Whole genome sequencing of six diagnostic brucellaphages, Tbilisi (Tb), Firenze (Fz), Weybridge (Wb), S708, Berkeley (Bk) and R/C, was followed with genomic comparisons including recently described genomes of the Tb phage from Mexico (TbM) and Pr phage to elucidate genomic diversity and candidate host range determinants. Comparative whole genome analysis revealed high sequence homogeneity among these brucellaphage genomes and resolved three genetic groups consistent with defined host range phenotypes. Group I was composed of Tb and Fz phages that are predominantly lytic for Brucella abortus and Brucella neotomae; Group II included Bk, R/C, and Pr phages that are lytic mainly for B. abortus, Brucella melitensis and Brucella suis; Group III was composed of Wb and S708 phages that are lytic for B. suis, B. abortus and B. neotomae. We found that the putative phage collar protein is a variable locus with features that may be contributing to the host specificities exhibited by different brucellaphage groups. The presence of several candidate host range determinants is illustrated herein for future dissection of the differential host specificity observed among these phages.

METHODS

Polymerase chain reaction (PCR) offers great promise for the rapid, sensitive and specific diagnosis of tuberculous meningitis (TBM). However, the isolation of DNA of high quantity and quality from cerebrospinal fluid (CSF) samples is critical for successful PCR assays.

OBJECTIVE

To develop and use a single-tube method for the isolation of PCR-compatible DNA from Mycobacterium tuberculosis using Chelex-100 chelating resin, which does not require organic solvents or detergents.

METHODS

The study focused on the standardisation of a suitable Chelex protocol and its evaluation in 32 CSF samples from TBM and non-TBM subjects. A simultaneous comparison was made with the conventional phenol/chloroform extraction method.

RESULTS

PCR was found to be more sensitive, more rapid and less technically demanding with the Chelex protocol than the conventional phenol/chloroform extraction method (sensitivity 84.2% vs. 73.6%).

CONCLUSIONS

The single-tube method and the simplicity of the procedure permits early and reliable diagnosis of TBM and makes it an attractive method for routine laboratory assays.

Tolbutamide (TBM) was found to form an inclusion complex with beta cyclodextrin (beta-CD) in solution and in solid state. Inclusion complex formation between tolbutamide and beta-cyclodextrin in aqueous solution was studied by phase solubility and spectral shift methods. The apparent stability constant Ks calculated by these techniques, in water, were estimated as 195.7 and 236.5 M(-1), respectively. The phase solubility studies revealed a B(S)-type diagram with an inclusion complex of 1:2 molar ratio. The solid inclusion complexes of TBM and beta-CD were prepared at a molar ratio of 1:2 by kneading, coprecipitation, freeze-drying, and spray-drying methods. In addition, the physical mixture was prepared. Characterization of TBM: beta-CD inclusion was performed using differential scanning calorimetry (DSC), Raman spectroscopy, and X-ray diffractometry and by application of a so-called ether wash method. All the inclusion systems investigated led to a significant improvement in the dissolution over free TBM, and the dissolution rate of the active material was observed to be independent of the preparation method.

In order to evaluate the determinants of the metabolic cost for cycle ergometry, we analyzed the relationship between VO2 and leg mass (LM) and total body mass (TBM) in 71 randomly-selected sedentary subjects (34 men), aged 20 to 80. Participants performed constant work rate (WR) tests at 0, 25, and 50 W (at 60 rpm) for 6 minutes in a randomized sequence: gross VO2, gross efficiency, and work efficiency were related to TBM and LM as assessed by dual energy x-ray absorptiometry. We found that gross VO2 and gross efficiency were more strongly related to LM than TBM but work efficiency values were independent of both (P>0.05). Significantly higher values of VO2TBM were found in subjects with large LM/TBM ratios and vice-versa; VO2/LM, however, did not change with anthropometric characteristics. Gross VO2 (mL/min) was predicted by a LM-based equation (10.6 [WR, W] + 16.8 [LM, kg] +75) with a mean error below 5%: this equation predicted the cost more accurately than previous TBM-based formulations (P<0.01). We conclude that leg mass actually provides the preferred frame of reference for predicting the oxygen cost for cycle ergometry at 60 rpm in sedentary subjects, independent of age or gender.

The effects of several concentrations of puromycin on the nucleoproteins of HeLa cells grown in monolayers were studied by cytochemical and biochemical techniques. The earliest change at all concentrations of puromycin was a decrease in a granular form of ribonucleoprotein (RNP) that is demonstrable in the normal HeLa cell by the toluidine blue-molybdate (TBM) stain. The other types of RNP revealed by the TBM method were unaltered although the cell volume decreased markedly. Treatment with high concentrations of the antimetabolite resulted in pre-prophase inhibition of mitotic division and led to production of inclusions containing RNP in the cytoplasm; lower concentrations resulted in metaphase arrest. Biochemical analyses confirmed the cytochemical observations and indicated that synthesis of RNA and protein was inhibited to the same extent.

We examined the ultrastructure of the bovine glomerular basement membrane (GBM) and tubular basement membrane (TBM) using ultra-high-resolution scanning electron microscopy after conductive staining without metal coating. Purified basement membranes (BMs) were obtained by sonication and acellular BMs by detergent treatments. Purified GBMs (PGBMs) and acellular GBMs (AGBMs) showed similar meshwork structures composed of regular round or oval pores and branching strands. Pore diameters were 10.2 +/- 2.4 nm (mean +/- SD) in PGBMs and 9.8 +/- 3.1 nm in AGBMs. Purified TBMs (PTBMs) and acellular TBMs (ATBMs) exhibited heterogeneous meshwork structures in which compact meshes in the cristae were combined with coarse meshes in the invaginations on the epithelial surfaces. Pore diameters were 12.6 +/- 5.2 nm in PTBMs and 11.4 +/- 4.0 nm in ATBMs which were significantly larger than those in GBMs. The width of the strands ranged from 3 to 15 nm in all BMs. A 4 M guanidine hydrochloride extraction of the acellular BMs revealed large polygonal networks in the invaginations of the TBM and twisted strands which were considered to be type IV collagen fibrils. Ultra-high-resolution scanning electron microscopy and conductive staining were useful for the study of three-dimensional architectures of BMs and revealed heterogeneous meshwork structures in the GBM and TBM which were probably caused by a different ratio of the major components.

OBJECTIVE

To study tuberculin reactivity in childhood tuberculous meningitis both in clinical and histopathological (HP) context.

METHODS

Children with tuberculous meningitis (TBM) were given tuberculin test by Mantoux technique, which was read at the end of 72 hours after the placement of skin test. Histopathological examination of the punch biopsy specimen of the tuberculin test site was performed and histopathological grading of the tuberculin reaction was compared with clinical reaction and clinical parameters.

RESULTS

Of the 50 children studied, 68% of them were malnourished and 42% had BCG scar. Tuberculin test was positive in 22 (44%) cases. Spearman analysis showed negative correlation between stage of TBM and the size of tuberculin reaction. BCG status did not affect the size of tuberculin reaction. Histopathological grade of the tuberculin reaction was found to be directly proportional to the size of the tuberculin reaction and it was not affected by the stage of TBM.

CONCLUSIONS

Tuberculin positivity is low in TBM irrespective of the nutritional status. At least some degree of inflammatory reaction can be seen at the site of tuberculin administration. In tuberculin negative cases, varying grades of cellular response in the absence of clinical induration can be seen in histopathology.

Tubulointerstitial nephritis antigen (TIN-ag) is an extracellular matrix basement protein which was originally identified as a target antigen involved in anti-tubular basement membrane (TBM) antibody-mediated interstitial nephritis (TIN). Further investigations elucidated that TIN-ag plays a role in renal tubulogenesis and that TIN-ag is defected in hereditary tubulointerstitial disorder such as juvenile nephronophthisis. We previously isolated and characterized 54 kDa glycoprotein as TIN-ag. cDNA encoding rabbit and mouse TIN-ag has recently been identified. In the present study, the cDNA of the human homologue of TIN-ag was cloned and its nucleotide sequence was determined (Accession No. AB022277; the DDBJ nucleotide sequence database). Deduced amino acid sequence (476 aa) exhibited the presence of a signal peptide (1-18 aa), cysteine residues termed follistatin module, six potential glycosylation sites, and an ATP/GTP-binding site. Homology search revealed approximately 85% homology with both rabbit and mouse TIN-ag, and also some ( approximately 40%) similarity with C. elegans. Human TIN-ag contained a sequence similar to several classes of extracellular matrix molecules in amino terminal region and to cathepsin family of cysteine proteinases in the carboxyl terminal region. Northern blot analysis revealed exclusive expression of this molecule in human adult and fetal kidney tissues. Using a monoclonal antibody recognizing human TIN-ag, protein expression ( approximately 50 kDa) was identified in cultured COS-1 cells transfected with human TIN-ag cDNA. The human TIN-ag was mapped to chromosome 6p11.2-12 by fluorescence in situ hybridization. These results may provide further evidence for understanding TIN-ag molecule and clues for gene analysis of juvenile nephronophthisis.

A decrease in the concentration of heparan sulphate proteoglycan (HSPG) in the glomerular basement membrane (GBM) is supposed to cause the increased GBM permeability in the congenital nephrotic syndrome (CNS). Therefore, we analysed the glycosaminoglycan (GAG) content and composition of the GBM and tubular basement membrane (TBM) from 3 patients with CNS of the Finnish type (FCNS) and 16 control infants. The GAG content, determined by spectrophotometric assay after papain digestion, was not significantly different in FCNS patients compared with controls. In addition, the GAG composition was comparable in the two groups, with heparan sulphate (HS) constituting at least 75% of the total GAG content. The urinary GAG content (expressed as mg GAG/mmol creatinine) was age dependent, but similar in both groups. Indirect immunofluorescence studies on kidney tissue from normal human infants, using monoclonal or polyclonal antibodies against the core protein of human GBM HSPG, showed linear staining of almost all renal basement membranes. A monoclonal antibody directed against the HS chain of HSPG showed strong GBM and a weak TBM staining. Kidney tissue from three patients with FCNS displayed no discernible differences in the distribution or quality of staining with the same antibodies. These biochemical and immunohistochemical results are in contrast to the decrease in anionic sites (by polyethyleneimine staining) and the replacement of GBM HS by chondroitin sulphate, observed by others in CNS of the diffuse mesangial sclerosis type.

The purpose of this study was to investigate the prevalence of attention deficit hyperactivity disorder (ADHD) in children who recovered from tuberculous meningitis (TBM) as part of an ongoing TBM research project. During this study, each TBM group subject underwent a thorough clinical-neurological examination, and a test battery which included the child behaviour check list (CBCL) Teacher's Report Form and Conners Rating Scale. The parents and teachers of each of the 21 TBM group and 21 control group subjects completed the above-mentioned questionnaires. All 21 TBM group subjects displayed symptoms of ADHD. The TBM group was significantly more hyperactive and unable to sustain attention than the control group. Furthermore, TBM group subjects were perceived as being significantly more unpopular, obsessive, compulsive and aggressive than the control group subjects. With regard to the frequency of externalizing behaviour, the TBM group subjects displayed significantly more externalizing behaviours as well as symptoms of attention deficit and hyperactivity. No significant differences between parents' and teachers' ratings were found. We conclude that ADHD is a common long-term complication of TBM.

High doses of rifampin may help patients with tuberculous meningitis (TBM) to survive. Pharmacokinetic pharmacodynamic evaluations suggested that rifampin doses higher than 13 mg/kg given intravenously or 20 mg/kg given orally (as previously studied) are warranted to maximize treatment response. In a double-blind, randomized, placebo-controlled phase II trial, we assigned 60 adult TBM patients in Bandung, Indonesia, to standard 450 mg, 900 mg, or 1,350 mg (10, 20, and 30 mg/kg) oral rifampin combined with other TB drugs for 30 days. The endpoints included pharmacokinetic measures, adverse events, and survival. A double and triple dose of oral rifampin led to 3- and 5-fold higher geometric mean total exposures in plasma in the critical early days (2 ± 1) of treatment (area under the concentration-time curve from 0 to 24 h [AUC0-24], 53.5 mg · h/liter versus 170.6 mg · h/liter and 293.5 mg · h/liter, respectively; P < 0.001), with proportional increases in cerebrospinal fluid (CSF) concentrations and without an increase in the incidence of grade 3 or 4 adverse events. The 6-month mortality was 7/20 (35%), 9/20 (45%), and 3/20 (15%) in the 10-, 20-, and 30-mg/kg groups, respectively (P = 0.12). A tripling of the standard dose caused a large increase in rifampin exposure in plasma and CSF and was safe. The survival benefit with this dose should now be evaluated in a larger phase III clinical trial. (This study has been registered at ClinicalTrials.gov under identifier NCT02169882.).

The purpose of this study was to compare the maximal exercise performance of 10 men during friction braked cycle ergometry of 20 s duration when resistive forces reflected total body mass (TBM) or fat free mass (FFM). Fat mass was calculated from the sum of skinfold thicknesses. Increases (P < 0.05) in peak power output (PPO) were found between TBM and FFM (1,015+/-165 W TBM vs 1,099+/-172 W FFM). Decreases (P < 0.05) were observed for the time taken to reach PPO (3.8+/-1.4 s TBM vs 2.9+/-1 s FFM). Pedal velocity increased (P < 0.05) during the FFM protocol (129.4+/-8.2 rpm TBM vs 136.3+/-8 rpm FFM). Rating of perceived exertion (RPE) was also (P < 0.05) greater for FFM (18.4+/-1.6 TBM vs 19.8+/-0.4 FFM). No changes were found for Mean Power Output (MPO), fatigue index (FI) or Work Done (WD) between trials. These findings suggest that high intensity resistive force loading protocols may need to be reconsidered. Results from this study indicate that the active tissue component of body composition needs consideration in resistive force selection when ascertaining maximal cycle ergometer power profiles.

Regional gray matter (GM) atrophy in multiple sclerosis (MS) at disease onset and its temporal variation can provide objective information regarding disease evolution. An automated pipeline for estimating atrophy of various GM structures was developed using tensor based morphometry (TBM) and implemented on a multi-center sub-cohort of 1008 relapsing remitting MS (RRMS) patients enrolled in a Phase 3 clinical trial. Four hundred age and gender matched healthy controls were used for comparison. Using the analysis of covariance, atrophy differences between MS patients and healthy controls were assessed on a voxel-by-voxel analysis. Regional GM atrophy was observed in a number of deep GM structures that included thalamus, caudate nucleus, putamen, and cortical GM regions. General linear regression analysis was performed to analyze the effects of age, gender, and scanner field strength, and imaging sequence on the regional atrophy. Correlations between regional GM volumes and expanded disability status scale (EDSS) scores, disease duration (DD), T2 lesion load (T2 LL), T1 lesion load (T1 LL), and normalized cerebrospinal fluid (nCSF) were analyzed using Pearson׳s correlation coefficient. Thalamic atrophy observed in MS patients compared to healthy controls remained consistent within subgroups based on gender and scanner field strength. Weak correlations between thalamic volume and EDSS (r=-0.133; p<0.001) and DD (r=-0.098; p=0.003) were observed. Of all the structures, thalamic volume moderately correlated with T2 LL (r=-0.492; P-value<0.001), T1 LL (r=-0.473; P-value<0.001) and nCSF (r=-0.367; P-value<0.001).

It remains unknown whether migraine headache has a progressive component in its pathophysiology. Quantitative MRI may provide valuable insight into abnormal changes in the migraine interictum and assist in identifying disrupted brain networks. We carried out a data-driven study of structural integrity and functional connectivity of the resting brain in migraine without aura. MRI scanning was performed in 36 patients suffering from episodic migraine without aura and 33 age-matched healthy subjects. Voxel-wise analysis of regional brain volume was performed by registration of the T1-weighted MRI scans into a common study brain template using the tensor-based morphometry (TBM) method. Changes in functional synchronicity of the brain networks were assessed using probabilistic independent component analysis (ICA). TBM revealed that migraine is associated with reduced volume of the medial prefrontal cortex (mPFC). Among 375 functional brain networks, resting-state connectivity was decreased between two components spanning the visual cortex, posterior insula, and parietal somatosensory cortex. Our study reveals structural and functional alterations of the brain in the migraine interictum that may stem from underlying disease risk factors and the "silent" aura phenomenon. Longitudinal studies will be needed to investigate whether interictal brain changes are progressive and associated with clinical disease trajectories.

Acute tubulo-interstitial nephritis (ATIN) is an important cause of acute renal failure resulting from a variety of insults, including immune complex-mediated tubulo-interstitial injury, but drugs such as non-steroidal anti-inflammatory drugs (NSAIDs) are a far more frequent cause. Overall, as an entity, ATIN remains under-diagnosed, as symptoms resolve spontaneously if the medication is stopped. We report on a 14-year-old boy who developed acute renal failure 2 weeks after aortic valve surgery. He was put on aspirin following surgery and took ibuprofen for fever for nearly a week prior to presentation. He then presented to the emergency department feeling quite ill and was found to have a blood urea nitrogen (BUN) concentration of of 147 mg/dl, creatinine of 15.3 mg/dl and serum potassium of 8.7 mEq/l. Dialysis was immediately initiated. A kidney biopsy showed inflammatory infiltrate consistent with ATIN. However, in the tubular basement membrane (TBM), very intense granular deposits of polyclonal IgG and C3 were noted. He needed dialysis for 2 weeks and was treated successfully with steroids for 6 months. His renal recovery and disappearance of proteinuria took a year. In conclusion, this is a first report of NSAIDs-associated ATIN, showing deposits of granular immune complex present only in the TBM and not in the glomeruli.

(i)

OBJECTIVE

The study was carried out with the aim to evaluate a polymerase chain reaction (PCR) based on the amplication of a 169 bp DNA fragment specific for the Mycobacterium tuberculosis complex for the rapid diagnosis of tuberculous meningitis (TBM). (ii)

METHODS

A total of 105 CSF specimens from clinically suspected cases of TBM were studied. Clinical details of the cases and cytochemical parameters of the CSF specimens were recorded. In addition to the 105 specimens, 10 CSF specimens from cases other than TBM, 4 non-mycobacterial culture isolates (1 strain of E. coli, 1 strain of Proteus species and 2 strains of Salmonella species) and 1 sample of sterile distilled water were processed as negative controls. For positive control standard culture of Mycobacterium tuberculosis H37Rv was processed with every batch of specimens. Besides PCR, smear for AFB by the Ziehl Neelsen Carbol Fuchsin (ZNCF) and the fluoro chrome method and culture on LJ medium was also carried out. (iii)

RESULTS

By PCR, 31.42% specimens were found positive, whereas by conventional culture on LJ medium only 3.8% specimens were positive. Only 1.9% specimens were found to be smear positive by the fluorochrome staining method, while none was positive by the ZNCF method. The PCR results showed complete correlation with the clinical findings of the patients. (iv)

CONCLUSIONS

The PCR was found to be superior to the currently available techniques for the diagnosis of tuberculous meningitis in terms of sensitivity, specificity and rapidity and could play a critical role in the diagnosis of suspected cases.

BACKGROUND

The protective role of BCG vaccination against tuberculous meningitis (TBM) is still controversial in India, largely due to the presence of other predisposing factors such as age, nutritional and socioeconomic status, and household contact. Very few Indian studies have focused on the interaction between BCG and these factors on the occurrence of TBM.

METHODS

We did an unmatched hospital-based case-control study with prospective enrolment. Children with TBM diagnosed on the basis of predefined criteria were enrolled as cases. For each case, two children admitted on the same day and who did not have any neurological symptoms were enrolled as controls. Demographic data and information on predisposing factors for tuberculosis were collected for both cases and controls and the presence of a BCG scar was charted. Cases and controls were compared by univariate followed by multivariate analysis to obtain significant independent predictors for the occurrence of TBM. To assess the interaction between other predisposing factors and protective efficacy of BCG, a stratified analysis was also done.

RESULTS

A total of 91 cases and 182 controls were enrolled over a one-year study period, of which 37 cases and 111 controls had a BCG scar. The crude odds ratio for the occurrence of TBM in the absence of a BCG scar was 2.28 (range: 1.32-3.94). The time elapsed since vaccination was significantly longer in the cases. Also, the proportion with a household contact was significantly higher in the cases, the mean age of the cases was higher than that of the controls, and the mean weight and height for age percentage were significantly lower. The cases had a significantly lower socioeconomic status. On multivariate analysis, the significant independent predictors for the occurrence of TBM were positive household contact with tuberculosis (adjusted OR 4.26; 95% CCI 2.26-8.04), absent BCG scar (adjusted OR 1.98; 95%ClI 1.09-3.57) and rural residence (adjusted OR 2.07; 95% ClI 1.02-4.17).

CONCLUSIONS

Vaccination with BCG was found to be protective even after controlling for the effect of other variables. Stratified analysis showed that protection due to BCG failed to reach significance for those>> 5 years of age, if the weight was <6 0% of that expected for age, in the presence of a household contact with tuberculosis, and in socioeconomic classes III, IV and V.

Early diagnosis of tuberculous meningitis (TBM) is essential for a positive outcome; but present microbiological diagnostic techniques are insensitive, slow, or laborious. We evaluated the standard BDProbeTec ET strand displacement amplification method (the standard ProbeTec method) for the detection of Mycobacterium tuberculosis complex organisms in parallel with the ProbeTec method with a modified pretreatment procedure with 101 prospectively collected cerebrospinal fluid specimens from 94 patients with suspected TBM. By the modified method, the sample-washing step was omitted. A definitive diagnosis was attained by culture. Thirteen specimens from 12 patients were culture positive for M. tuberculosis complex organisms; three specimens (23%) were microscopy positive for acid-fast bacilli. Among the culture-positive specimens, the standard ProbeTec method was positive for 8 (61.5%) and the modified assay was positive for 10 (76.9%). The overall specificity by both procedures was 98.8% compared to the results of culture. After discrepancy analysis, conducted by reviewing the patients' previous laboratory data, the specificity increased to 100%. If the cutoff value for respiratory specimens was adjusted from the recommended value of 3,400 to 1,000, the sensitivity of the modified procedure increased to 84.7%, with unchanged specificity. Results were obtained in 3 to 4 h. The new pretreatment procedure with the ProbeTec assay described here provides a rapid, simple, and sensitive tool for the diagnosis of TBM.

A kidney tubular basement membrane (TBM) component that is bound by antibodies from individuals with anti-TBM antibody-associated tubulointerstitial nephritis (TIN) was purified and characterized (TIN antigen). TIN antigen was prepared from rabbit TBM by extraction with guanidine and purified by ion-exchange, gel filtration, and reversed-phase chromatography. Based upon yields of protein and antibody reactivity, TIN antigen accounts for about 9% of the mass of TBM and thus is a major component of this basement membrane. A predominant 58-kDa form comprises about 90% of purified TIN antigen, and a 50-kDa form accounts for the remainder. The two forms share the amino-terminal sequence Ser-Ile-Phe-Gln-Gly-Gln-Tyr-X-Arg-Ser-Phe-Gly- and give similar tryptic peptide maps, indicating that they are structurally related. Their amino acid compositions overall are similar to laminin and entactin/nidogen. The absence of hydroxyproline and hydroxylysine and the low levels of glycine in TIN antigen indicate that it is noncollagenous. No similarities were found between other known proteins and sequences of tryptic peptides and the amino terminus of TIN antigen, suggesting that it is distinct from other characterized basement membrane components. A goat polyclonal antibody toward rabbit TIN antigen showed the same kidney distribution as human antibodies and was completely inhibited in enzyme-linked immunosorbent assay by purified TIN antigen. These data further support the idea that TIN antigen is the primary target for anti-TBM antibodies associated with TIN. This research presents methods to prepare TIN antigen for biochemical studies and investigations of its role in anti-TBM autoimmune TIN.

Computed tomography (CT) was performed in 14 cases of tuberculous meningitis (TBM), 12 of which were examined during the acute phase of the disease. CT findings in these cases included internal hydrocephalus (6/12), internal combined with external hydrocephalus (2/12), focal lesions consistent with localized encephalitis (3/12), diffuse brain edema (1/12), and middle cerebral artery infarction (1/12). In comparison to 32 cases of nonspecific bacterial meningitis, internal hydrocephalus was found significantly more often in TBM than in nonspecific meningitis (p less than 0.01) making CT an additional tool for the differentiation of these conditions in doubtful cases. In addition, CT features of 2 cases of cerebral tuberculoma are presented.

BACKGROUND

Tuberculous meningitis (TBM) is closely associated with miliary tuberculosis and a pathogenetic relationship is suspected, although it has been proposed that the two processes are unrelated.

OBJECTIVE

To describe miliary tuberculosis of the central nervous system (CNS) on MRI in children with TBM.

METHODS

A retrospective descriptive study of 32 paediatric TBM patients referred for MRI. The presence of miliary nodules in the CNS was recorded. Lesions were categorized according to their distribution, enhancement pattern, size and signal characteristics.

RESULTS

A miliary distribution of nodules was present in 88% of patients. All patients with a miliary distribution had leptomeningeal nodules and 18% of these patients had deep parenchymal nodules in addition. At least one tuberculoma with central T2 hypointensity was identified in 39% of patients.

CONCLUSIONS

The high prevalence of miliary leptomeningeal nodules in the CNS of children with TBM is significant because it points to a pathogenetic relationship that has long been suspected on epidemiological grounds. Our findings challenge the concept that miliary tuberculosis is only an incidental finding in TBM patients and suggest that it plays an integral part in the pathogenesis.

Antibodies against Mycobacterium tuberculosis antigens were detected by enzyme-linked immunosorbent assay in cerebrospinal fluid (CSF) samples obtained from 442 patients with tuberculous meningitis (TBM) and 102 control patients. Antibodies were found in the CSF of 87% of patients with clinical (culture-negative) TBM, 72% of patients with culture-positive TBM, and 65% of patients with autopsy-proven TBM. That anti-M. tuberculosis antibodies were detected in the CSF of patients with clinically diagnosed cases more frequently than in patients with culture-positive cases suggests that the detection of antibodies in CSF tends to decrease as bacillary load increases. Of the patients with clinical TBM who were coinfected with human immunodeficiency virus (HIV), 70% exhibited anti-M. tuberculosis antibody in CSF, which suggests that antibody responses in this group were substantially weaker than those in HIV-negative patients with clinical TBM. Some groups showed a stronger response to certain antigens, which suggests that antigen recognition patterns may be specific for the stage of disease.

The early diagnosis of tuberculous meningitis (TBM) is very important. In this study, the efficiency of the polymerase chain reaction (PCR), one of the most reliable and sensitive DNA-based assays, was compared with conventional methods (acid-fast microscopy and culture) for the detection of M. tuberculosis in cerebrospinal fluid(CSF) specimens from patients suspected of TBM. Of the 29 CSF specimens from highly-probable TBM patients (based on clinical features), 25 were positive by PCR (86.2%), whereas only one of 29 was acid-fast microscopy (AFM) positive (3.4%), and 5 out of 29 were culture-positive (17.2%). No positive results were found by AFM, culture or PCR in the non-tuberculous control group. The results of this study indicate that the application of PCR should be extremely useful in the diagnosis of TBM.

Mycobacterium tuberculosis (MTB) strains were isolated from cerebrospinal fluids collected from individual tuberculous meningitis (TBM) patients from 1996 to 2007 (n = 184) and characterised based on IS6110-restriction fragment length polymorphism (RFLP), spoligotyping, Mycobacterium interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) and large sequence polymorphisms (LSPs). Beijing strains were found to possess the highest transmissibility and proportion in clustered isolates. Beijing strain predomination and stability, at 56% of the genotypic proportion, as well as association with drug resistance in TBM patients, was demonstrated. The proportion of Beijing sublineages revealed that the modern Beijing sublineage showed an increasing trend, whereas the ancestral Beijing sublineage showed a decreasing trend across the three periods. In contrast, there were neither clustered nor multidrug-resistance (MDR) isolates from the Euro-American (EuA) lineage, and the lineage genotypic proportion trend was also decreased. Based on LSPs, only the Beijing, Indo-Oceanic and Euro-American lineages were identified from TBM patients in Thailand. TBM mortality rates were not associated with either drug resistance or significantly different among MTB lineages. This study may support the Beijing genotype strain as most pathogenic causing TBM, with the EuA lineage genotype as the most benign of the strain genotypes tested. The analysis of drug susceptibility also revealed the trend of increasing drug resistance, especially MDR, in TBM patients in Thailand.