Expression of variable viruses as herpes simplex glycoprotein D and varicella zoster gE glycoprotein using a novel plasmid based expression system in insect cell.

Several prokaryotic and eukaryotic expression systems have been used for in vitro production of viruses' proteins. However eukaryotic expression system was always the first choice for production of proteins that undergo post-translational modification such as glycosylation. Recombinant baculoviruses have been widely used as safe vectors to express heterologous genes in the culture of insect cells, but the manipulation involved in creating, titrating, and amplifying viral stocks make it time consuming and laborious. Therefore, to facilitate rapid expression in insect cell, a plasmid based expression system was used to express herpes simplex type 1 glycoprotein D (HSV-1 gD) and varicella zoster glycoprotein E (VZV gE). Recombinant plasmids were generated, transfected into insect cells (SF9), and both glycoproteins were expressed 48 h post-infection. A protein with approximately molecular weight of 64-kDa and 98-kDa for HSV-1 gD and VZV gE respectively was expressed and confirmed by SDS. Proteins were detected in insect cells cytoplasm and outer membrane by immunofluorescence. The antigenicity and immunoreactivity of each protein were confirmed by immunoblot and ELISA. Results suggest that this system can be an alternative to the traditional baculovirus expression for small scale expression system in insect cells.

Pan-human coronavirus and human bocavirus SYBR Green and TaqMan PCR assays; use in studying influenza A viruses co-infection and risk of hospitalization.

PURPOSE: Influenza A viruses, human coronaviruses (hCoV) and human bocavirus (hBoV) are emerging respiratory viruses. This study investigated the association between influenza A viruses co-infection with hBoV and hCoV and severity and the sensitivity of a real-time polymerase chain reaction (RT-PCR) assay for identification of 15 coronaviruses. METHODOLOGY: Published sequences for the 15 human coronaviruses were used to design a consensus PCR targeting the replicase open reading frame 1b. A previously published PCR targeting the NS1 Gene of all known human bocavirus strains was also utilized. A series of 217 samples from patients aged 37.7 (SD ± 30.4)] with seasonal influenza A viruses (SeasFluA) identified between 06/2011 and 06/2012 in NW England were tested for hCoV and hBoV using RT-PCR. Association between co-infection and disease outcome was assessed using logistic regression. RESULTS: The limit of detection of hCoV RT-PCR assay was 2 copies/µl of human coronavirus RNA template, a sensitivity comparable to a previously published SYBR green assay for human coronaviruses. A total of 12 hCoV and 17 hBoV were identified in the 217 influenza A positive samples. A higher proportion (61.5%; 8/13) of SeasFluA/hBoV co-infections were identified in patients that were admitted either to a general ward or the intensive care unit compared to 44.3% (66/149) of single SeasFlu A virus infections (OR 2.5 95% CI 0.67-9.34, p = 0.17). In a stratified analysis, there was a trend towards higher association between FluA, hCoV and hBoV with increasing age (especially in patients aged 24-45 years and >65 year old). CONCLUSION: Our hCoV RT-PCR protocol appeared to be of adequate analytical sensitivity for diagnosis. More and larger studies are needed to confirm the role of hCoV, hBoV in causing severe disease when they co-infect with influenza A viruses.

Single, dual and multiple respiratory virus infections and risk of hospitalization and mortality.

Respiratory virus infections cause a significant number of hospitalization and deaths globally. This study investigated the association between single and multiple respiratory virus infections and risk of admission to a general ward, intensive care unit or death in patients aged 0-105 years (mean ± s.d. = 24·4 ± 24·1 years), from North West England, that were tested for respiratory virus infections between January 2007 and June 2012. The majority of infections were in children aged ⩽5 years. Dual or multiple infections occurred in 10·4% (1214/11 715) of patients, whereas single infection occurred in 89·6% (10 501/11 715). Rhinovirus was the most common co-infecting virus (occurring in 69·5%; 844/1214 of co-infections). In a multivariate logistic regression model, multiple infections were associated with an increased risk of admission to a general ward [odds ratio (OR) 1·43, 95% confidence interval (CI) 1·2-1·7, P < 0·0001]. On the other hand, patients with respiratory syncytial virus (RSV) and human parainfluenza virus types 1-3 (hPIV1-3), as a single infection, had a higher risk of being admitted to a general ward (OR 1·49, 95% CI 1·28-1·73, P < 0·0001 and OR 1·34, 95% CI 1·003-1·8, P = 0·05, respectively); admitted to an intensive-care unit or dying (OR 1·5, 95% CI 1·20-2·0, P = 0·001 and OR 1·60, 95% CI 1·02-2·40, P = 0·04, respectively). This result emphasizes the importance of RSV, hPIV and mixed infections and calls for research on vaccines, drugs and diagnostic tests targeting these respiratory viruses.

Mutations associated with severity of the pandemic influenza A(H1N1)pdm09 in humans: a systematic review and meta-analysis of epidemiological evidence.

Mutations in the haemagglutinin (HA), non-structural protein 1 (NS1) and polymerase basic protein 2 (PB2) of influenza viruses have been associated with virulence. This study investigated the association between mutations in these genes in influenza A(H1N1)pdm09 virus and the risk of severe or fatal disease. Searches were conducted on the MEDLINE, EMBASE and Web of Science electronic databases and the reference lists of published studies. The PRISMA and STROBE guidelines were followed in assessing the quality of studies and writing-up. Eighteen (18) studies, from all continents, were included in the systematic review (recruiting patients 0 - 77 years old). The mutation D222G was associated with a significant increase in severe disease (pooled RD: 11 %, 95 % CI: 3.0 % - 18.0 %, p = 0.004) and the risk of fatality (RD: 23 %, 95 % CI: 14.0 %-31.0 %, p = < 0.0001). No association was observed between the mutations HA-D222N, D222E, PB2-E627K and NS1-T123V and severe/fatal disease. The results suggest that no virus quasispecies bearing virulence-conferring mutations in the HA, PB2 and NS1 predominated. However issues of sampling bias, and bias due to uncontrolled confounders such as comorbidities, and viral and bacterial coinfection, should be born in mind. Influenza A viruses should continue to be monitored for the occurrence of virulence-conferring mutations in HA, PB2 and NS1. There are suggestions that respiratory virus coinfections also affect virus virulence. Studies investigating the role of genetic mutations on disease outcome should make efforts to also investigate the role of respiratory virus coinfections.

Epstein-Barr virus infection in adult renal transplant recipients.

Epstein-Barr virus (EBV) DNAemia in the first year posttransplantation has been studied extensively. There is a paucity of information on prevalence and sequelae of EBV infection in adult renal transplantation beyond the first year. This single-center study examines the relationship between EBV DNAemia and demographic, immunosuppressive, hematologic and infection-related parameters in 499 renal transplant recipients between 1 month and 33 years posttransplant. Participants were tested repeatedly for EBV DNAemia detection over 12 months and clinical progress followed for 3 years. Prevalence of DNAemia at recruitment increased significantly with time from transplant. In multivariate adjusted analyses, variables associated with DNAemia included EBV seronegative status at transplant (p = 0.045), non-White ethnicity (p = 0.014) and previous posttransplant lymphoproliferative disease (PTLD) diagnosis (p = 0.006), while low DNAemia rates were associated with mycophenolate mofetil use (p < 0.0001) and EBV viral capsid antigen positive Epstein-Barr nuclear antigen-1 positive serostatus at transplant (p = 0.044). Patient and graft survival, rate of kidney function decline and patient reported symptoms were not significantly different between EBV DNAemia positive and negative groups. EBV DNAemia is common posttransplant and increases with time from transplantation, but EBV DNAemia detection in low-risk (seropositive) patients has poor specificity as a biomarker for future PTLD risk.

Management of suspected viral encephalitis in children - Association of British Neurologists and British Paediatric Allergy, Immunology and Infection Group national guidelines.

In the 1980s the outcome of patients with herpes simplex encephalitis was shown to be dramatically improved with aciclovir treatment. Delays in starting treatment, particularly beyond 48 h after hospital admission, are associated with a worse prognosis. Several comprehensive reviews of the investigation and management of encephalitis have been published. However, their impact on day-to-day clinical practice appears to be limited. The emergency management of meningitis in children and adults was revolutionised by the introduction of a simple algorithm as part of management guidelines. In February 2008 a group of clinicians met in Liverpool to begin the development process for clinical care guidelines based around a similar simple algorithm, supported by an evidence base, whose implementation is hoped would improve the management of patients with suspected encephalitis.

Using automated extraction of hepatitis B tests for surveillance: evidence of decreasing incidence of acute hepatitis B in England.

Surveillance of acute hepatitis B in England is necessary to estimate incidence, determine routes of transmission and inform public health actions. Here we describe an automated process to extract information on testing for markers of hepatitis B infection in English sentinel laboratories between 2002 and 2008. The resulting data were used to identify individuals with acute infections, describe their characteristics and estimate the incidence of infection. Two-thirds of acute infections were in males. Heterosexual exposure and injecting drug use were the main risks reported. Annual incidence was estimated at 1.3/100 000 person-years overall (1.7 and 0.6 for males and females, respectively) and declined each year. Automated extraction of hepatitis B markers, including quantitative results where available, can help to classify HBV status more accurately for surveillance. HBV incidence in England is at its lowest level in recent years.

Comparative performance of a novel herpes simplex virus type 2-specific enzyme-linked immunosorbent assay using a targeted chain oligopeptide, peptide 55.

Herpes simplex virus (HSV) glycoprotein G (gG2) has been used as the basis of many serological assays for the detection of HSV type 2 (HSV-2)-specific antibodies. In the present study, an enzyme-linked immunosorbent assay (ELISA), the Pathozyme Viro HSV-2 immunoglobulin G (IgG) ELISA (Omega Diagnostics, Alva, United Kingdom), based on an immunodominant epitope of gG2 presented in a branched-chain format (peptide 55), was compared with two commercially available gG2-specific assays, the Bioelisa HSV-2 IgG assay (Biokit, S.A., Barcelona, Spain) and the HerpesSelect HSV-2 IgG assay (Focus Diagnostics, Cypress, CA). A panel of 218 well-characterized serum samples was tested. Thirty-one samples were determined to be HSV-2 IgG antibody positive and 164 samples were determined to be negative with all three kits. The levels of concordance between the tests were 95.9% between the Omega and HerpeSelect assays, 90.8% between the Omega and Bioelisa assays, and 94.5% between the HerpeSelect and Bioelisa assays. Twenty-three samples gave discordant results. Western blot results showed that of these, the results for 77% were correctly identified by the Omega assay, the results for 68% were correctly identified by the HerpeSelect assay, and the results for 13.6% were correctly identified by the Bioelisa assay. Although there was a high level of agreement between the results obtained by the three assays and no false-positive results were detected by any of the three kits, confirmation of the results for samples with discordant results by Western blotting suggested that the peptide 55-based Omega assay is the most sensitive and specific assay among the assays evaluated.

Age-related pattern of KI and WU polyomavirus infection.

BACKGROUND: The role of two recently identified polyomaviruses, KI and WU, in the causation of respiratory disease has not been established. OBJECTIVES: To determine the prevalence of KI and WU viruses (KIV and WUV) in 371 respiratory samples and evaluate their contribution to respiratory disease. STUDY DESIGN: Specimens were screened for KIV and WUV using single, multiplex or real time PCR; co-infection with other respiratory viruses was evaluated. RESULTS: Of the 371 samples analysed, 10 (2.70%) were positive for KIV and 4 (1.08%) were positive for WUV yielding an overall case prevalence of KIV and WUV infection of 3.77%. KIV and WUV were identified in patients aged<15 years (11 patients) with upper or lower respiratory tract infection and >45 years (3 patients) with upper respiratory tract infection. Co-infections were found in 5 (50%) and 3 (75%) of the KIV and WUV positive samples, respectively. CONCLUSIONS: This study supports previous conclusions that KIV and WUV detection in the respiratory tract may be coincidental and reflect reactivation of latent or persistent infection with these viruses. The age distribution of KIV and WUV infection in this study mirrors that found for the other human polyomaviruses, BK and JC.

Detection of BK virus and JC virus DNA in urine samples from immunocompromised (HIV-infected) and immunocompetent (HIV-non-infected) patients using polymerase chain reaction and microplate hybridisation.

BACKGROUND: The majority of the human population is infected with two human polyomaviruses BK virus (BKV) and JC virus (JCV) during childhood. After initial infection both viruses persist within renal system. Reactivation of both viruses may be linked with immunodeficiency or immunosuppressive therapy. OBJECTIVE: To evaluate the relationship between immunodeficiency and viruria, prevalence of BK and JC viruria over time was investigated in a cohort of HIV seropositive individuals at different stages of disease. The excretion in this group was compared with virus excretion in their HIV seronegative partners and in an unselected cohort of patients attending a Genito-Urinary Medicine (GUM) clinic. STUDY DESIGN: The excretion of BKV and JCV DNA in multiple urine samples from HIV-infected patients at different stages of disease and their HIV-negative partners, and in single samples from a cohort of patients at a GUM clinic was investigated. A microplate hybridisation method was developed to increase both the sensitivity and specificity of detection of the PCR product. The method was also applied to estimate the DNA copy numbers of BKV and JCV in urine samples. RESULTS: Within the HIV group, the level of immunosuppression (CD4+ category) was not associated with JCV viruria. By contrast, there was a modest correlation between immunodeficiency as indicated by a decline in CD4+ count and BKV viruria. Shedding of both BKV and JCV DNA together in urine samples of HIV-infected patients was much higher than in control groups (P = 0.02), indicating that HIV infection may associate with polyomavirus reactivation. The incidence of flu-like syndrome was much higher in HIV-infected asymptomatic individuals than acquired immunodeficiency syndrome (AIDS)-related complex (ARC)/AIDS patients. In general, the concentration of BKV DNA viruria (DNA copy number) was dependent to CD4+ counts (P = 0.008) while concentration of JCV DNA was independent to CD4+ cell count (P = 0.54). The prevalence of BKV and JCV DNA in patients who were infected with C. trachomatis was 9/50 (18%) and 11/50 (22%), respectively. BKV and JCV DNA was detected in 3/19 (15%) and 2/19 (10%) of patients who were infected with N. gonorrhoea. Results suggested that persons infected with C. trachomatis were more likely to show BKV and JCV viruria. CONCLUSION: These results confirm that shedding of BK and JC viruses in urine is not exclusively found in immunosupression, it may also occur in healthy individuals. The frequency of virus excretion is however, apparently increased in HIV-infected patients, although no firm statistical difference could be established. One of the interesting aspects of these findings was the relatively high incidence of BKV and JCV viruria in both control groups, i.e. HIV-negative partners of HIV-infected patients and patients attending a GUM clinic.

BK virus DNA in CSF of immunocompetent and immunocompromised patients.

AIM: To investigate the possible aetiological role of BK and JC viruses in immunocompetent and immunocompromised children with suspected encephalitis and meningoencephalitis. METHODS: The polymerase chain reaction and microplate hybridisation method was employed for the detection of polyomavirus DNA in 266 CSF specimens collected from immunocompetent and immunocompromised patients. RESULTS: BK virus DNA was detected in three (2.1%) CSF samples taken from patients aged 2-5 years; two were patients with acute lymphocytic leukaemia without overt neurological symptoms, the other was a patient with suspected encephalitis. BK virus DNA was also detected in two (1.6%) CSF samples taken from older children in the age range 10-16 years; both children had suspected encephalitis. JC virus DNA was not found in any CSF sample from either age group. CONCLUSIONS: Detection of BK virus in the CSF of immunocompromised and immunocompetent patients with suspected neurological disease suggests that this virus may have had a pathogenic role in the aetiology of this condition.

BKV-DNA and JCV-DNA in CSF of patients with suspected meningitis or encephalitis.

BACKGROUND: Few studies have looked for the polyoma viruses JC or BK virus in the central nervous system (CNS) of patients without neurological symptoms or with neurological symptoms other than progressive multifocal leukoencephalopathy (PML). PCR-microplate hybridization method was employed for the detection of BKV-DNA or JCV-DNA in cerebrospinal fluid (CSF) specimens from patients with suspected meningitis or encephalitis. MATERIALS AND METHODS: A total of 181 CSF specimens from 151 patients with suspected meningitis or encephalitis was examined for BKV or JCV using PCR-microplate hybridization method. None of the patients had (clinically diagnosed) PML. A control group consisting of 20 CSF specimens from normal subject was also included. RESULTS: BKV DNA was found in five out of 131 (3.8%) and JCV DNA in two out of 131 (1.5%) of the patients with suspected meningitis or encephalitis by PCR ELISA. BKV or JCV DNA was not detected in CSF samples of any of 19 HIV positive patients. BKV and JCV DNAs were detected respectively in two CSF samples in which Mycobacterium tuberculosis (TB) PCR was also positive. Another patient who was positive for JCV PCR died with a diagnosis of cerebral lymphoma. Among the BK virus infected patients there was a patient with a previous history of hemolytic uremia and acute renal failure. Neither BKV nor JCV DNA was found in any of the 20 CSF samples from normal patients undergoing lumbar puncture for myelography as a part of an investigation of lower back pain. CONCLUSION: These results suggest that BK virus may be associated with neurological diseases either in immunocompetent or immunocompromised patients. Detection of BKV and JCV DNA in the CSF of the patients suspected to have either meningitis or encephalitis suggests that these viruses may have an etiological role. Thus, diagnostic tests for BK and JC viruses should be included in the investigative program for meningitis or encephalitis patients.

European proficiency testing program for molecular detection and quantitation of hepatitis B virus DNA.

External quality control of hepatitis B virus (HBV) DNA detection remains an important issue. This study reports and compares the results obtained from two different proficiency panels for both the qualitative and quantitative assessment of HBV DNA. The panels were designed by the European Union Quality Control Concerted Action, prepared by Boston Biomedica, Inc., and distributed in May 1999 (panel 1) and February 2000 (panel 2). Each contained two negative samples and six positive samples with 10(3) to 10(7) copies/ml (panel 1) or 10(3) to 2 x 10(6) copies of HBV DNA per ml (panel 2). For panel 1, 42 laboratories submitted 20 qualitative (all in-house PCRs) and 37 quantitative (87% commercial assays) data sets. For panel 2, 51 laboratories submitted 25 qualitative (all in-house PCRs) and 47 quantitative (94% commercial assays) data sets. Five data sets (8.8%) in panel 1 and two data sets (2.8%) in panel 2 contained totals of six and two false-positives, respectively, corresponding to false-positive result rates of 5.3% for panel 1 and 1.4% for panel 2. The false-negative result rates of 10.5% for panel 1 and 17.4% for panel 2 were dependent on the detection levels of the assays employed as well as panel composition. In the qualitative analysis of all data sets, 47.4% (panel 1) and 51.4% (panel 2) had all samples correct. An adequate or better score (all correct or only the weak-positive sample missed) was obtained with 77.2% of the panel 1 samples and 68.1% of the panel 2 samples. In the quantitative analysis, 57.1% (panel 1) and 42.6% (panel 2) of the data sets achieved an adequate or better score (positive results within the acceptable range of the geometric mean +/- 0.5 log(10) of all positive results). These results demonstrate that while the qualitative performance of HBV detection has considerably improved compared to that of a previously published HBV proficiency study, the detection levels of many commercial quantitative assays are still too high to allow adequate quantitation of all relevant clinical samples.

Multicenter proficiency testing of nucleic acid amplification methods for the detection of enteroviruses.

A multicenter study of molecular detection of enteroviruses was conducted using a proficiency panel. Of 70 data sets, 46 (66%) reported correct results for samples containing at least 1 50% infective dose per ml and for negative samples. Variation in performance between laboratories demonstrates the need for ongoing quality control.

Multicenter evaluation of a pathogenic mycobacterium screening probe.

The introduction of nucleic acid amplification assays into the clinical laboratory has reduced the time needed to diagnose diseases caused by members of the Mycobacterium tuberculosis complex (MTBC). However, several mycobacterial species other than those of the MTBC are known to cause disease, especially in immunocompromised individuals. A screening assay has been developed for the detection of the major pathogenic mycobacterial species. The assay utilizes pan-genus primers to amplify mycobacterial DNA and a screening probe (KY493) that detects all major pathogenic mycobacteria. A multicenter European study was conducted to assess the performance of the screening probe in the clinical laboratory. The screening probe was evaluated against individual probes specific for M. tuberculosis, M. avium, and M. intracellulare, a genus-specific probe with broader species coverage, and culture. The screening probe had a sensitivity equivalent to that of the species-specific probes; all specimens positive with any of the species-specific probes were also positive with the screening probes. Compared to culture, the sensitivity of the screening probe was 89% (154 of 173) for all culture-positive specimens tested. This value was 89.6% for the genus-specific probe. The screening probe was more specific than the genus-specific probe. Specificity was 93.9% (661 of 704) compared to culture results alone. The comparable specificity value for the genus-specific probe was 84.8%. When clinical data were taken into consideration, the sensitivity of the screening assay was similar to that of culture (81% versus 76.2%) but the positive predictive value of the test was lower (76.2% versus 100% for culture). However, the screening probe was more sensitive than smear and may be a useful tool in the rapid diagnosis of mycobacterial disease.

The epidemiology of adenovirus infections in Greater Manchester, UK 1982-96.

Data relating to 3313 adenovirus isolates from patients in Greater Manchester, UK between 1982 and 1996 were analysed using chi2 tests and 95% confidence intervals. Of the 3098 isolates that were typed, 18.6% were serotype 2, 14.9% serotype 3, 12.1% serotype 1 and 10.9% serotype 41. There was evidence of a seasonal occurrence of serotype 7 (March-August), serotype 2 (January-April), serotype 4 (June-August) and subgenus F (September-November). Children less than 5 years old were the most common group of patients with adenovirus infection (61.3%) compared to 24.2% for adults and only 5.6% for school children (5-15 years). Gastric symptoms were the most common amongst infants (47.6%) followed by respiratory (27.5%) and general symptoms (12.9%). In adults, the overwhelming clinical condition was conjunctivitis (88.9%). Despite the traditional association with adenoviruses, remarkably few cases of pharyngoconjunctival fever were recorded (1.7%).

Impaired humoral responses to subgenus D adenovirusenovirus infections in HIV-positive patients.

HIV-positive patients are at increased risk of developing adenovirus infection, particularly of the gastrointestinal tract and with unusual subgenus D strains. To investigate humoral immunity to these strains of adenoviruses, the humoral immune response was examined in longitudinal samples of serum against isolates collected from a prospective study of HIV-positive patients with subgenus D adenovirus infection. Of 10 HIV-positive patients developing adenovirus infection, 3 had chronic infection (8->27 months) with one serotype, 3 had chronic infection (>/=10 months) with changing serotypes and 4 had acute and self-limiting adenovirus infection (<1 month). Fifty-one sera were tested, and samples collected before adenovirus infection were available in 8 patients. Neutralising assays were performed against the patient's own isolate (adenoviruses 9, 17, 19, 19/23, 19/37, 23, 26, 23/26, 43 and 46) and common circulating strains of adenovirus 1-5. Indirect immunofluorescence tests were carried out against the autologous isolate and complement-fixation tests were undertaken using a standard assay. Immunofluorescence test antibodies were detected (titre >/=160) in all patients, and present to high titre (>/=320) in 8/10 patients. Complement-fixing antibodies were not detected in significant titre. Of particular note, there was no significant neutralising antibody response to the patient's own isolate after acute infection. Neutralising antibody to adenovirus 3 (titre 20) was transiently detected in two patients. In the remaining patients neutralising antibody directed against adenoviruses 1-5 was not detected. Persistent carriage of subgenus D adenoviruses in HIV-positive patients is probably the result of failure of cell-mediated immune responses to clear primary infection. Nevertheless, there is marked impairment of B cell responses resulting in poor neutralising and complement-fixing antibody production even though immunofluorescence test determined antibodies are produced in high titre. These possibly reflect impairment of effective B cell priming mechanisms within the germinal centres of lymph nodes, or the polyclonal activation of B cells driven by HIV infection.

Needlestick injuries and hepatitis B virus vaccination in health care workers.

An analysis of 2646 needlestick injuries in hospitals in the Greater Manchester area between April 1992 and April 1999 was carried out. Ten per cent of members of staff injured in these incidents had never been vaccinated against hepatitis B virus (HBV) and 27% of those who had been vaccinated had no anti-HBs (< 10 IU/L). Although few health care workers were at risk of transmission of HBV through needlestick incidents in this study (0.6% (12/2084) of all source patients were HBsAg positive; 9 HBeAg positive, 7 anti-HBe positive), the large number of members of staff who were not protected from HBV infection indicates a need for occupational health departments to reinforce HBV vaccination policies.

TB PCR in the early diagnosis of tuberculous meningitis: evaluation of the Roche semi-automated COBAS Amplicor MTB test with reference to the manual Amplicor MTB PCR test. .

SETTING: Cecilia Makiwane Hospital, Mdantsane, Eastern Cape, Republic of South Africa. OBJECTIVE: To assess the role of the semi-automated Roche COBAS AMPLICOR(TM)Mycobacterium tuberculosis PCR test in the diagnosis of tuberculous meningitis (TBM). DESIGN: Eighty-three specimens of cerebrospinal fluid (CSF) were collected prospectively from 69 patients with suspected TBM. The COBAS AMPLICOR TB PCR test was compared with the manual AMPLICOR(TM)TB PCR test, clinical and cerebrospinal fluid (CSF) findings, direct ZN smear and radiometric TB culture. RESULTS: CSF from 7/40 (17.5%) patients treated for TBM were positive by TB COBAS AMPLICOR(TM). The sensitivity of the test was not significantly different (p=0.375) from the manual TB AMPLICOR(TM)PCR test. The comparative sensitivities of the TB COBAS AMPLICOR(TM)PCR and the manual AMPLICOR PCR for detecting cases of definite and probable TBM from CSF collected within 9 days of commencing antituberculosis treatment were 40% and 60% respectively. All 29 patients not treated for TBM were negative by COBAS AMPLICOR(TM), giving a specificity of 100%. CONCLUSION: The COBAS AMPLICOR(TM)TB PCR test is a rapid and highly specific diagnostic test for TBM. However, there was a non-significant trend favouring slightly greater sensitivity using the manual AMPLICOR(TM)TB PCR test.