immunodeficiency syndrome.

A report entitled "Kaposi's Sarcoma and Pneumocytosis Carinii Pneumonia among homosexual men in New York City and California" in the MMWR in July 1981 alerted the world to the appearance of a completely new disease. The opportunistic infections and cancers occurring in these patients had previously only been seen in patients who were immunosuppressed. Homosexual men were the first as a major risk group to be identified. Others quickly followed. The pattern of occurrence clearly indicated an infectious agent as the likely cause, and within two years the virus had been identified in Europe and the USA. In Europe it was named Lymphadenopathy Associated Virus (LAV) by Montagnier its discover, and in the USA, Human T cell Lymphotrophic Virus III (HTLV III). It is now known as Human Immunodeficiency Virus (HIV).

Prolonged IgM antibodies and histopathological evidence of chronicity in hepatitis A.

The case of a young man with hepatitis A and a chronic course is presented. The patient received a short course of steroid therapy for recurrence of symptoms following acute hepatitis A. Thereafter, liver enzymes have remained marginally elevated for 4 years and annual liver biopsies have shown evidence of chronicity. HAV IgM Ab persisted for 1034 days with subsequent development of HAV IgG Ab. The possibility of other viruses in the aetiology and the role of steroids in the development of chronicity are discussed.

Changes in incidence of hepatitis B in Ireland from 1970-1987.

This paper analyses data on 2,226 cases of Hepatitis B virus (HBV) infection detected during the years 1970 to 1987. Of those where information on risk-group was available (1,301), infection among intravenous drug-abusers accounted for the largest proportion (49%). Most became infected during and since an outbreak of hepatitis B and Delta hepatitis which originated in this group in 1980. A comparison of the data before and after the start of the outbreak among drug-abusers shows a marked increase in the number of HBV infections in non drug users, including haemophiliacs, homosexuals and health-care staff, and a dramatic decrease in hepatitis B following blood transfusion. A larger group (165 cases), many of whom are long-term healthy hepatitis B surface antigen carriers, were patients in institutions for the mentally handicapped (IMH). Most were detected recently during pre-vaccination sampling programmes. Others affected included visitors to and from high-incidence areas, tattooed persons, dialysis patients, persons born to infected mothers, and members of the security forces dealing with drug-abusers. In all, 8.4% of the hepatitis B cases detected were found to be carriers and 67% of these remained carriers in 1987 The mean duration of carriage was 3.25 years. Intravenous drug-abusers and IMH patients constituted the two largest groups of carriers. The running-three-yearly mean incidence of new cases of hepatitis B has levelled off below the peak of 1981. Although the number of cases among drug-abusers has apparently decreased, the number of cases among non drug-abusing groups has increased by 50%. (ABSTRACT TRUNCATED AT 250 WORDS)

The serology of delta hepatitis and the detection of IgM anti-HD by EIA using serum derived delta antigen.

A sensitive and specific capture assay for IgM antibody to hepatitis D virus (HDV) was developed employing serum-derived delta antigen (HDAg). In a retrospective and prospective study of an outbreak of hepatitis B (HB), 135 hepatitis B surface antigen (HBsAg) positive drug-abusers with acute hepatitis and 18 HBsAg carriers, attending various hospitals and clinics in Dublin, were found to be infected with HDV. Serological follow-up was available from 24 of those with acute hepatitis allowing a comparison of the duration and level of IgM anti-HD with the more commonly used markers, HDAg and anti-delta (anti-HD), and an assessment of the usefulness of each. HDV and HB serology was grossly altered by human immunodeficiency virus (HIV) in two patients, with severe clinical manifestation in one. All 135 patients with HDV co-infection had delta antigenaemia. In co-infections with optimum sampling times, the mean duration of delta antigenaemia was 21 days. IgM anti-HD was always found between HDAg and sero-conversion to anti-delta and was the only 'window' marker present in five cases. The mean duration of IgM anti-HD was four weeks (optimum at 2.8 weeks) and was of moderate or low titre and occurred simultaneously with HDAg in 78%. In HDV-infected HBsAg carriers, high-titre IgM anti-HD (greater than 1/10,000) persisted for the duration of the study and is a useful indicator of chronic HDV infection. IgM anti-HD was not found in 202 random blood donors nor in 205 patients with non-B hepatitis or other disorders.(ABSTRACT TRUNCATED AT 250 WORDS)