Diagnosis of Tuberculosis by Trained African Giant Pouched Rats and Confounding Impact of Pathogens and Microflora of the Respiratory Tract

ABSTRACT 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.

[1]  S. Molin,et al.  Volatile metabolites from actinomycetes. , 2002, Journal of agricultural and food chemistry.

[2]  W. Takken,et al.  Cultured skin microbiota attracts malaria mosquitoes , 2009, Malaria Journal.

[3]  M. Drancourt,et al.  Dissection of phylogenetic relationships among 19 rapidly growing Mycobacterium species by 16S rRNA, hsp65, sodA, recA and rpoB gene sequencing. , 2004, International journal of systematic and evolutionary microbiology.

[4]  Edward T. Richardson,et al.  Rapid Identification of Mycobacterium tuberculosis and Nontuberculous Mycobacteria by Multiplex, Real-Time PCR , 2009, Journal of Clinical Microbiology.

[5]  Using giant African pouched rats to detect tuberculosis in human sputum samples: 2010 findings , 2011, The Pan African medical journal.

[6]  S. Wilton,et al.  Detection and identification of multiple mycobacterial pathogens by DNA amplification in a single tube. , 1992, PCR methods and applications.

[7]  I. Bastian,et al.  A review of the diagnosis and treatment of smear-negative pulmonary tuberculosis. , 2000, The international journal of tuberculosis and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease.

[8]  A. Poling,et al.  Using giant African pouched rats to detect tuberculosis in human sputum samples: 2009 findings. , 2010, The American journal of tropical medicine and hygiene.

[9]  U. Reischl,et al.  Detection and Differentiation of Mycobacterium tuberculosis and Nontuberculous Mycobacterial Isolates by Real-Time PCR , 2003, Journal of Clinical Microbiology.

[10]  H. Reichenbach,et al.  Identification and synthesis of volatiles released by the myxobacterium Chondromyces crocatus , 2004 .

[11]  H. Sheehan,et al.  The staining of tubercle bacilli with Sudan black B. , 1949, The Journal of pathology and bacteriology.

[12]  D. van Soolingen,et al.  Nontuberculous Mycobacteria, Zambia , 2009, Emerging infectious diseases.

[13]  Graham Bothamley,et al.  Breath biomarkers of active pulmonary tuberculosis. , 2010, Tuberculosis.

[14]  M. McNeil,et al.  Rapid Identification of Nocardia farcinica Clinical Isolates by a PCR Assay Targeting a 314-Base-Pair Species-Specific DNA Fragment , 2004, Journal of Clinical Microbiology.

[15]  Jeroen S. Dickschat,et al.  Bacterial volatiles: the smell of small organisms. , 2007, Natural product reports.

[16]  M. Barer,et al.  Resuscitation-promoting factors reveal an occult population of tubercle Bacilli in Sputum. , 2010, American journal of respiratory and critical care medicine.

[17]  K. Wilkins,et al.  Volatile Organic Metabolites from Selected Streptomyces Strains , 2009 .

[18]  E. Desmond,et al.  Opacification of Middlebrook agar as an aid in identification of Nocardia farcinica , 1993, Journal of clinical microbiology.

[19]  M. Nei,et al.  Extensive Gains and Losses of Olfactory Receptor Genes in Mammalian Evolution , 2007, PloS one.

[20]  J. Wahlfors,et al.  Use of multiplex PCR for simultaneous detection of four bacterial species in middle ear effusions , 1997, Journal of clinical microbiology.

[21]  S. Chambers,et al.  The scent of Mycobacterium tuberculosis. , 2008, Tuberculosis.

[22]  Z. Premji,et al.  Coartem®: the journey to the clinic , 2009, Malaria Journal.

[23]  A. Karstaedt,et al.  Nocardial infection as a complication of HIV in South Africa. , 2000, Journal of Infection.

[24]  Lauren E. Manning,et al.  The scent of Mycobacterium tuberculosis--part II breath. , 2009, Tuberculosis.

[25]  G. Strobel,et al.  Isolation and characterization of Muscodor albus I-41.3s, a volatile antibiotic producing fungus , 2005 .

[26]  E. Tóth,et al.  Fly-attracting volatiles produced by Rhodococcus fascians and Mycobacterium aurum isolated from myiatic lesions of sheep. , 2002, Journal of microbiological methods.

[27]  L. Tell,et al.  Comparison of Four Rapid DNA Extraction Techniques for Conventional Polymerase Chain Reaction Testing of Three Mycobacterium spp. that Affect Birds , 2003, Avian diseases.

[28]  T. Demeester,et al.  Specificity and sensitivity of objective diagnosis of gastroesophageal reflux disease. , 1987, Surgery.

[29]  F. Lwilla,et al.  African pouched rats for the detection of pulmonary tuberculosis in sputum samples. , 2009, The international journal of tuberculosis and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease.

[30]  A Lazarus,et al.  Management of tuberculosis , 2000 .

[31]  S. Goodison,et al.  16S ribosomal DNA amplification for phylogenetic study , 1991, Journal of bacteriology.

[32]  Eduardo Gotuzzo,et al.  Rapid molecular detection of tuberculosis and rifampin resistance. , 2010, The New England journal of medicine.

[33]  C. Hackett,et al.  Production of Volatile Organic Compounds by Trichoderma in Media Containing Different Amino Acids and Their Effect on Selected Wood Decay Fungi , 2000 .

[34]  J. Crump,et al.  Invasive Disease Caused by Nontuberculous Mycobacteria, Tanzania , 2009, Emerging infectious diseases.

[35]  K. Yazawa,et al.  Identification of Nocardia farcinica by a PCR primer amplifying a specific DNA band for the bacterium. , 2007, Nihon Ishinkin Gakkai zasshi = Japanese journal of medical mycology.

[36]  H. Vandendool,et al.  A GENERALIZATION OF THE RETENTION INDEX SYSTEM INCLUDING LINEAR TEMPERATURE PROGRAMMED GAS-LIQUID PARTITION CHROMATOGRAPHY. , 1963, Journal of chromatography.

[37]  S. Egwaga,et al.  The quality of sputum smear microscopy diagnosis of pulmonary tuberculosis in Dar es Salaam, Tanzania. , 2008, Tanzania health research bulletin.

[38]  S. Gharbia,et al.  Multispacer Sequence Typing for Mycobacterium tuberculosis Genotyping , 2008, PloS one.

[39]  C. Hackett,et al.  Effect of substrate composition on production of volatile organic compounds from Trichoderma spp. inhibitory to wood decay fungi , 1997 .

[40]  P. Farmer,et al.  The Cambridge Declaration: towards clinical trials for drug-resistant tuberculosis. , 2009, The international journal of tuberculosis and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease.