Detection of 65 kD heat shock protein in cerebrospinal fluid of tuberculous meningitis patients

Background  

Diagnosis of tuberculous meningitis (TBM) is difficult. Rapid confirmatory diagnosis is essential to initiate required therapy. There are very few published reports about the diagnostic significance of 65 kD heat shock protein (hsp) in TBM patients, which is present in a wide range of Mycobacterium tuberculosis species and elicits a cellular and humoral immune response. In the present study we have conducted a prospective evaluation for the demonstration of 65 kD hsp antigen in cerebrospinal fluid (CSF) of TBM patients, by indirect ELISA method using monoclonal antibodies (mAb) against the 65 kD hsp antigen, for the diagnosis of TBM.     

Tumour Necrosis Factor-alpha Production and Immune Cell Activation in Tuberculous Meningitis

The local production of tumour necrosis factor-alpha (TNFalpha) was evaluated in the cerebrospinal fluid (CSF) from ten patients with tuberculous meningitis (TBM). The degree of intrathecal immune activation was also studied by assessing the CSF levels of beta(2)-microglobulin (beta(2)-M) and adenosine deaminase activity (ADA). Results indicate that elevated CSF concentrations of TNFalpha, beta(2)-M and ADA were found in all TBM patients. Moreover, TNFalpha is produced and selectively concentrated for a long period of time, while beta(2)-M and ADA values progressively decline during the course of TBM. Our findings suggest that in TBM patients, after an early activation of immune cells, there is an enhanced and continuous production of TNFalpha at the site of infection.     

A dot-immunobinding assay for the laboratory diagnosis of tuberculous meningitis and its comparison with enzyme-linked immunosorbent assay.

In an attempt to establish an alternative to standard bacteriological methods in the laboratory diagnosis of tuberculous meningitis (TBM), a simple dot-immunobinding assay (Dot-Iba) was standardized to detect Mycobacterium tuberculosis antigen 5 and antimycobacterial antibody in cerebrospinal fluid (CSF) specimens of patients with TBM. Sensitivity and specificity of Dot-Iba was compared with conventional enzyme-linked immunosorbent assay (ELISA) and standard bacteriological techniques. The Dot-Iba showed excellent correlation with indirect ELISA for the detection of antimycobacterial antibody in CSF and showed 60% sensitivity and 100% specificity in culture-negative patients with TBM. However Dot-Iba was less sensitive for the detection of antigen 5 in CSFs and showed false negative results (60%) in culture-positive patients with TBM.     

A dot-immunobinding assay for the laboratory diagnosis of tuberculous meningitis and its comparison with enzyme-linked immunosorbent assay

V.V. RADHAKRISHNAN AND A. MATHAI. 1991. In an attempt to establish an alternative to standard bacteriological methods in the laboratory diagnosis of tuberculous meningitis (TBM), a simple dot-immunobinding assay (Dot-Iba) was standardized to detect Mycobacterium tuberculosis antigen 5 and antimycobacterial antibody in cerebrospinal fluid (CSF) specimens of patients with TBM. Sensitivity and specificity of Dot-Iba was compared with conventional enzyme-linked immunosorbent assay (ELISA) and standard bacteriological techniques. The Dot-Iba showed excellent correlation with indirect ELISA for the detection of antimycobacterial antibody in CSF and showed 60% sensitivity and 100% specificity in culture-negative patients with TBM. However Dot-Iba was less sensitive for the detection of antigen 5 in CSFs and showed false negative results (60%) in culture-positive patients with TBM.     

Presentation and outcome of tuberculous meningitis in a high HIV prevalence setting

Background

Mycobacterium tuberculosis is a common, devastating cause of meningitis in HIV-infected persons. Due to international rollout programs, access to antiretroviral therapy (ART) is increasing globally. Starting patients with HIV-associated tuberculous meningitis (TBM) on ART during tuberculosis (TB) treatment may increase survival in these patients. We undertook this study to describe causes of meningitis at a secondary-level hospital in a high HIV/TB co-infection setting and to determine predictors of mortality in patients with TBM.

Methods

A retrospective review of cerebrospinal fluid findings and clinical records over a six-month period (March 2009–August 2009). Definite, probable and possible TBM were diagnosed according to published case definitions.

Results

TBM was diagnosed in 120/211 patients (57%) with meningitis. In 106 HIV-infected patients with TBM, six-month all-cause mortality was lower in those who received antiretroviral therapy (ART) during TB treatment; hazard ratio = 0.30 (95% CI = 0.08–0.82). Factors associated with inpatient mortality in HIV-infected patients were 1) low CD4⁺ count at presentation; adjusted odds ratio (AOR) = 1.4 (95% confidence interval [CI] = 1.03–1.96) per 50 cells/µL drop in CD4⁺ count and, 2) higher British Medical Research Council TBM disease grade (2 or 3 versus 1); AOR = 4.8 (95% CI = 1.45–15.87).

Interpretation

Starting ART prior to or during TB treatment may be associated with lower mortality in patients with HIV-associated TBM. Advanced HIV and worse stage of TBM disease predict in-hospital mortality in patients presenting with TBM.     

Predominance of Vgamma9/Vdelta2 T lymphocytes in the cerebrospinal fluid of children with tuberculous meningitis: reversal after chemotherapy.

BACKGROUND: We analyzed the gammadelta T cell composition and responses in the peripheral blood and cerebrospinal fluid (CSF) of children affected by tuberculous meningitis (TBM) and in control children. MATERIALS AND METHODS: Peripheral blood and CSF samples were stimulated with different phosphoantigens and IL-2, and expansion of Vgamma9/Vdelta2 T cells assessed by FACS analysis. Vgamma9/Vdelta2 lines were obtained by culturing CSF or peripheral blood mononuclear cells (PBMC) in vitro with phosphoantigens and IL-2 for 2 months, and tested for proliferation and cytokine production in response to phosphoantigens. Vdelta2(D)Jdelta junctional sequence length was assessed by PCR. RESULTS: The repertoire of gammadelta T cells from the CSF of TBM patients was characterized by the predominance of Vgamma9/Vdelta2 T lymphocytes, which accounted for >80% of gammadelta T cells. Vgamma9/Vdelta2 cells from the CSF of TBM children responded to different synthetic and natural (mycobacterial) phosphoantigens and produced discrete amounts of IFN-gamma and TNF-alpha. The in vitro expansion of Vgamma9/Vdelta2 T cells from CSF and peripheral blood of TBM patients prominently decreased following chemotherapy, and similarly, the proportion of ex vivo unstimulated Vgamma9/Vdelta2 T cells in CSF of TBM patients decreased to levels detected in the CSF of control subjects. Vdelta2 CDR3 TCR analysis showed that the remaining Vdelta2 cells in the CSF of TBM patients were still polyclonal. CONCLUSIONS: These findings are consistent with an involvement of Vgamma9/Vdelta2 T cells in TBM. http://link. springer-ny.com/link/service/journals/00020/bibs/5n5p301. html     

Long-term mortality in patients with tuberculous meningitis: a Danish nationwide cohort study

Background

With high short-term mortality and substantial excess morbidity among survivors, tuberculous meningitis (TBM) is the most severe manifestation of extra-pulmonary tuberculosis (TB). The objective of this study was to assess the long-term mortality and causes of death in a TBM patient population compared to the background population.

Methods

A nationwide cohort study was conducted enrolling patients notified with TBM in Denmark from 1972–2008 and alive one year after TBM diagnosis. Data was extracted from national registries. From the background population we identified a control cohort of individuals matched on gender and date of birth. Kaplan-Meier survival curves and Cox regression analysis were used to estimate mortality rate ratios (MRR) and analyse causes of death.

Findings

A total of 55 TBM patients and 550 individuals from the background population were included in the study. Eighteen patients (32.7%) and 107 population controls (19.5%) died during the observation period. The overall MRR was 1.79 (95%CI: 1.09–2.95) for TBM patients compared to the population control cohort. TBM patients in the age group 31–60 years at time of diagnosis had the highest relative risk of death (MRR 2.68; 95%CI 1.34–5.34). The TBM patients had a higher risk of death due to infectious disease, but not from other causes of death.

Conclusion

Adult TBM patients have an almost two-fold increased long-term mortality and the excess mortality stems from infectious disease related causes of death.     

Diagnostic Value of T-Cell Interferon-γ Release Assays on Cerebrospinal Fluid for Tuberculous Meningitis

Diagnosis of tuberculous meningitis (TBM) remains a challenge. This study aimed to evaluate the performance of T-SPOT.TB test on cerebrospinal fluid mononuclear cells (CSFMCs) for suspected TBM patients. 43 consecutive patients with suspected TBM were enrolled in the study from June 2011 to September 2014. T-SPOT.TB was performed on both CSFMCs and peripheral blood mononuclear cells (PBMCs). The final diagnosis of TBM was independent of the T-SPOT.TB result. The diagnostic sensitivity, specificity, predictive value, and likelihood ratio of T-SPOT.TB on CSFMCs and PBMCs were analyzed. Of the 43 patients, 12 (27.9%) were finally diagnosed with TBM, 28 (65.1%) with non-TBM, and 3 (7.0%) with indeterminate diagnoses. Of 40 cases with definite diagnoses, the sensitivity and specificity were 92.0% and 96.0% for T-SPOT.TB on CSFMCs, and 83.0% and 82.0% for T-SPOT.TB on PBMCs, respectively. The positive predictive value (PPV) and negative predictive value (NPV) of T-SPOT.TB on CSFMCs were 85.0% and 96.0%, respectively. The PPV and NPV were 67.0% and 92.0% for T-SPOT.TB on PBMCs. The difference of T-SPOT.TB between CSFMCs and PBMCs was not significant so far as sensitivity, specificity, PPV, and NPV were concerned (P>0.05 for each). However, T-SPOT.TB on CSFMC and CSFMC: PBMC in TBM cases seemed higher than that in non-TBM cases. Our study further showed that T-SPOT.TB on CSFMCs might be a rapid and accurate diagnostic test for TBM. CSFMC: PBMC T-SPOT.TB ratio might be useful for the early diagnosis of TBM.     

Characterization of tubular basement membrane antigens in human kidney

Tubular basement membrane (TBM) was prepared from normal human kidneys and solubilized with various enzymes. Collagenase digestion released antigenic moieties from the TBM. All four anti-TBM antibodies we studied, three from patients with idiopathic tubulo-interstitial nephritis (TIN) and one from a renal allograft recipient, distinctively reacted with collagenase-digested (CD) TBM during enzyme-linked immunoassay and could discriminate among sera of normal controls or of other nephritis patients, including anti-glomerular basement membrane (anti-GBM) nephritis. When digested with pronase, trypsin, or pepsin, antigenicity of the TBM decreased. We studied the TBM antigens with immunoprecipitation and immunoblotting. After incubation of radio-iodinated CDTBM with anti-TBM sera, immunoprecipitates were identified by single-dimension SDS polyacrylamide gel electrophoresis or two-dimension gel electrophoresis, followed by autoradiography. All four antibodies had identical results on immunoprecipitation; under nonreducing conditions, they gave two protein bands with m.w. of 54,000 and 48,000 and with pI 7.0 to 8.0 and 6.5 to 7.0. Electrophoresis performed under reducing conditions disclosed only one band at the m.w. of 48,000 and pI of 6.5, suggesting that the 54-kDa component is composed of peptides linked by interchain disulfide bonds. Immunoblot analysis showed that the anti-TBM antibodies were heterogeneous; three antibodies from the idiopathic TIN patients reacted with the 54-kDa band, but the one from the renal allograft recipient reacted with neither band. This finding suggests that there are two antigenic determinants on the 54-kDa component. One such determinant that was resistant to denaturation with SDS was detected by the first three antibodies, and the other that was sensitive to such denaturation bound to the last antibody. The 48-kDa component seemed not to be immunoreactive after incubation with SDS. We studied TBM antigens reactive with anti-GBM antibodies. By immunoblotting, all four sera from patients with anti-GBM nephritis stained TBM proteins of 45 to 50 kDa and 25 to 27 kDa at pH 8.0 to 9.0; this was similar to the staining pattern of CDGBM with the same sera, but the highly cationic (pH greater than 9.0) components were specifically detected in the CDGBM. By inhibition ELISA, the binding of the anti-GBM sera to denatured CDTBM decreased with preincubation of the sera with CDGBM, suggesting that the anti-GBM antibodies recognize the same epitope(s) on the GBM and the TBM.     

Characterization of anti-tubular basement membrane antibodies in rats

Autoimmune tubulointerstitial nephritis was induced in Brown-Norway (BN) rats by immunization with bovine (Bov) tubular basement membrane (TBM) in complete Freund's adjuvant. Serum antibodies thus produced reacted to a greater extent with Bov than BN TBM antigens by indirect immunofluorescence and by radioimmunoassay with particulate (P) and collagenase-solubilized (CS) TBM. The quantities of antibodies reactive with CS TBM correlated with the intensity of tubulointerstitial pathologic changes. Antibodies eluted from kidneys reactive with BN TBM by indirect immunofluorescence were 508 times more concentrated in the kidney than in the serum, compared with 15 times for Bov TBM-reactive antibodies. The reactivity of eluted antibodies to P BN TBM was inhibited by 70% after absorption with BN CS TBM. A major CS TBM antigen of 42,000 m.w. was identified by polyacrylamide gel electrophoresis. This antigen was present in both Bov and BN TBM, and may be important in triggering autoantibody formation in this model. Lewis rats immunized under the same conditions produced antibodies reactive with BN TBM by immunofluorescence but failed to develop immune deposits in TBM of their own kidneys. Analysis of serum anti-TBM antibodies in Lewis rats revealed a selective lack of reactivity with either homologous or autologous CS TBM. These results suggest that the ability to make an immune response to one or more elements of CS TBM plays a major role in the development of autoimmune tubulointerstitial nephritis in rats.     

Differential diagnosis of tuberculous meningitis from partially-treated pyogenic meningitis by cell ELISA

Background  

Tuberculous meningitis (TBM) is a major global health problem, and it is sometimes difficult to perform a differential diagnosis of this disease from other diseases, particularly partially-treated pyogenic meningitis (PTPM). In an earlier study, we demonstrated the presence of a 30-kD protein antigen in cerebrospinal fluid (CSF) of TBM patients. We have also shown that lymphocytes from CSF of TBM patients respond differently to this antigen than do those from PTPM patients. The purpose of this study was to develop an assay that can discriminate between TBM and PTPM.     

Pretreatment intracerebral and peripheral blood immune responses in Vietnamese adults with tuberculous meningitis: diagnostic value and relationship to disease severity and outcome

Tuberculous meningitis (TBM) is the most devastating form of tuberculosis. Both intracerebral and peripheral blood immune responses may be relevant to pathogenesis, diagnosis, and outcome. In this study, the relationship between pretreatment host response, disease phenotype, and outcome in Vietnamese adults with TBM was examined. Before treatment, peripheral blood IFN-gamma ELISPOT responses to the Mycobacterium tuberculosis Ags ESAT-6, CFP-10, and purified protein derivative (PPD) were a poor diagnostic predictor of TBM. Cerebrospinal fluid IL-6 concentrations at presentation were independently associated with severe disease presentation, suggesting an immunological correlate of neurological damage before treatment. Surprisingly however, elevated cerebrospinal fluid inflammatory cytokines were not associated with death or disability in HIV-negative TBM patients at presentation. HIV coinfection attenuated multiple cerebrospinal fluid inflammatory indices. Low cerebrospinal fluid IFN-gamma concentrations were independently associated with death in HIV-positive TBM patients, implying that IFN-gamma contributes to immunity and survival. Collectively, these results reveal the effect of HIV coinfection on the pathogenesis of TBM and suggest that intracerebral immune responses, at least in HIV-negative cases, may not be as intimately associated with disease outcome as previously thought.     

A putative urinary biosignature for diagnosis and follow-up of tuberculous meningitis in children: outcome of a metabolomics study disclosing host–pathogen responses

Introduction

Tuberculous meningitis (TBM) is a severe manifestation of tuberculosis, presenting with high morbidity and mortality in children. Existing diagnostic methods for TBM are invasive and time-consuming and the need for highly sensitive and selective diagnosis remains high on the TBM agenda.

Objective

Our aim was to exploit metabolomics as an approach to identify metabolites as potential diagnostic predictors for children with TBM through a non-invasive means.

Methods

Urine samples selected for this study were from three paediatric groups: patients with confirmed TBM (n = 12), patients clinically suspected with TBM but later confirmed to be negative (n = 19) and age-matched controls (n = 29). Metabolomics data were generated through gas chromatography–mass spectrometry analysis and important metabolites were identified according to standard statistical procedures used for metabolomics data.

Results

A global metabolite profile that characterized TBM was developed from the data, reflecting the host and microbial responses. Nine different logistic regression models were fitted to selected metabolites for the best combination as predictors for TBM. Four metabolites—methylcitric, 2-ketoglutaric, quinolinic and 4-hydroxyhippuric acids—showed excellent diagnostic ability and provided prognostic insight into our TBM patients.

Conclusions

This study is the first to illustrate holistically the metabolic complexity of TBM and provided proof-of-concept that a biosignature of urinary metabolites can be defined for non-invasive diagnosis and prognosis of paediatric TBM patients. The biosignature should be developed and validated through future prospective studies to generate a medical algorithm for diagnosis in the initial stages of the disease and for monitoring of treatment strategies.

     

The clinical benefit of adjunctive dexamethasone in tuberculous meningitis is not associated with measurable attenuation of peripheral or local immune responses

Outcome from tuberculous meningitis (TBM) is believed to be dependent on the severity of the intracerebral inflammatory response. We have recently shown that dexamethasone improved survival in adults with TBM and postulated that the clinical effect would be associated with a measurable systemic and intracerebral impact on immunological markers of inflammation. Prolonged inflammatory responses were detected in all TBM patients irrespective of treatment assignment (placebo or dexamethasone). The inflammatory response in the cerebrospinal fluid was characterized by a leukocytosis (predominantly CD3(+)CD4(+) T lymphocytes, phenotypically distinct from those in the peripheral blood), elevated concentrations of inflammatory and anti-inflammatory cytokines, chemokines, and evidence of prolonged blood-brain barrier dysfunction. Dexamethasone significantly modulated acute cerebrospinal fluid protein concentrations and marginally reduced IFN-gamma concentrations; other immunological and routine biochemical indices of inflammation were unaffected. Peripheral blood monocyte and T cell responses to Mycobacterium tuberculosis Ags were also unaffected. Dexamethasone does not appear to improve survival from TBM by attenuating immunological mediators of inflammation in the subarachnoid space or by suppressing peripheral T cell responses to mycobacterial Ags. These findings challenge previously held theories of corticosteroid action in this disease. An understanding of how dexamethasone acts in TBM may suggest novel and more effective treatment strategies.     

Potential application of dot-immunobinding assay as a rapid diagnostic test in tuberculous meningitis

A simple dot-immunobinding assay (Dot-Iba) in nitrocellulose paper was developed for the detection of specific IgG antibody to Mycobacterium tuberculosis antigen 5 and mycobacterial antigen in cerebrospinal fluid of patients with tuberculous meningitis (TBM). The assay gave 77.1% sensitivity for the detection of IgG antibody to M. tuberculosis antigen 5 and 48.6% sensitivity for the detection of mycobacterial antigen in patients with TBM.     

GalaxyTBM: template-based modeling by building a reliable core and refining unreliable local regions

ABSTRACT: BACKGROUND: Protein structures can be reliably predicted by template-based modeling (TBM) when experimental structures of homologous proteins are available. However, it is challenging to obtain structures more accurate than the single best templates by either combining information from multiple templates or by modeling regions that vary among templates or are not covered by any templates. RESULTS: We introduce GalaxyTBM, a new TBM method in which the more reliable core region is modeled first from multiple templates and less reliable, variable local regions, such as loops or termini, are then detected and re-modeled by an ab initio method. This TBM method is based on "Seok-server," which was tested in CASP9 and assessed to be amongst the top TBM servers. The accuracy of the initial core modeling is enhanced by focusing on more conserved regions in the multiple-template selection and multiple sequence alignment stages. Additional improvement is achieved by ab initio modeling of up to 3 unreliable local regions in the fixed framework of the core structure. Overall, GalaxyTBM reproduced the performance of Seok-server, with GalaxyTBM and Seok-server resulting in average GDT-TS of 68.1 and 68.4, respectively, when tested on 68 single-domain CASP9 TBM targets. For application to multi-domain proteins, GalaxyTBM must be combined with domain-splitting methods. CONCLUSION: Application of GalaxyTBM to CASP9 targets demonstrates that accurate protein structure prediction is possible by use of a multiple-template-based approach, and ab initio modeling of variable regions can further enhance the model quality.     

Noninvasive estimation of bone mass in ancient vertebrae

Histomorphometry is useful in the assessment of trabecular bone mass (TBM), and thus, in the estimation of the prevalence and intensity of osteopenia in ancient population groups. However, it is a destructive method. It is therefore necessary to explore the accuracy of nondestructive approaches, such as radiography, bone mineral density (BMD) assessed by double-energy X-ray absorptiometry (DEXA), bone density (BD), or optical density (OD) in the diagnosis of osteopenia. We selected 51 vertebrae out of a total sample composed of 333 T12, L1, and L2 vertebrae belonging to adult pre-Hispanic inhabitants from El Hierro. These vertebrae underwent histomorphometrical analysis, a fine-grained film radiography with assessment of trabecular pattern following standard methods, OD, DEXA-assessed BMD, and BD. The presence of biconcave vertebrae and wedge-shaped vertebrae was also assessed by measuring anterior height (a), posterior height (p), and height at the middle point of the vertebral body (m), and further calculating the indices 2m/(a + p) ("spine score") and a/p. Significant correlations were observed between TBM and BMD (r=0.43), TBM and BD (r=0.49), TBM and OD (r=0.52), BMD and OD (r=0.51), and BMD and BD (r=0.36), but not between TBM and the indices 2m/(a + p) and a/p. In the stepwise multiple correlation analysis between TBM and BMD, BD, and OD, OD entered into first place and BD into second place, whereas BMD became displaced; the multiple correlation coefficient was 0.63, with a standard error of 3.78. A BMD greater than 0.60 g/cm2, or a bone density greater than 0.60 g/cm3, excluded osteopenia (TBM <15%) with a specificity greater than 90%, whereas a BMD value less than 0.35 g/cm2, a BD less than 0.35 g/cm3, or optical density >1.6 excluded a normal bone mass (TBM >20%) with a specificity greater than 90%. Based on radiographic criteria on the total sample, we also conclude that the overall prevalence of vertebral fractures in the adult pre-Hispanic population of El Hierro of any age is 7.5%.     

C-reactive proteins, immunoglobulin profile and mycobacterial antigens in cerebrospinal fluid of patients with pyogenic and tuberculous meningitis.

Cerebrospinal fluid (CSF) samples were collected from 12 patients with pyogenic meningitis (PM), 19 with tuberculous meningitis (TBM), 20 with clinically suspected but not definitely proved cases of tuberculous meningitis (STBM) and 12 normal controls. C-reactive proteins, immunoglobulins G, A, M and mycobacterial antigens were estimated in the CSF samples. Seven out of 51 (13.7%) samples obtained from the patient groups were positive for CRP. Immunoglobulins M and A were significantly raised in the PM group. When the TBM and STBM groups were compared with the controls a highly significant increase was obtained for all immunoglobulins. Mycobacterial antigens/epitopes were identified in 36.8% samples with TBAGB1 and TB68-H monoclonals and in 26.3% with WTB72-A2. In case of patients with suspected TBM, 6.6% were positive with TBAGB1 and WTB72-A2 and 13.3% with TB68-H. However, non-tuberculous patients also reacted with WTB72-A2 (10.5%) and TB68-H (21.0%). This is, to the authors' knowledge, the first report on the presence of CRP in the CSF. Technique for immunoglobulins in CSF is also updated in this paper. We infer that the monoclonal antibody TBAGB1 and immunoglobulins G and A may be safely considered as diagnostic markers of TBM. Estimation of CRP in CSF samples may be made to give a preliminary or additional diagnosis of meningitis regardless of its aetiology.     

Spontaneous tandem-base mutations (TBM) show dramatic tissue,age, pattern and spectrum specificity

To supplement a previous analysis of spontaneous tandem-base mutations (TBM) in the lacI gene of Big Blue((R)) mice, 2658 additional mutants were sequenced from 13 tissues and 44 spontaneous TBM were identified (tripling the sample size). Previous findings were confirmed and generalized and several new observations were made. TBM differ from single and other double mutations in that TBM frequency varies dramatically with tissue type. In certain tissues, most notably male germ cells, no TBM are observed despite screening as many as 26 million plaque forming units. TBM are most frequent in kidney and liver (3.45 and 2x10(-6), respectively), accounting for 7.6 and 4.8% of all mutational events in kidney and liver, respectively. There is a trend for elevated TBM frequency in thymic lymphomas in p53-deficient mice. TBM are more frequent in old age in both liver and kidney. TBM differ from single mutations and other double mutations because they display a marked difference in pattern and dramatic tissue specificity for target sequence. Five of the 78 possible TBM outcomes comprise 79% of those observed, and mutations at GG/CC predominate. TBM in mice were compared with TBM found in human mutation databases. TBM are also rare in the human germline (one in 5133 germline mutations reported in five human mutation databases). In general, the types of somatic TBM are similar in mice and humans except for an excess of TG/CA to CA/TG TBM in humans (TBM related to ultraviolet light-induced skin cancer were excluded). TBM may be the result of unknown mechanisms that may have some similarities in mice and humans.     

Evaluation of the MODS culture technique for the diagnosis of tuberculous meningitis

Background

Tuberculous meningitis (TBM) is a devastating condition. The rapid instigation of appropraite chemotherapy is vital to reduce morbidity and mortality. However rapid diagnosis remains elusive; smear microscopy has extremely low sensitivity on cerebrospinal fluid (CSF) in most laboratories and PCR requires expertise with advanced infrastructure and has sensitivity of only around 60% under optimal conditions. Neither technique allows for the microbiological isolation of M. tuberculosis and subsequent drug susceptibility testing. We evaluated the recently developed microscopic observation drug susceptibility (MODS) assay format for speed and accuracy in diagnosing TBM.

Methodology/Principal Findings

Two hundred and thirty consecutive CSF samples collected from 156 patients clinically suspected of TBM on presentation at a tertiary referal hospital in Vietnam were enrolled into the study over a five month period and tested by Ziehl-Neelsen (ZN) smear, MODS, Mycobacterial growth Indicator tube (MGIT) and Lowenstein-Jensen (LJ) culture. Sixty-one samples were from patients already on TB therapy for >1day and 19 samples were excluded due to untraceable patient records. One hundred and fifty samples from 137 newly presenting patients remained. Forty-two percent (n = 57/137) of patients were deemed to have TBM by clinical diagnostic and microbiological criteria (excluding MODS). Sensitivity by patient against clinical gold standard for ZN smear, MODS MGIT and LJ were 52.6%, 64.9%, 70.2% and 70.2%, respectively. Specificity of all microbiological techniques was 100%. Positive and negative predictive values for MODS were 100% and 78.7%, respectively for HIV infected patients and 100% and 82.1% for HIV negative patients. The median time to positive was 6 days (interquartile range 5–7), significantly faster than MGIT at 15.5 days (interquartile range 12–24), and LJ at 24 days (interquartile range 18–35 days) (P<0.01).

Conclusions

We have shown MODS to be a sensitive, rapid technique for the diagnosis of TBM with high sensitivity, ease of performance and low cost (0.53 USD/sample).