Bovine Tuberculosis at the Wildlife-Livestock-Human Interface in Hamer Woreda,South Omo,Southern Ethiopia

Bovine tuberculosis (BTB) is endemic in cattle in the Ethiopian Highlands but no studies have been done so far in pastoralists in South Omo. This study assessed the prevalence of bovine tuberculosis (BTB) at an intensive interface of livestock, wildlife and pastoralists in Hamer Woreda (South Omo), Ethiopia. A cross-sectional survey including a comparative intradermal skin testing (CIDT) was conducted in 499 zebu cattle and 186 goats in 12 settlements. Sputum samples from 26 symptomatic livestock owners were cultured for TB. Fifty-one wildlife samples from 13 different species were also collected in the same area and tested with serological (lateral flow assay) and bacteriological (culture of lymph nodes) techniques. Individual BTB prevalence in cattle was 0.8% (CI: 0.3%–2%) with the >4 mm cut-off and 3.4% (CI: 2.1%–5.4%) with the >2 mm cut-off. Herd prevalence was 33.3% and 83% when using the >4 and the >2 mm cut-off respectively. There was no correlation between age, sex, body condition and positive reactors upon univariate analysis. None of the goats were reactors for BTB. Acid fast bacilli (AFB) were detected in 50% of the wildlife cultures, 79.2% of which were identified as Mycobacterium terrae complex. No M. bovis was detected. Twenty-seven percent of tested wildlife were sero-positive. Four sputum cultures (15.4%) yielded AFB positive colonies among which one was M. tuberculosis and 3 non-tuberculous mycobacteria (NTM). The prevalence of M. avium-complex (MAC) was 4.2% in wildlife, 2.5% in cattle and 0.5% in goats. In conclusion, individual BTB prevalence was low, but herd prevalence high in cattle and BTB was not detected in goats, wildlife and humans despite an intensive contact interface. On the contrary, NTMs were highly prevalent and some Mycobacterium spp were more prevalent in specific species. The role of NTMs in livestock and co-infection with BTB need further research.     

Predictive value of the acid fast smear for detection of Mycobacterium tuberculosis in respiratory specimens in a reference center of HIV/AIDS in Rio de Janeiro, Brazil

In order to evaluate the predictive value of acid fast bacilii (AFB) smear for the diagnosis of Mycobacterium tuberculosis in respiratory specimens in a setting with a high prevalence of AIDS and an unknown prevalence of nontuberculous mycobacteria (NTM), we retrospectively examined specimens cultured for mycobacteria between 1 September 1993 and 30 September 1994 and medical records of patients with positive culture in a General Hospital, AIDS reference in Rio de Janeiro, Brazil. Seventy three per cent (1517/2077) of samples were respiratory specimens and mycobacteria were recovered from 20.6% (313/1517) of these. M. tuberculosis was identified in 94.2% (295/313) and NTM in 5.8% (18/313). The yield of positive AFB smear and of positive culture was 6.1% (93/1517) and 20.6% (313/1517), respectively. The positive predictive value (PPV) of AFB for M. tuberculosis was 98.4% in expectorated sputum and 96.4% in bronchoalveolar lavage. Forty four percent (130/295) of specimens with positive culture for M. tuberculosis and 66.7% (12/18) for NTM were from patients HIV positive. The conclusion was that in our study population, the PPV of AFB for M. tuberculosis in respiratory specimens was high and the prevalence of NTM was low despite the high prevalence of HIV positive.     

Assessment of the N-PCR Assay in Diagnosis of Pleural Tuberculosis: Detection of M.tuberculosis in Pleural Fluid and Sputum Collected in Tandem

Background

The nonspecific clinical presentation and paucibacillary nature of tuberculous pleuritis remains a challenge for diagnosis. Diagnosis of tuberculous pleural effusion depends on the demonstration of the presence of tubercle bacilli in the sputum, pleural fluid, or pleural biopsy specimen, or demonstration of granuloma in pleura by histological examination. We examined the clinical utility of the diagnosis of pleural tuberculosis using the in house N-PCR assay, AFB smear microscopy and culture. Besides pleural fluid the inclusion of sputum in the efficacy of diagnosis of pleural tuberculosis was scrutinized.

Methodology/Principal Findings

Pleural fluid and sputum samples of 58 tuberculous and 42 non-tuberculous pleural effusion patients were processed for AFB smear microscopy, culture and the N-PCR assay. Mycobacteria were detected exclusively in tuberculous pleural effusion samples. None of the non-tuberculous pleural effusion samples were positive for mycobacteria. Comparative analysis showed that the N-PCR assay had the highest sensitivity. Inclusion of sputum along with pleural fluid increased N-PCR sensitivity from 51.7 to 70.6% (p<0.0001).This improved sensitivity was reflected in AFB smear microscopy and isolation by culture. The sensitivity enhanced on inclusion of sputum from 3.4 (p = 0.50) to 10.3% (p = 0.038) for AFB smear microscopy and for isolation of mycobacteria from 10.3(p = 0.03) to 22.4% (p = 0.0005). Thirteen isolates were obtained from 58 pleural tuberculosis patients. Eleven mycobacterial isolates were identified as M.tuberculosis and two as M.fortuitum and M.chelonae. Complete concordance was seen between the biochemical identification of isolates and the N-PCR identification of mycobacterial species prior to isolation.

Conclusions/Significance

To the best of our knowledge this is the first PCR based report on utility of sputum for diagnosis of pleural tuberculosis. The present study demonstrates that a combination of pleural fluid with sputum sample and N-PCR improved the diagnosis of pleural tuberculosis.     

Comparison of the mycobacteria growth indicator tube with radiometric and solid culture for isolation of mycobacteria from clinical specimens and susceptibility testing of Mycobacterium tuberculosis

We compared the mycobacteria growth indicator tube (MGIT) system with the BACTEC 460 TB and Loewenstein-Jensen (LJ) systems for the recovery of mycobacteria (acid-fast bacilli [AFB]) from 600 clinical specimens. A total of 50 AFB (32 Mycobacterium tuberculosis complex, 10 M. avium complex, 3 M. gordonae, 3 M. xenopi, 1 M. terrae and 1 M. fortuitum) were detected. MGIT recovered 50 isolates of AFB (100% sensitivity), and BACTEC 460 TB and LJ recovered 49 (98% sensitivity) and 19 (38% sensitivity) AFB isolates, respectively. The mean times to detect mycobacteria were 10, 10 and 25 days for MGIT, BACTEC 460, and LJ slants. All isolates of M. tuberculosis complex were tested for susceptibility to streptomycin, isoniazid, rifampin, and ethambutol with the MGIT and BACTEC 460 TB. Both systems yielded identical susceptibility data with different mean times to report (5.38 days for MGIT versus 7.33 days for BACTEC 460 TB, P<0.05). The results suggest that MGIT is equivalent to BACTEC 460 TB in its ability to support the growth of mycobacteria, but significantly more efficient than LJ. MGIT may also be used for susceptibility testing of primary antituberculosis drugs.     

Nucleic acid amplification of Mycobacterium tuberculosis complex DNA from archival fine needle aspiration smear scrapings vs. fresh fine needle aspirates of tuberculous lymphadenitis

OBJECTIVE: To assess the efficacy of the nucleic acid amplification (NAA) technique for Mycobacterium tuberculosis (MTB) complex from archival fine needle aspirate (FNA) smear scrapings of confirmed cases of extrapulmonary tuberculosis (EPTB) for a retrospective diagnosis of EPTB as compared to NAA from fresh FNA material from the same cases. STUDY DESIGN: Smear scrapings from 51 cases; 33 cases of tuberculous lymphadenitis (from patients who had undergone NAA 1 year before for MTB from fresh FNA material); 13 negative controls from nontuberculous, archival FNA smears; and 5 known acid-fast bacilli (AFB)-positive sputum smears, were subjected to NAA using the IS6110 primer sequence of M tuberculosis. Ziehl-Neelsen staining was done in all the smears. RESULTS: Of the 33 cases of tuberculous lymphadenitis, 15 (45.4%) were AFB positive and 18 (64.5%) AFB negative. MTB NAA was positive in 73.3% (11 of 15 AFB-positive cases) in the freshly aspirated material and was observed in 60% (9 of 15 AFB-positive cases) when done on DNA extracted from the archival smear scrapings of the same cases. Similarly, in the 18 AFB-negative cases, MTB NAA positivity was 72.2% (13 of 18) on fresh material and 44.4% (8 of 18) on archival smear scrapings from the same AFB-negative cases. Overall NAA positivity was 51.5% for archival smear scrapings as compared to 71% for fresh FNA of the same cases. CONCLUSION: Low NAA sensitivity of MTB DNA in archival material of known tuberculous cases limits the routine use of NAA based retrospective molecular diagnosis of MTB complex.     

Concentration of Lymph Node Aspirate Improves the Sensitivity of Acid Fast Smear Microscopy for the Diagnosis of Tuberculous Lymphadenitis in Jimma,Southwest Ethiopia

Background

Tuberculous lymphadenitis (TBLN) is the most common form of extrapulmonary tuberculosis. The cytomorphological features of lymph node smears have reduced specificity for the diagnosis of tuberculosis. The diagnosis of TBLN with direct smear microscopy lacks sensitivity due to the limited number of bacilli in lymph node aspirate. Therefore, we aimed to assess whether the concentration of lymph node aspirate improves the sensitivity of acid fast smear microscopy for the diagnosis of tuberculous lymphadenitis.

Methods

A cross-sectional comparative study was conducted on 200 patients clinically suspected for tuberculous lymphadenitis in Jimma, Ethiopia. Lymph node aspirate was collected. The first two drops were used for cytomorphological study and direct acid fast staining. The remaining aspirate was treated with N-acetyl-L-cysteine (NALC) and concentrated by centrifugation at 3000 g for 15 minutes. The sediment was used for acid fast staining and culture. Differentiation of M. tuberculosis complex (MTBC) from non-tuberculous mycobacteria (NTM) was done by para-nitrobenzoic acid susceptibility test.

Result

Complete data were available for 187 study subjects. 68% (127/187) were positive for M. tuberculosis on culture. Four isolates, 2.1% (4/187), were identified as NTM. The detection rate of direct smear microscopy was 25.1% and that of the concentration method 49.7%. Cytomorphologically, 79.7% of cases were classified as TBLN. The sensitivity of direct smear microscopy was 34.6%, for concentrated smear microscopy 66.1%, and for cytomorphology 89.8%. Two AFB positive cases on concentration method were non-tuberculosis mycobacteria (NTM). The concentration method yielded a positive result from seven cases diagnosed as suppurative abscess by cytology. Both for the direct and concentration methods the highest rate of AFB positivity was observed in smears showing caseous necrosis alone. Smear positivity rate decreased with the appearance of epithelioid cell aggregates.

Conclusion

The concentration of lymph node aspirates for acid fast smear microscopy had significantly higher sensitivity than direct microscopy.     

Rapid immunochromatographic serum assay of nontuberculous mycobacterial infections

A rapid immunochromatographic serologic assay (Dot assay) is proposed to be applied on patients infected with nontuberculous mycobacteria (NTM). This assay could evidentiate the infecting species and allow the beginning of the treatment. The test is based on the principle of immunoblotting chromatography, a rapid membrane-based assay, capable of diagnosing NTM infections in serum, in less than 1 hour, with no need of special equipment or skilled staff. The secreted extracellular antigens have been isolated from the unheated culture filtrates of the clinically significant NTM (M. avium, MAI, M. kansasii, M. xenopi, M. chelonaei, M. scrofulaceum, M. marinum, M. fortuitum, M. abscesus, M. szulgai). The patients have been tested against these antigens, as well as from M. tuberculosis H37Rv, due to the possibility of co-infection with tuberculous bacilli. A number of 385 tests on patient sera have been performed (10, with NTM suspected infection, with or without M. tuberculosis co-infection, 5 with confirmed diagnosis of NTM infection, 10 with TB, 10 with other respiratory diseases). The preliminary results presented in this paper support the fact that the rapid immunochromatographic serum assay, combined with clinical and radiographic evidence, could evidentiate the infecting NTM species and allow the start of an earlier treatment, but must be confirmed on a higher number of patients.     

Increase in Nontuberculous Mycobacteria Isolated in Shanghai,China: Results from a Population-Based Study

Background

In China, the prevalence of nontuberculous mycobacteria (NTM) in isolates from mycobacterial culture-positive patients with pulmonary tuberculosis (TB) is largely unknown.

Methods

We used conventional biochemical and 16S rRNA gene sequencing to identify species of mycobacteria in specimens from patients suspected of having TB. Drug-susceptibility testing was performed on NTM isolates using the proportion method. We also determined the independent risk factors associated with infection with NTM compared with infection with Mycobacterium tuberculosis.

Results

The overall rate of NTM isolated from mycobacterial culture-positive patients was 5.9% in this population, with a significantly increasing trend from 3.0% in 2008 to 8.5% in 2012 (P for trend <0.001). The organism most frequently identified was M. kansasii (45.0%), followed by M. intracellulare (20.8%) and M. chelonae/abscessus (14.9%). The overall proportion of isolates resistant to the four first-line anti-TB agents were 64.6% for isoniazid, 77.6% for streptomycin, 63.3% for rifampicin and 75.1% for ethambutol. The risk factors most often associated with NTM infection were older age (P for trend <0.001), being a resident of Shanghai (adjusted odds ratio [aOR], 1.48; 95% CI, 1.10–2.00), having been treated for tuberculosis (aOR, 1.64; 95% CI, 1.18–2.29), having a cavity on chest X-ray (aOR, 1.51; 95% CI, 1.16–1.96), and being sputum smear–negative (aOR, 1.59; 95% CI, 1.16–2.18).

Conclusions

The prevalence of NTM isolated in Shanghai increased between 2008 and 2012, thus clinicians should consider NTM as a possible cause of TB-like disease. Accurate species identification is imperative so that proper treatment can be administered for diseases caused by the diversity of NTM species.     

Species-specific immunocytochemical localization of Mycobacterium tuberculosis complex in fine needle aspirates of tuberculous lymphadenitis using antibody to 38 kDa immunodominant protein antigen

OBJECTIVE: To examine immunocytochemical localization of Mycobacterium tuberculosis (MTB) complex antigen in fine needle aspiration (FNA) smears of tuberculous lymphadenitis (TBLN) using species-specific monoclonal antibody MTSS to 38-kDa immnunodominant protein antigen as a diagnostic adjunct to conventional cytomorphology and its advantage over Ziehl-Neelsen (ZN) microscopy. Study Design FNA smears from 340 cases-174 TBLN; 34 negative controls from nontuberculous, positive controls of 13 known acid-fast bacilli (AFB)-positive sputum smears; 50 blind controls; and 69 other controls (smears from stock cultures of bacterial, atypical mycobacteria and fungal species) were subjected to ZN and immunocytochemical staining using MTSS by the streptavidin-biotin method. RESULTS: Immunocytochemical staining was positive in 59 of 61 (96.7%) archival and 110 of 113 (97.3%) fresh FNA smears; ZN positivity for AFB was observed in 27 of 61 (44.2%) archival and 48 of 113 (42.4%) fresh FNA smears of TBLN. CONCLUSION: The immunostaining using MTSS showed a definite advantage over conventional ZN staining for detection and specific diagnosis of TBLN in FNA smears with 0% false positive results. Immunostaining of cytosmears with species specific antibody to MTB would prove to be a good diagnostic adjunct to morphologic diagnosis.     

Comparison of NaOH-N-acetyl cysteine and sulfuric acid decontamination methods for recovery of mycobacteria from clinical specimens

We compared the NaOH-N-acetyl cysteine (NaOH-NALC) and the sulfuric acid decontamination procedure in the detection of mycobacteria using the Mycobacteria Growth Indicator Tube (MGIT). In total 219 sputum specimens were collected from 142 Zambian patients and subjected to mycobacterial culture. One half of the specimen was decontaminated with NaOH-NALC and the other half was decontaminated with sulfuric acid. From the 438 samples a total of 261 (60%) cultures yielded growth of mycobacteria, consisting of 22 different species. The sulfuric acid method was more successful than the NaOH-NALC method in recovering mycobacteria in MGITs (146 versus 115 respectively, p = 0.001). Of the 146 positive mycobacterial cultures recovered after sulfuric acid decontamination 28 were Mycobacterium tuberculosis, 84 nontuberculous mycobacteria (NTM) and 34 acid fast bacterial isolates which could not be identified to the species level. The 115 mycobacteria recovered by the NaOH-NALC method consisted of 34 M. tuberculosis strains, 55 NTM and 26 acid fast bacteria that could not be identified. The most frequently isolated NTM were Mycobacterium lentiflavum and Mycobacterium intracellulare. Comparing the two decontamination methods the recovery of NTM in the sulfuric acid group was significant higher than in the NaOH-NALC group (p = 0.001). In contrast, no significant difference was found for the recovery of M. tuberculosis. These results show that the decontamination method used affects the recovery of nontuberculous mycobacteria in particular.     

Mycobacterial growth in human macrophages: variation according to donor, inoculum and bacterial strain

The microbicidal capacity of the macrophage is frequently evaded by mycobacteria, leading to tuberculosis (TB). We investigated a number of parameters affecting the rate of growth of mycobacteria in human monocyte-derived macrophages (MDM). The results show a great deal of variation in the growth of both Mycobacterium bovis BCG and M. tuberculosis H37Rv, using a large number of human macrophage donors, (132 and 40, respectively), but no correlation was seen with the TB status of the MDM donor. Clumping of the mycobacteria resulted in more vigorous growth in MDM, suggesting that inoculum size could affect disease progression. The growth rates of 17 clinical isolates of M. tuberculosis were measured in macrophages derived from three donors and no consistent or marked differences between isolates were observed over the 5-day period of growth measurement. However, all 17 clinical strains grew consistently faster than H37Rv in the same experiments.     

Utility of repeat fine needle aspiration in acute suppurative lesions. Follow-up of 263 cases

OBJECTIVE: To determine the clinical value of repeat fine needle aspiration (FNA) as a follow-up strategy in the management of patients in India, clinically suspected of having tuberculosis (TB) but showing a cytologic picture of acute suppuration. STUDY DESIGN: Repeat aspirates from 263 patients presenting with lymph node or soft tissue masses were analyzed. The previous FNA of these cases had shown acute inflammatory exudate but no epithelioid granuloma or acid-fast bacilli (AFB). RESULTS: The repeat FNA helped to detect 55% additional cases of TB within a period of 8 weeks; 67% of them were diagnosed in the second and third weeks. Diagnostic yield rose to 59% after the third FNA. AFB were detected in 34 (13.3%) cases that showed a low bacterial load. In addition, nontubercular lesions, such as epidermal inclusion cyst (4), cysticercosis (3), sialadenitis (2) and metastatic carcinoma (8), were diagnosed. CONCLUSION: All cases showing acute suppuration without granulomas or AFB on the first FNA should be reevaluated by follow-up FNA and staining for AFB. This will enhance the diagnostic yield of tuberculosis in developing countries, where molecular diagnostics are too costly or unavailable. This procedure is cost effective as compared to biopsy and culture. In addition to tuberculosis, many unexpected nontubercular lesions may also be unmasked. Repeat FNA reduces sampling and screening errors, improves sensitivity and helps to study the evolution of epithelioid granulomas.     

Mycobacteria mobility shift assay: a method for the rapid identification of Mycobacterium tuberculosis and nontuberculous mycobacteria

The identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-perform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteria.     

Mycobacterium bovis identification by a molecular method from post-mortem inspected cattle obtained in abattoirs of Mato Grosso do Sul, Brazil

The presence of Mycobacterium bovis in bovine carcasses with lesions suggestive of tuberculosis was evaluated. Seventy-two carcass samples were selected during slaughter inspection procedures in abattoirs in the state of Mato Grosso do Sul, Brazil. Seventeen (23.6%) of samples showed colonies suggestive of mycobacteria that were confirmed to be acid-fast bacilli by Ziehl-Neelsen staining. Polymerase chain reaction (PCR) using primers specific for M. bovis identified M. bovis in 13 (76.5%) isolates. The PCR-restriction enzyme pattern analysis using gene encoding for the 65-kDa protein and two restriction enzymes identified the remaining four isolates that were represented by two M. tuberculosis complex and two nontuberculous mycobacteria. The results are indicative of infection of slaughter cattle by M. bovis and other mycobacteria in the state of Mato Grosso do Sul.     

Transmission of Mycobacterium tuberculosis between Farmers and Cattle in Central Ethiopia

Background

Transmission of Mycobacterium tuberculosis (M. tuberculosis) complex could be possible between farmers and their cattle in Ethiopia.

Methodology/Principal Findings

A study was conducted in mixed type multi-purposes cattle raising region of Ethiopia on 287 households (146 households with case of pulmonary tuberculosis (TB) and 141 free of TB) and 287 herds consisting of 2,033 cattle belonging to these households to evaluate transmission of TB between cattle and farmers. Interview, bacteriological examinations and molecular typing were used for human subjects while comparative intradermal tuberculin (CIDT) test, post mortem and bacteriological examinations, and molecular typing were used for animal studies. Herd prevalence of CIDT reactors was 9.4% and was higher (p<0.01) in herds owned by households with TB than in herds owned by TB free households. Animal prevalence was 1.8% and also higher (p<0.01) in cattle owned by households with TB case than in those owned by TB free households. All mycobacteria (141) isolated from farmers were M. tuberculosis, while only five of the 16 isolates from cattle were members of the M. tuberculosis complex (MTC) while the remaining 11 were members of non-tuberculosis mycobacteria (NTM). Further speciation of the five MTC isolates showed that three of the isolates were M. bovis (strain SB1176), while the remaining two were M. tuberculosis strains (SIT149 and SIT53). Pathology scoring method described by “Vordermeier et al. (2002)” was applied and the average severity of pathology in two cattle infected with M. bovis, in 11 infected with NTM and two infected with M. tuberculosis were 5.5, 2.1 and 0.5, respectively.

Conclusions/Significance

The results showed that transmission of TB from farmers to cattle by the airborne route sensitizes the cows but rarely leads to TB. Similarly, low transmission of M. bovis between farmers and their cattle was found, suggesting requirement of ingestion of contaminated milk from cows with tuberculous mastitis.     

Nontuberculous mycobacteria in respiratory samples from patients with pulmonary tuberculosis in the state of Rond?nia, Brazil

The main cause of pulmonary tuberculosis (TB) is infection with Mycobacterium tuberculosis (MTB). We aimed to evaluate the contribution of nontuberculous mycobacteria (NTM) to pulmonary disease in patients from the state of Rondônia using respiratory samples and epidemiological data from TB cases. Mycobacterium isolates were identified using a combination of conventional tests, polymerase chain reaction-based restriction enzyme analysis of hsp65 gene and hsp65 gene sequencing. Among the 1,812 cases suspected of having pulmonary TB, 444 yielded bacterial cultures, including 369 cases positive for MTB and 75 cases positive for NTM. Within the latter group, 14 species were identified as Mycobacterium abscessus, Mycobacterium avium, Mycobacterium fortuitum, Mycobacterium intracellulare, Mycobacterium gilvum, Mycobacterium gordonae, Mycobacterium asiaticum, Mycobacterium tusciae, Mycobacterium porcinum, Mycobacterium novocastrense, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium phlei and Mycobacterium holsaticum and 13 isolates could not be identified at the species level. The majority of NTM cases were observed in Porto Velho and the relative frequency of NTM compared with MTB was highest in Ji-Paraná. In approximately half of the TB subjects with NTM, a second sample containing NTM was obtained, confirming this as the disease-causing agent. The most frequently observed NTM species were M. abscessus and M. avium and because the former species is resistant to many antibiotics and displays unsatisfactory cure rates, the implementation of rapid identification of mycobacterium species is of considerable importance.     

Paleopathological and biomolecular study of tuberculosis in a medieval skeletal collection from England

Nine human skeletons of medieval date from a rural English burial site show signs of skeletal tuberculosis. They were subject to polymerase chain reaction (PCR) assays aimed at detecting traces of DNA from infecting mycobacteria, with the purpose both of confirming the paleopathological diagnosis of tuberculosis and determining in individual cases whether disease was due to M. tuberculosis or M. bovis. In all nine cases, evidence for M. tuberculosis complex DNA was found, and in all instances it appeared that disease was due to M. tuberculosis rather than M. bovis. The significance of the findings for understanding tuberculous infection in rural agrarian communities in medieval England is discussed.     

Identification of Mycobacterium tuberculosis complex based on amplification and sequencing of the oxyR pseudogene from stored Ziehl-Neelsen-stained sputum smears in Brazil

A cross-sectional analysis of stored Ziehl-Neelsen (ZN)-stained sputum smear slides (SSS) obtained from two public tuberculosis referral laboratories located in Juiz de Fora, Minas Gerais, was carried out to distinguish Mycobacterium bovis from other members of the Mycobacterium tuberculosis complex (MTC). A two-step approach was used to distinguish M. bovis from other members of MTC: (i) oxyR pseudogene amplification to detect MTC and, subsequently, (ii) allele-specific sequencing based on the polymorphism at position 285 of this gene. The oxyR pseudogene was successfully amplified in 100 of 177 (56.5%) SSS available from 99 individuals. No molecular profile of M. bovis was found. Multivariate analysis indicated that acid-fast bacilli (AFB) results and the source laboratory were associated (p < 0.05) with oxyR pseudogene amplification. SSS that were AFB++ SSS showed more oxyR pseudogene amplification than those with AFB0, possibly due to the amount of DNA. One of the two source laboratories presented a greater chance of oxyR pseudogene amplification, suggesting that differences in sputum conservation between laboratories could have influenced the preservation of DNA. This study provides evidence that stored ZN-SSS can be used for the molecular detection of MTC.     

Species Spectrum of Nontuberculous Mycobacteria Isolated from Clinical Specimens in Kuwait

Specific identification of mycobacteria is of clinical relevance since treatment varies according to the Mycobacterium species causing infection. All mycobacterial isolates are currently identified as M. tuberculosis (MTB) or nontuberculous mycobacteria (NTM) based on p-nitro-α-acetylamino-β-hydroxypropiophenone (NAP) test, and the species spectrum of NTM-causing infections in Kuwait remains unknown. This study identified all NTM strains isolated in Kuwait from 1 October 2003 to 31 March 2004 to the species level. The mycobacteria were cultured from various clinical specimens using the BACTEC 460 TB system and NAP test was performed to differentiate MTB from NTM strains. The INNO-LiPA MYCOBACTERIA v2 assay (LiPA) was used for species-specific identification of NTM strains and some randomly selected MTB strains. The LiPA results for selected isolates were confirmed by DNA sequencing of the 16S-23S internal transcribed spacer region. A total of 325 isolates of Mycobacterium species originating from 305 individual patients were recovered during the study period, with 307 and 18 isolates identified as MTB and NTM, respectively. The LiPA correctly identified all 18 MTB isolates analyzed. Seven different NTM species were identified among 18 NTM isolates originating from 14 patients, with M. fortuitum causing the majority of NTM infections in Kuwait. One patient was infected with two NTM species. Rapid species-specific identification of NTM may help with appropriate treatment regimens for proper patient management. The DNA sequencing data reported in this study are deposited in EMBL under accession numbers AM709724 to AM709731.     

Identification of antigens specific to non-tuberculous mycobacteria: the Mce family of proteins as a target of T cell immune responses

The lack of an effective TB vaccine hinders current efforts in combating the TB pandemic. One theory as to why BCG is less protective in tropical countries is that exposure to non-tuberculous mycobacteria (NTM) reduces BCG efficacy. There are currently several new TB vaccines in clinical trials, and NTM exposure may also be relevant in this context. NTM exposure cannot be accurately evaluated in the absence of specific antigens; those which are known to be present in NTM and absent from M. tuberculosis and BCG. We therefore used a bioinformatic pipeline to define proteins which are present in common NTM and absent from the M. tuberculosis complex, using protein BLAST, TBLASTN and a short sequence protein BLAST to ensure the specificity of this process. We then assessed immune responses to these proteins, in healthy South Africans and in patients from the United Kingdom and United States with documented exposure to NTM. Low level responses were detected to a cluster of proteins from the mammalian cell entry family, and to a cluster of hypothetical proteins, using ex vivo ELISpot and a 6 day proliferation assay. These early findings may provide a basis for characterising exposure to NTM at a population level, which has applications in the field of TB vaccine design as well as in the development of diagnostic tests.