Tubercle and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease最新文献

A total of 832 respiratory specimens not including the sputum (402 bronchial lavages, 241 bronchial brushing specimens, 136 pumping lavages, 41 pleural effusions, and 12 others) from 462 patients were assayed using theRoche Amplicor Mycobacterium tuberculosis test for amplification and identification of M. tuberculosis, M. avium andM. intracellulare (Amplicor PCR). The results were compared with those obtained using conventional microscopy and cultivation methods. Each patient had little or no sputum and showed an abnormal chest X-ray shadowing of unknown cause. No patients had previously undergone antituberculous therapy. Of the specimens obtained, 24 were both PCR and culture positive, 786 were both PCR and culture negative, 11 were PCR positive and culture negative, and 11 were PCR negative and culture positive. Based on these results, the sensitivity and specificity of Amplicor PCR were determined to be 68.67% and 98.6%, respectively, when compared with culture of respiratory specimens not including the sputum. After correcting for discrepancies due to differences in patient clinical data, the sensitivity of Amplicor PCR was found to be 68.6%, and the specificity to be 99.9%; the corresponding values for culture were 66.7% and 100%, and those for smear were 9.8% and 100%. Thus, Amplicor PCR was shown to possess a similar sensitivity to culture and to be a highly specific technique for the diagnosis of tuberculosis in the respiratory system using non-sputum specimens within hours in patients showing little or no sputum and abnormal chest X-ray shadowing of an indeterminant cause.

Setting: Cecilia Makiwane Hospital, Mdantsane, Eastern Cape, Republic of South Africa.

Objective: To assess the role of the semi-automated Roche COBAS AMPLICOR^TMMycobacterium 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^TMTB 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^TMPCR test. The comparative sensitivities of the TB COBAS AMPLICOR^TMPCR 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^TMTB 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^TMTB PCR test.

Setting: One important aspect of macrophage function is the production of inflammatory and anti-inflammatory cytokines, which in turn affect the survival of intracellular organisms such as mycobacteria.

Objective: To determine the relationship between phagocytosis of mycobacteria and expression of intracellular cytokines.

Design: Phagocytosis and cytokine production were studied simultaneously within human monocyte-derived macrophages (MDMs) from healthy donors using fluorescent labelling of M. bovis BCG and flow cytometry.

Results: At a range of infection ratios (5:1, 1:1, 0.2:1) TNF- α, IL-10, IL-6 and IL-12 were all produced in a dose-dependent manner. At an infection ratio representative of the in vivo situation (1:1), cytokine production was induced in both MDMs containing intracellular M. bovis BCG and in uninfected bystander MDMs. Phagocytosis increased over time, but there was considerable donor variation: the proportions of cells containing one or more mycobacterium were 15.4±14.8% (mean±SD) at 4 h and 32.7±21.1% at 24 h (n=19). Analysis of cytokine production by MDMs not containing mycobacteria (bystander cells) at 4 h revealed that these uninfected cells produced 79±6.6% of the TNF- α, 53.9±40.0% of the IL-10 and 64.2±12.4% of the IL-12. By 20 h these proportions had decreased to 57±13.5%, 30.9±7.4% and 45.5±13.3% respectively.

Conclusion: Both infected and bystander MDMs can be stimulated to produce cytokines in response toM. bovis BCG, indicating that the ability of MDMs to produce cytokines is not necessarily dependent on the ability to phagocytose mycobacteria.

The diagnosis of tuberculosis (TB) principally rests on the sputum examination and culture. However, the sensitivity of sputum smear for acid-fast bacteria is only ≊50% and sputum culture has a relatively long turnaround time. As a result, a number of studies have been conducted in an attempt to find a rapid and accurate diagnostic test for TB. They include serological assays against various mycobacterial antigens. Here we review the merits and deficiencies of the serological tests for TB. In general, serological assays have a high negative predictive value, making them potentially useful as a screening test to rule out active TB although in HIV-positive individuals, low sensitivity and low negative predictive value compromises the accuracy of the seroassays in this group of individuals. In populations where the prevalence of latent TB infection is high, the relatively low positive predictive value of the tests reduces their specificity for active TB. Furthermore, the higher costs and greater training required in performing these tests makes it important that future studies also assess whether their use affects patient outcomes in management of TB.

Setting: Mycobacterium avium is the major cause of disseminated infection in patients with late stage AIDS.

Objective: In order to identify M. avium genes that may be involved in bacterial uptake and intracellular survival, a phoA -based reporter system was used to identify genes that encoded surface-expressed or exported proteins.

Design: PhoA (alkaline phosphatase) is only active if the protein is exported across the cell membrane into the periplasm. Consequently, detectable PhoA activity requires the fusion of a promoterless phoA gene with a DNA fragment containing a functional promoter and export leader sequence. A M. avium promoter library was constructed in the phoA reporter plasmid pJEM11 and screened in M. smegmatis for expression of active PhoA.

Results: More than 100 independent PhoA⁺recombinants were isolated, of which 15 were sequenced. Most of these exhibited varying degrees of homology with published M. avium, M. tuberculosis, M. bovis and M. leprae sequences. Based on sequence homology, one M. avium sequence was identified as a homologue of the M. tuberculosis phosphate transport gene phoS2 (Ag88). Another M. avium sequence was homolog with a putative M. tuberculosis cutinase gene. Both of these M. avium genes were cloned and sequenced. Several other M. avium sequences were homologous with, as yet, unidentified M. tuberculosis genes.

Conclusion: PhoA fusion technology is applicable to the study of atypical slow growing mycobacteria. Most of the M. avium exported proteins identified in this study are highly homologous with genes from M. tuberculosis and M. leprae. In addition, parallels in gene organization were identified between M. avium and members of the M. tuberculosis complex.

Setting: This study was carried out at the MRC Laboratories, The Gambia.

Objectives: To characterize the antigen-specific IFNγ production and cytotoxic T cell (CTL) responses of patients during active tuberculosis, treatment, and following recovery.

Design: PBMC were isolated from 37 patients with tuberculosis and incubated with either PPD, live M. bovis BCG, or no antigen and IFNγ production measured after 7 days. CTL activity against these antigens was determined using autologous antigen-pulsed monocyte-derived macrophages as target cells. A subset of these patients (7–18 depending on antigen and assey used) were tested 2 months into drug treatment and 3 months after discharge. A group of blood bank donors (n= 21) were also tested to evaluate IFNγ responses in endemic controls; a subset (n= 16) were also tested for CTL activity.

Results: The ability to produce IFNγ in response to mycobacterial antigens correlated with the Mantoux skin test status of the patient. IFNγ production to live M. bovis BCG was diminished at diagnosis but returned after 2 months of drug treatment, and was sustained after completion of drug therapy. The CTL responses to both PPD and live M. bovis BCG were reduced during the period of drug treatment compared to those at diagnosis, but returned to the original levels after recovery.

Conclusions: Drug treatment induced marked alterations in the immune responses of tuberculosis patients with induction of IFNγ production in response to stimulation with live M. bovis BCG. This may indicate activation of both CD4 and CD8 T cells.

By subtractive hybridization, we isolated genes, differentially expressed in virulent strain (dev), that are expressed at higher levels in the virulent Mycobacterium tuberculosis H37Rv strain in comparison to its avirulent counterpart, H37Ra, and consequently may be associated with the virulence phenotype of M. tuberculosis. A two-component system, devR-devS, was identified by DNA sequencing of a dev clone. DevR, the predicted gene product of devR, is a response regulator (RR) in the NarL/ UhpA subfamily of two-component systems. The devS gene product displayed homology with histidine protein kinases (HPKs) including UhpB, NarX and NarQ. The devR-devS locus is preceded by gene Rv3134c that encodes a putative alanine–aline- rich protein. This locus was conserved in M. tuberculosis and M. bovis BCG but not in other mycobacteria. A devR -lacZ transcription fusion demonstrated β-galactosidase activity in M. smegmatis and in M. tuberculosis. The devR and devS genes were cotranscribed and the levels of their transcripts were lower in two isolates of the avirulent H37Ra strain in comparison to the virulent H37Rv strain of M. tuberculosis. The level of DevR protein was also lower in one of the H37Ra strains in comparison to the H37Rv strain. However, in a third isolate of H37Ra, RNA and protein expression was equivalent to that in the H37Rv strain. Electron microscopic immunogold analysis of M. tuberculosis grown in laboratory medium and within human monocytes revealed specific labelling for DevR protein within the bacteria and the phagosomal lumen of infected monocytes. These findings collectively suggest a potential role for devR-devS in the regulation of genetic programmes unique to the tubercle bacillus.

Three VNTR loci were previously cloned from Mycobacterium tuberculosis in our laboratory. The VNTR sequences were used as queries to search for similar sequences in the GenBank database by the BLAST program. Direct and tandem repeats were identified visually. The search revealed 45 more loci of direct and tandem repeats. Comparison of the sequences to the ones in the genome sequence database of the M. tuberculosis CDC1551 strain revealed 22 different loci. Combining these results with previously reported experimental work, at least 24 loci should be polymorphic enough to be detected by simple PCR. The repeats are present both inside coding sequences and in intergenic regions on the 5′ or 3′ ends of genes. M. tuberculosis contains several VNTR. Studies of their functions may be useful for understanding the differences of phenotypes between strains.

Setting: Northern and Southern areas of Vietnam. Objective: To study the correlation between DNA fingerprinting of 168 Mycobacterium tuberculosis strains isolated from patients with a particular historical past (political separation of Vietnam for 20 years) and data about geographical origin, drug susceptibility, HIV infection and BCG vaccination status.

Methods: Comparison of restriction fragment length polymorphism (RFLP) patterns produced by Southern hybridization of PvuII-digested chromosomal DNA.

Results: The number of IS 6110 copies for the 168 strains ranges from 0 to 23. Strains originating from the North or the South differ strongly with respect to the number of copies of IS 6110. Indeed, the strains originating from the north have predominantly from 3 to 14 IS 6110 copies while the southern strains have predominantly from 15 to 23 IS 6110 copies. Furthermore, strains isolated in the North are dispersed into 6 groups whereas 80% of the strains isolated in the South form a single group. Moreover, the prevalence of drug resistance is higher in strains isolated in the South than in the North. No noticeable correlation is observed between RFLP patterns, drug susceptibility, or HIV infection.

Conclusion: The IS 6110 fingerprints of 168 M. tuberculosis strains isolated in Vietnam showed a high range of polymorphism. Only a few strains have been found with no IS6110 (1.8%). The differences between the strains from the North and South, having more than six IS 6110, suggests that they derived from ancestral strains that would be distinguishable by the number of IS 6110 and their transposition sites throughout the genome. The genomic structure of the population of strains from South Vietnam resembles that of the Beijing strain population. This could account for a similar evolution of M. tuberculosis due to a selection by BCG-induced immunity in the two populations.