Pulmonary tuberculosis in Harare, Zimbabwe: analysis by spoligotyping

BACKGROUND: Over the last 10 years there has been a fourfold increase in cases of tuberculosis in Harare, Zimbabwe. The use of molecular epidemiology to understand tuberculosis transmission in this epidemic has been hampered by the availability of suitable culture facilities. A study was therefore undertaken to explore the potential of spoligotyping, a polymerase chain reaction based technique that does not require tuberculosis culture. METHODS: Adults attending a chest clinic with clinical or radiological pulmonary tuberculosis and one smear positive sputum were enrolled over one month. Demographic, socioeconomic, and clinical data were gathered using a standardised questionnaire. Molecular fingerprinting of genomic DNA recovered from sputum was performed by spoligotyping. RESULTS: Sixty one subjects (median age 28 years (range 18-73); 61% men) were recruited and 57 provided adequate sputum samples. Recent rural-urban migration or immigration was not common; 40% of subjects lived in crowded living conditions. DNA suitable for spoligotyping was recovered from 28 patients and 20 different genotypes of Mycobacterium tuberculosis were identified. Fifteen patients were infected with an M tuberculosis strain shared by one or more individuals. Patients infected with a shared spoligotype were not closely linked geographically within Harare, but were more likely to live in overcrowded conditions (69% versus 23%; odds ratio 6.85 (95% CI 1.2 to 47), p = 0.026). Analysis of the patients' original rural family homes revealed two geographically related spoligotype clusters. CONCLUSIONS: Spoligotyping may yield valuable molecular typing information in populations where tuberculosis culture is not available. This novel technique requires further development and evaluation in larger epidemiological studies.     

Pathophysiology of antigen 85 in patients with active tuberculosis: antigen 85 circulates as complexes with fibronectin and immunoglobulin G

Antigen 85 (Ag85) complex proteins are major secretory products of Mycobacterium tuberculosis and induce strong cellular and humoral immune responses in infected experimental animals and human beings. We have previously shown that nanogram doses of these 30- to 32-kDa fibronectin-binding proteins inhibit local expression of delayed hypersensitivity by a T-cell fibronectin-dependent mechanism. Circulating levels of Ag85 might be expected to be elevated in patients with active tuberculosis and possibly to play a role in systemic anergy in these patients. To test this hypothesis, Ag85 was measured in serum and urine by a monoclonal antibody-based dot immunobinding assay in 56 patients and controls with known skin test reactivity. Median serum Ag85 levels were 50- to 150-fold higher in patients with active tuberculosis than in patients with active M. avium-intracellulare disease or other nontuberculous pulmonary disease or in healthy controls (P < 0.001). The median and range of serum Ag85 in patients with active tuberculosis was not significantly different between skin test-positive and -negative subjects. Patients with active M. avium disease could be distinguished from those with disease due to M. tuberculosis by monoclonal anti-Ag85 antibodies of appropriate specificities. No increases in urinary Ag85 were detected in any patient, regardless of the Ag85 level in serum. Chromatographic analysis and immunoprecipitation studies of serum revealed that Ag85 existed in the serum of these patients complexed to either fibronectin or immunoglobulin G (IgG). Uncomplexed circulating Ag85 was demonstrable in serum from fewer than 20% of patients with active tuberculosis. In patients with active tuberculosis, Ag85 is therefore likely to circulate primarily as complexes with plasma fibronectin and IgG rather than in unbound form. The existence of Ag85 complexes with plasma proteins would account for its lack of urinary clearance.     

Clinical utility of an amplification test based on ligase chain reaction in pulmonary tuberculosis

We evaluated the sensitivity and specificity of a new semiautomated direct amplification test (DAT), the LCx-MTB, for the diagnosis of pulmonary tuberculosis (TB) and assessed its positive predictive value by focusing on patients with high clinical and radiologic suspicion of pulmonary TB. Respiratory tract specimens from 32 consecutive patients with high suspicion of active pulmonary TB (case patients) and from 204 control patients were cultured for Mycobacterium tuberculosis and tested by LCx-MTB. Sensitivity and specificity of LCx-MTB when compared with culture was, respectively, 80 and 98%. Pulmonary TB was confirmed in the 32 case patients without knowledge of the LCx results: 18 patients were smear- and culture-positive for M. tuberculosis, and all gave at least one specimen that was LCx-positive. Eight patients were smear-negative culture-positive, and seven gave at least one LCx-positive specimen. LCx-MTB was negative in all the specimens obtained from six patients with smear- and culture-negative TB. A positive LCx-MTB result in a smear negative specimen was 100% predictive that at least one of the case patients' specimens would yield M. tuberculosis. As a consequence, knowledge of the LCx-MTB results at the time of specimen collection could have hastened the start of the antituberculosis therapy in three (21%) smear-negative case patients and could have avoided unnecessary invasive diagnostic procedures in four (29%). We conclude that the sensitivity of LCx-MTB in detecting M. tuberculosis DNA in respiratory tract specimens is similar to other DATs, that LCx-MTB is a reliable test for confirmation of TB in smear-positive patients and that LCx-MTB could be beneficial as a diagnostic step in smear-negative patients with a high suspicion of pulmonary TB.     

Transmission of Mycobacterium tuberculosis from patients smear-negative for acid-fast bacilli

BACKGROUND: The microscopic examination of sputum for acid-fast bacilli, is a simple and rapid test that is used to provide a presumptive diagnosis of infectious tuberculosis. While patients with tuberculosis with sputum smears negative for acid-fast bacilli are less infectious than those with positive smears, both theoretical and empirical evidence suggest that they can still transmit Mycobacterium tuberculosis. We aimed to estimate the risk of transmission from smear-negative individuals. METHODS: As part of an ongoing study of the molecular epidemiology of tuberculosis in San Francisco, patients with tuberculosis with mycobacterial isolates with the same DNA fingerprint were assigned to clusters that were assumed to have involved recent transmission. Secondary cases with tuberculosis, whose mycobacterial isolates had the same DNA, were linked to their presumed source case to estimate transmission from smear-negative patients. Sensitivity analyses were done to assess potential bias due to misclassification of source cases, unidentified source cases, and HIV-1 co-infection. FINDINGS: 1574 patients with culture-positive tuberculosis were reported and DNA fingerprints were available for 1359 (86%) of these patients. Of the 71 clusters of patients infected with strains that had matching fingerprints, 28 (39% [95% CI 28-52]) had a smear-negative putative source. There were 183 secondary cases in these 71 clusters, of whom a minimum of 32 were attributed to infection by smear-negative patients (17% [12-24]). The relative transmission rate of smear-negative compared with smear-positive patients was calculated as 0.22 (95% CI 0.16-0.32). Sensitivity analyses and stratification for HIV-1 status had no impact on these estimates. INTERPRETATION: In San Francisco, the acid-fast-bacilli smear identifies the most infectious patients, but patients with smear-negative culture-positive tuberculosis appear responsible for about 17% of tuberculosis transmission.     

Regulation of nuclear factor-kappa B and its inhibitor I kappa B-alpha/MAD-3 in monocytes by Mycobacterium tuberculosis and during human tuberculosis

Blood monocytes from patients with active tuberculosis are activated in vivo, as evidenced by an increase in the stimulated release of proinflammatory cytokines, such as TNF-alpha, and the spontaneous expression of IL-2R. Further, monocytes from patients demonstrate an augmented susceptibility to a productive infection with HIV-1 in vitro. Mycobacterium tuberculosis and its components are strong signals to activate monocytes to production of cytokines. In this study we examined the basis of activation of monocytes during active tuberculosis and by M. tuberculosis. We found a constitutive degradation of I kappa B-alpha, the major cytoplasmic inhibitor of nuclear factor kappa B (NF-kappa B), in freshly isolated PBMC and monocytes from patients with tuberculosis. In contrast, I kappa B-alpha levels in PBMC and monocytes from healthy subjects or from patients with nontuberculous pulmonary conditions were intact. Further, by electrophoretic mobility shift assay, NF-kappa B was activated in monocytes from tuberculous patients. The expression of I kappa B-alpha gene, which is responsive to activation by NF-kappa B, was up-regulated in PBMC and monocytes from patients, but not in mononuclear cells from healthy subjects or those with nontuberculous lung diseases. By contrast, the expression of other adherence-associated early genes, such as IL-8 and IL-1 beta, was not up-regulated in PBMC of tuberculous patients. Further, M. tuberculosis and its tuberculin, purified protein derivative, induced the degradation of I kappa B-alpha and the expression of I kappa B-alpha mRNA, and purified protein derivative induced the activation of NF-kappa B in monocytes.     

Acquired drug resistance in Mycobacterium tuberculosis isolates recovered from compliant patients with human immunodeficiency virus-associated tuberculosis

We describe five compliant patients with human immunodeficiency virus (HIV)-associated tuberculosis (TB) that relapsed, with acquisition of resistance by the original Mycobacterium tuberculosis strains. Both the first and second isolates from each patient had the same IS (insertion sequence) 6110-based DNA fingerprint patterns. Three of the five patients developed TB that was resistant to rifampin alone; no mutation in the region of the rpoB gene was detected by a line probe assay in two of the isolates from these patients. We discuss several factors presumably associated with acquired drug resistance in HIV-infected patients, including exogenous reinfection, drug interactions, malabsorption of drugs, and the presence of a large organism burden.     

Rapid diagnosis of Mycobacterium tuberculosis bacteremia by PCR

A method based on DNA amplification and hybridization has been used for the rapid detection of Mycobacterium tuberculosis in blood samples from 38 hospitalized patients (15 human immunodeficiency virus [HIV] positive and 23 HIV negative) in whom localized or disseminated forms of tuberculosis were suspected. In 32 of these patients, the diagnosis of tuberculosis was eventually confirmed by conventional bacteriological or histological procedures. M. tuberculosis DNA was detected with the PCR technique in the peripheral blood mononuclear cells from 9 of 11 (82%) HIV-infected patients and in 7 of 21 (33%) HIV-negative patients (P < 0.01), while M. tuberculosis blood cultures were positive in 1 of 8 (12.5%) and 1 of 18 (5.5%) patients, respectively. PCR was positive in all cases with disseminated disease in both HIV-negative and HIV-positive patients and also in the HIV-positive patients with extrapulmonary tuberculosis. Seven samples from patients with documented illness other than tuberculosis and 12 specimens from healthy volunteers, including seven volunteers with a recent positive purified protein derivative test, were used as controls and had a negative PCR. These results suggest that detection of M. tuberculosis DNA in peripheral blood mononuclear cells may be a useful tool for rapid diagnosis of disseminated and extrapulmonary forms of tuberculosis, especially in an HIV-positive population.     

Isolation of murine telomere-proximal sequences by affinity capture and PCR

We describe a method of selectively enriching for murine telomere-proximal sequences using affinity capture followed by PCR amplification. The telomeric fragments were selected from NotI-digested and lambda exonuclease-resected mouse genomic DNA by annealing to a biotinylated riboprobe containing multiple copies of the telomere repeat (TTAGGG)n. The resultant DNA-RNA hybrids were selectively retained on a matrix with covalently bound avidin. The captured DNA was then specifically released by ribonuclease action, and PCR amplification was performed using mouse repeat primers. The PCR products were cloned and used to screen a mouse genomic cosmid library, and the resultant cosmid clones were analyzed by fluorescence in situ hybridization. Ten of 70 clones analyzed gave telomere-proximal hybridization signals, indicating an at least 500-fold enrichment for telomere-proximal sequences.     

Early detection of Mycobacterium tuberculosis in BACTEC cultures by ligase chain reaction

The LCx Mycobacterium tuberculosis ligase chain reaction system (Abbott Diagnostic Division, Abbott Park, Ill.) was used to detect M. tuberculosis in 150 consecutive BACTEC vials on the day on which a positive growth index (GI) was recorded. By LCx, M. tuberculosis DNA was detected in BACTEC vials on average 2.6 days before the presence of acid-fast bacilli could be confirmed by microscopic examination. A total of 106 of 108 M. tuberculosis isolates were detected without centrifugation from bottles presenting very low GIs (average, 70; median, 33). No false-positive result was obtained from nontuberculous mycobacteria or from isolates with contaminants.     

Heterogeneous antibody responses in tuberculosis

Antibody responses during tuberculosis were analyzed by an enzyme-linked immunosorbent assay with a panel of 10 protein antigens of Mycobacterium tuberculosis. It was shown that serum immunoglobulin G antibodies were produced against a variety of M. tuberculosis antigens and that the vast majority of sera from tuberculosis patients contained antibodies against one or more M. tuberculosis antigens. The number and the species of serologically reactive antigens varied greatly from individual to individual. In a given serum, the level of specific antibodies also varied with the antigen irrespective of the total number of antigens recognized by that particular serum. These findings indicate that person-to-person heterogeneity of antigen recognition, rather than recognition of particular antigens, is a key attribute of the antibody response in tuberculosis.     

Clinical review of 74 cases with miliary tuberculosis

Seventy-four cases of miliary tuberculosis were studied retrospectively. The mean age of the patients was 45.3 years. Twenty-two patients suffered from another underlying diseases. Six were infected with human immunodeficiency virus. Twelve had been treated with corticosteroids. Fever was present in 97.3 per cent of patients. Elevation of serum alkaline phosphatase was found in 67.6 per cent of cases. The skin reaction to tuberculin was positive in 61.2 per cent. Nodular shadows were found in the chest X-ray in 98.6 per cent of cases. The nodules were smaller than 2 mm in diameter in 52.7 per cent of cases. Other findings were enlargement of mediastinal lymph node (17.6%), cavities (23.0%), pleural effusion (27.0%), and consolidation (35.1%). Sputum cultures and urine cultures were positive for Mycobacterium tuberculosis in 76.8 per cent and 58.6 per cent of cases respectively. The diagnosis was confirmed by histopathological findings in some cases. The rate of positive biopsies was 61.5 per cent by bone marrow aspiration, 83.3 per cent by lymph node biopsy, 100 per cent by liver and lung biopsy. Antituberculosis therapy was successful in most of the patients. Seven patients died of miliary tuberculosis, 4 of them had adult respiratory distress syndrome.     

Purification and characterisation of isocitrate dehydrogenase and malate dehydrogenase from Mycobacterium tuberculosis and evaluation of their potential as suitable antigens for the serodiagnosis of tuberculosis

SETTING: Enzymes from Mycobacterium tuberculosis are potent antigens and might thus be of interest in the serodiagnosis of tuberculosis. OBJECTIVE: The purpose of the study was to purify and characterize the two enzymes isocitrate dehydrogenase (IDH) and malate dehydrogenase (MDH) from, M. tuberculosis and to evaluate their potential in the serodiagnosis of tuberculosis. DESIGN: The two enzymes were analysed for specificity by electrophoresis and then purified by means of affinity chromatography using reactive dyes and ion exchange chromatography. The two isolated enzyme fractions were analysed by ELISA, using antisera against related organisms. They were then tested as antigens in ELISA together with sera from tuberculous patients and controls. RESULTS: The electrophoretical analyses showed that the two enzymes each differed markedly from the corresponding enzymes of other mycobacteria. The serological analyses, however, could not distinguish between either IDH or MDH from other mycobacteria, but organisms of other genera, such as Nocardia, gave much weaker responses. When IDH and MDH were tested with sera from tuberculous patients and controls the former gave clearly higher optical density values than the latter. CONCLUSION: The enzymes/antigens IDH and MDH may be of value in developing a serological test for tuberculosis. The latter fraction seemed particularly capable of discriminating patients from controls.     

Diminished adherence and/or ingestion of virulent Mycobacterium tuberculosis by monocyte-derived macrophages from patients with tuberculosis

The interaction between the macrophage and Mycobacterium tuberculosis is mediated by a variety of macrophage membrane-associated proteins. Complement receptors have been implicated in the adherence of M. tuberculosis to macrophages. In the present work, the adherence and/or ingestion of M. tuberculosis H37Rv to human monocyte-derived macrophages (MDM) from patients with tuberculosis (TB) and healthy controls was measured by microscopical examination, [3H]uracil incorporation, and CFU. The adherence and/or ingestion was enhanced by fresh serum and inhibited by heat inactivation, EDTA treatment, and anti-CR1 and anti-CR3 antibodies. Comparison of MDM from TB patients and healthy controls showed that the former exhibited a significantly decreased capacity to adhere and/or ingest M. tuberculosis, as determined by the number of CFU and 3H incorporation. The expression of CR1 (CD35) and CR3 (CD11b/CD18) on MDM from TB patients and healthy controls, as determined by flow cytometry, did not show significant differences. These results suggest that the lower ingestion of M. tuberculosis by MDM from TB patients is not due to defects in complement receptors, and therefore, there might be other molecules involved in the adherence and/or ingestion process that render MDM from TB patients ingest less mycobacteria than those from healthy controls.     

The identification of Mycobacterium marinum genes differentially expressed in macrophage phagosomes using promoter fusions to green fluorescent protein

Mycobacterium marinum, like Mycobacterium tuberculosis, is a slow-growing pathogenic mycobacteria that is able to survive and replicate in macrophages. Using the promoter-capture vector pFPV27, we have constructed a library of 200-1000 bp fragments of M. marinum genomic DNA inserted upstream of a promoterless green fluorescent protein (GFP) gene. Only those plasmids that contain an active promoter will express GFP. Macrophages were infected with this fusion library, and phagosomes containing fluorescent bacteria were isolated. Promoter constructs that were more active intracellularly were isolated with a fluorescence-activated cell sorter, and inserts were partially sequenced. The promoter fusions expressed intracellularly exhibited homology to mycobacterial genes encoding, among others, membrane proteins and biosynthetic enzymes. Intracellular expression of GFP was 2-20 times that of the same clones grown in media. Several promoter constructs were transformed into Mycobacterium smegmatis, Mycobacterium bovis BCG and Mycobacterium tuberculosis. These constructs were positive for GFP expression in all mycobacterial strains tested. Sorting fluorescent bacteria in phagosomes circumvents the problem of isolating a single clone from macrophages, which may contain a mixed bacterial population. This method has enabled us to isolate 12 M. marinum clones that contain promoter constructs differentially expressed in the macrophage.     

Stability of Mycobacterium tuberculosis IS6110 restriction fragment length polymorphism patterns and spoligotypes determined by analyzing serial isolates from patients with drug-resistant tuberculosis

The stability of Mycobacterium tuberculosis IS6110 fingerprint patterns and spoligotypes has been assessed by analyzing serial isolates from patients with drug-resistant tuberculosis. Altogether, 165 M. tuberculosis isolates obtained from 56 patients have been analyzed. The time spans between the first and the last or a changed isolate from one patient ranged from 1 to 772 days. Among the 56 patients, 5 (9%) were infected with isolates with changes in their IS6110 fingerprint patterns. According to the total number of strains analyzed, 5% of the subsequent isolates showed variations in their IS6110 restriction fragment length polymorphism patterns compared to the pattern of the first isolates. Up to 10 isolates from one patient sampled at time intervals of up to 772 days with no changes in their IS6110 patterns have been analyzed. A statistically significant correlation could be found between changes in insertion sequence (IS) patterns and the increased time intervals over which the isolates were obtained, whereas changes in IS patterns are not correlated to changes in the drug resistance of the isolates. In contrast to the observed variations in IS6110 fingerprint patterns, no changes in the spoligotypes of the isolates analyzed could be found. In conclusion, our results confirm that the IS6110 fingerprint patterns of M. tuberculosis isolates have high degrees of stability. Compared to IS6110, the direct repeat (DR) region, which is the basis for spoligotyping, has a lower rate of change. Partial deletions, e.g., deletions induced by homologous recombination between the repetitive DR elements, could not be detected in this study.     

Recombinant expression and characterization of the major beta-lactamase of Mycobacterium tuberculosis

New antibiotic regimens are needed for the treatment of multidrug-resistant tuberculosis. Mycobacterium tuberculosis has a thick peptidoglycan layer, and the penicillin-binding proteins involved in its biosynthesis are inhibited by clinically relevant concentrations of beta-lactam antibiotics. beta-Lactamase production appears to be the major mechanism by which M. tuberculosis expresses beta-lactam resistance. beta-Lactamases from the broth supernatant of 3- to 4-week-old cultures of M. tuberculosis H37Ra were partially purified by sequential gel filtration chromatography and chromatofocusing. Three peaks of beta-lactamase activity with pI values of 5.1, 4.9, and 4.5, respectively, and which accounted for 10, 78, and 12% of the total postchromatofocusing beta-lactamase activity, respectively, were identified. The beta-lactamases with pI values of 5.1 and 4.9 were kinetically indistinguishable and exhibited predominant penicillinase activity. In contrast, the beta-lactamase with a pI value of 4.5 showed relatively greater cephalosporinase activity. An open reading frame in cosmid Y49 of the DNA library of M. tuberculosis H37Rv with homology to known class A beta-lactamases was amplified from chromosomal DNA of M. tuberculosis H37Ra by PCR and was overexpressed in Escherichia coli. The recombinant enzyme was kinetically similar to the pI 5.1 and 4.9 enzymes purified directly from M. tuberculosis. It exhibited predominant penicillinase activity and was especially active against azlocillin. It was inhibited by clavulanic acid and m-aminophenylboronic acid but not by EDTA. We conclude that the major beta-lactamase of M. tuberculosis is a class A beta-lactamase with predominant penicillinase activity. A second, minor beta-lactamase with relatively greater cephalosporinase activity is also present.     

Infection by Mycobacterium tuberculosis promotes human alveolar macrophage apoptosis

The effect of Mycobacterium tuberculosis infection on the viability of healthy (control) human alveolar macrophages was evaluated by staining with ethidium homodimer and calcein to discriminate live from dead cells. Infection with M. tuberculosis H37Ra or H37Rv increased macrophage mortality at 6 days from the control level of 3.8% +/- 0.7% to 28.7% +/- 6.9% or 12.6% +/- 3.1%, respectively (P < 0.001 for comparisons of all conditions). A role for tumor necrosis factor alpha (TNF-alpha) in the M. tuberculosis-induced cytolysis of alveolar macrophages was demonstrated by increased cytotoxicity following the addition of exogenous TNF-alpha to the cultures and by enhancement of macrophage survival when M. tuberculosis-infected alveolar macrophages were treated with pentoxifylline or anti-TNF-alpha antibody. The cytolytic mechanism was determined to be apoptosis by the demonstration of a characteristic internucleosomal ladder of genomic DNA by agarose gel electrophoresis, by finding nuclear fragmentation and condensation by electron microscopy, and by in situ terminal transferase-mediated nick end labeling of fragmented DNA in alveolar macrophages infected with M. tuberculosis in vitro. The latter technique was employed to reveal extensive apoptosis within caseating granulomas from lung tissue samples from clinical tuberculosis cases. The induction of apoptosis in alveolar macrophages by M. tuberculosis may play a role in the macrophage-pathogen interaction of tuberculosis in vivo.     

Evaluation of PCR in detection of Mycobacterium tuberculosis from formalin-fixed, paraffin-embedded tissues: comparison of four amplification assays

We compared the sensitivities and specificities of four nested PCR assays for the detection of Mycobacterium tuberculosis from formalin-fixed, paraffin-embedded tissues. Thirty-seven autopsy samples from human immunodeficiency virus-positive patients were analyzed: 15 were M. tuberculosis positive, 11 served as negative controls, and 11 were Ziehl-Neelsen positive without cultural confirmation of M. tuberculosis. Three genomic sequences (mtp40, 65-kDa antigen gene, and IS6110) with different molecular masses and numbers of repetitions within the M. tuberculosis genome were targeted. On the IS6110 sequence, two fragments of different sizes (106 and 123 bp, respectively) were amplified with two separate pairs of primers. The highest sensitivity rates were obtained by amplifying the highly repetitive IS6110 insertion sequence, and the different primers tested showed a sensitivity ranging from 80 to 87%. Amplification of the large 223-bp fragment of the mtp40 sequence present in a single copy in the M. tuberculosis genome yielded a high rate of false-negative results, ranging from 66 to 80%. A poor sensitivity (from 47 to 60%) was also shown by PCR amplification of the 142-bp 65-kDa antigen gene. All the PCRs except that for the 65-kDa antigen gene showed a specificity of 100%. Moreover, different results were obtained with different dilutions of DNA, and DNA concentrations of 1 and 3 microg yielded the highest sensitivities depending upon which protocol was used. Application of the PCRs to the Ziehl-Neelsen-positive, culture-negative samples confirmed the sensitivities of the PCRs obtained with the control samples. In conclusion, PCR can successfully be used to detect M. tuberculosis from paraffin-embedded tissues and can be particularly useful in the validation of a diagnosis of tuberculosis in clinical settings in which the diagnosis is uncertain. However, the efficacy of PCR strictly depends on several amplification parameters such as DNA concentration, target DNA size, and the repetitiveness of the amplified sequence.