Mycobacterium genotypes in pulmonary tuberculosis infections and their detection by trained African giant pouched rats.

Tuberculosis (TB) diagnosis in low-income countries is mainly done by microscopy. Hence, little is known about the diversity of Mycobacterium spp. in TB infections. Different genotypes or lineages of Mycobacterium tuberculosis vary in virulence and induce different inflammatory and immune responses. Trained Cricetomys rats show a potential for rapid diagnosis of TB. They detect over 28 % of smear-negative, culture-positive TB. However, it is unknown whether these rats can equally detect sputa from patients infected with different genotypes of M. tuberculosis. A 4-month prospective study on diversity of Mycobacterium spp. was conducted in Dar es Salaam, Tanzania. 252 sputa from 161 subjects were cultured on Lowenstein-Jensen medium and thereafter tested by rats. Mycobacterial isolates were subjected to molecular identification and multispacer sequence typing (MST) to determine species and genotypes. A total of 34 Mycobacterium spp. isolates consisting of 32 M. tuberculosis, 1 M. avium subsp. hominissuis and 1 M. intracellulare were obtained. MST analyses of 26 M. tuberculosis isolates yielded 10 distinct MST genotypes, including 3 new genotypes with two clusters of related patterns not grouped by geographic areas. Genotype MST-67, shared by one-third of M. tuberculosis isolates, was associated with the Mwananyamala clinic. This study shows that diverse M. tuberculosis genotypes (n = 10) occur in Dar es Salaam and trained rats detect 80 % of the genotypes. Sputa with two M. tuberculosis genotypes (20 %), M. avium hominissuis and M. intracellulare were not detected. Therefore, rats detect sputa with different M. tuberculosis genotypes and can be used to detect TB in resource-poor countries.

Diagnosis of tuberculosis by trained African giant pouched rats and confounding impact of pathogens and microflora of the respiratory tract.

Trained African giant-pouched rats (Cricetomys gambianus) can detect Mycobacterium tuberculosis and show potential for the diagnosis of tuberculosis (TB). However, rats' ability to discriminate between clinical sputum containing other Mycobacterium spp. and nonmycobacterial species of the respiratory tract is unknown. It is also unknown whether nonmycobacterial species produce odor similar to M. tuberculosis and thereby cause the detection of smear-negative sputum. Sputum samples from 289 subjects were analyzed by smear microscopy, culture, and rats. Mycobacterium spp. were isolated on Lowenstein-Jensen medium, and nonmycobacterial species were isolated on four different media. The odor from nonmycobacterial species from smear- and M. tuberculosis culture-negative sputa detected by ≥2 rats ("rat positive") was analyzed by gas chromatography-mass spectrometry and compared to the M. tuberculosis odor. Rats detected 45 of 56 confirmed cases of TB, 4 of 5 suspected cases of TB, and 63 of 228 TB-negative subjects (sensitivity, 80.4%; specificity, 72.4%; accuracy, 73.9%; positive predictive value, 41.7%; negative predictive value, 93.8%). A total of 37 (78.7%) of 47 mycobacterial isolates were M. tuberculosis complex, with 75.7% from rat-positive sputa. Ten isolates were nontuberculous mycobacteria, one was M. intracellulare, one was M. avium subsp. hominissuis, and eight were unidentified. Rat-positive sputa with Moraxella catarrhalis, Streptococcus pneumoniae, Staphylococcus spp., and Enterococcus spp. were associated with TB. Rhodococcus, Nocardia, Streptomyces, Staphylococcus, and Candida spp. from rat-positive sputa did not produce M. tuberculosis-specific volatiles (methyl nicotinate, methyl para-anisate, and ortho-phenylanisole). Prevalence of Mycobacterium-related Nocardia and Rhodococcus in smear-negative sputa did not equal that of smear-negative mycobacteria (44.7%), of which 28.6% were rat positive. These findings and the absence of M. tuberculosis-specific volatiles in nonmycobacterial species indicate that rats can be trained to specifically detect M. tuberculosis.

Ability of Cricetomys rats to detect Mycobacterium tuberculosis and discriminate it from other microorganisms.

Trained African giant pouched rats (Cricetomys gambianus) have potential for diagnosis of tuberculosis (TB). These rats target volatile compounds of Mycobacterium tuberculosis (Mtb) that cause TB. Mtb and nontuberculous mycobacteria (NTM) species are related to Nocardia and Rhodococcus spp., which are also acid-fast bacilli and can be misdiagnosed as Mtb in smear microscopy. Diagnostic performance of C. gambianus on in vitro-cultured mycobacterial and related pulmonary microbes is unknown. This study reports on the response of TB detection rats to cultures of reference Mtb, clinical Mtb, NTM, Nocardia; Rhodococcus; Streptomyces; Bacillus; and yeasts. Trained rats significantly discriminated Mtb from other microbes (p < 0.008, Fisher's exact test). Detection of Mtb cultures was age-related, with exponential and early stationary phase detected more frequently than early log phase and late stationary phase (p < 0.001, Fisher's test) (sensitivity = 83.33%, specificity = 94.4%, accuracy = 94%). The detection of naturally TB-infected sputum exceeded that of negative sputum mixed with Mtb, indicating that C. gambianus are conditioned to detect odours of TB-positive sputum better than spiked sputum. Although further studies on volatiles from detectable growth phases of Mtb are vital for identification of Mtb-specific volatiles detected by rats, our study underline the potential of C. gambianus for TB diagnosis.

Pouched rats' detection of tuberculosis in human sputum: comparison to culturing and polymerase chain reaction.

Setting. Tanzania. Objective. To compare microscopy as conducted in direct observation of treatment, short course centers to pouched rats as detectors of Mycobacterium tuberculosis. Design. Ten pouched rats were trained to detect tuberculosis in sputum using operant conditioning techniques. The rats evaluated 910 samples previously evaluated by smear microscopy. All samples were also evaluated through culturing and multiplex polymerase chain reaction was performed on culture growths to classify the bacteria. Results. The patientwise sensitivity of microscopy was 58.0%, and the patient-wise specificity was 97.3%. Used as a group of 10 with a cutoff (defined as the number of rat indications to classify a sample as positive for Mycobacterium tuberculosis) of 1, the rats increased new case detection by 46.8% relative to microscopy alone. The average samplewise sensitivity of the individual rats was 68.4% (range 61.1-73.8%), and the mean specificity was 87.3% (range 84.7-90.3%). Conclusion. These results suggest that pouched rats are a valuable adjunct to, and may be a viable substitute for, sputum smear microscopy as a tuberculosis diagnostic in resource-poor countries.

Mycobacterium tuberculosis volatiles for diagnosis of tuberculosis by Cricetomys rats.

Tuberculosis (TB) diagnosis in regions with limited resources depends on microscopy with insufficient sensitivity. Rapid diagnostic tests of low cost but high sensitivity and specificity are needed for better point-of-care management of TB. Trained African giant pouched rats (Cricetomys sp.) can diagnose pulmonary TB in sputum but the relevant Mycobacterium tuberculosis (Mtb)-specific volatile compounds remain unknown. We investigated the odour volatiles of Mtb detected by rats in reference Mtb, nontuberculous mycobacteria, Nocardia sp., Streptomyces sp., Rhodococcus sp., and other respiratory tract microorganisms spiked into Mtb-negative sputum. Thirteen compounds were specific to Mtb and 13 were shared with other microorganisms. Rats discriminated a blend of Mtb-specific volatiles from individual, and blends of shared, compounds (P = 0.001). The rats' sensitivity for typical TB-positive sputa was 99.15% with 92.23% specificity and 93.14% accuracy. These findings underline the potential of trained Cricetomys rats for rapid TB diagnosis in resource-limited settings, particularly in Africa where Cricetomys rats occur widely and the burden of TB is high.

APOPO's tuberculosis research agenda: achievements, challenges and prospects.

This article describes Anti-Persoonsmijnen Ontmijnende Product Ontwikkeling (APOPO) recent use of specially trained African giant pouched rats as detectors of pulmonary tuberculosis in people living in Tanzania. It summarizes the achievements and challenges encountered over the years and outlines future prospects. Since 2008, second-line screening by the rats has identified more than 2000 tuberculosis-positive patients who were missed by microscopy at Direct Observation of Treatment--Short Course centres in Tanzania. Moreover, data that are reviewed herein have been collected with respect to the rats' sensitivity and specificity in detecting tuberculosis. Findings strongly suggest that scent-detecting rats offer a quick and practical tool for detecting pulmonary tuberculosis and within the year APOPO's tuberculosis-detection project will be extended to Mozambique. As part of its local capacity building effort, APOPO hires and trains Tanzanians to play many important roles in its TB detection project and provides research and training opportunities for Tanzanian students.

Using giant African pouched rats to detect tuberculosis in human sputum samples: 2010 findings.

Giant African pouched rats previously have detected tuberculosis (TB) in human sputum samples in which the presence of TB was not initially detected by smear microscopy. Operant conditioning principles were used to train these rats to indicate TB-positive samples. In 2010, rats trained in this way evaluated 26,665 sputum samples from 12,329 patients. Microscopy performed at DOTS centers found 1,671 (13.6%) of these patients to be TB-positive. Detection rats identified 716 additional TB-positive patients, a 42.8% increase in new-case detection. These previously unreported data, which extend to over 20,000 the number of patients evaluated by pouched rats in simulated second-line screening, suggest that the rats can be highly valuable in that capacity.

Using giant African pouched rats to detect tuberculosis in human sputum samples: 2009 findings.

In 2009, giant African pouched rats trained to detect tuberculosis (TB) evaluated sputum samples from 10,523 patients whose sputum had previously been evaluated by smear microscopy. Microscopists found 13.3% of the patients to be TB-positive. Simulated second-line screening by the rats revealed 620 new TB-positive patients, increasing the case detection rate by 44%. These data suggest that the rats may be useful for TB detection in developing countries, although further research is needed.