Evidence of Mycobacterium tuberculosis Complex bacteraemia in intradermal skin test positive cattle detected using phage-RPA

Bovine tuberculosis is a zoonotic infectious disease caused by Mycobacterium bovis 19 that affects cattle and can cause tuberculosis in a range of wildlife animals. A 20 bacteriophage-based method combined with PCR (phage-PCR) has been recently used to 21 detect and identify viable pathogenic mycobacteria in the peripheral blood mononuclear 22 cells (PBMCs) of animals suffering from paratuberculosis. To adapt this method for the 23 detection of M. bovis in blood, a new isothermal DNA amplification protocol using 24 Recombinase Polymerase Amplification (RPA) was developed and was found to be able to 25 detect M. bovis BCG within 48 h, with a limit of detection of approximately 10 cells per 26 ml of blood for artificially inoculated blood samples. When blood samples (2 ml) from a 27 Single Comparative Cervical Intradermal Tuberculin (SCCIT)- negative beef herd were 28 tested, Mycobacterium tuberculosis complex (MTC) cells were not detected from any 29 (45) of the blood samples. However when blood samples from SCCIT-positive animals 30 were tested, viable MTC bacteria were detected in 66 % (27/41) of samples. Of these 41 31 animals sampled, 32 % (13) had visible lesions. In the visible lesion (VL) group, 85 % 32 (11/13) had detectable levels of MTC whereas only 57 % (16/28) of animals which had 33 no visible lesions (NVL) were found to have detectable mycobacteraemia. These results 34 indicated that this simple, rapid method can be applied for the study of M. bovis 35 infections. The frequency with which viable mycobacteria were detected in the 36 peripheral blood of SCCIT-positive animals changes the paradigm of this disease

Evaluation of qPCR-Based Assays for Leprosy Diagnosis Directly in Clinical Specimens

The increased reliability and efficiency of the quantitative polymerase chain reaction (qPCR) makes it a promising tool for performing large-scale screening for infectious disease among high-risk individuals. To date, no study has evaluated the specificity and sensitivity of different qPCR assays for leprosy diagnosis using a range of clinical samples that could bias molecular results such as difficult-to-diagnose cases. In this study, qPCR assays amplifying different M. leprae gene targets, sodA, 16S rRNA, RLEP and Ag 85B were compared for leprosy differential diagnosis. qPCR assays were performed on frozen skin biopsy samples from a total of 62 patients: 21 untreated multibacillary (MB), 26 untreated paucibacillary (PB) leprosy patients, as well as 10 patients suffering from other dermatological diseases and 5 healthy donors. To develop standardized protocols and to overcome the bias resulted from using chromosome count cutoffs arbitrarily defined for different assays, decision tree classifiers were used to estimate optimum cutoffs and to evaluate the assays. As a result, we found a decreasing sensitivity for Ag 85B (66.1%), 16S rRNA (62.9%), and sodA (59.7%) optimized assay classifiers, but with similar maximum specificity for leprosy diagnosis. Conversely, the RLEP assay showed to be the most sensitive (87.1%). Moreover, RLEP assay was positive for 3 samples of patients originally not diagnosed as having leprosy, but these patients developed leprosy 5–10 years after the collection of the biopsy. In addition, 4 other samples of patients clinically classified as non-leprosy presented detectable chromosome counts in their samples by the RLEP assay suggesting that those patients either had leprosy that was misdiagnosed or a subclinical state of leprosy. Overall, these results are encouraging and suggest that RLEP assay could be useful as a sensitive diagnostic test to detect M. leprae infection before major clinical manifestations

Failure to Recognize Nontuberculous Mycobacteria Leads to Misdiagnosis of Chronic Pulmonary Tuberculosis

BACKGROUND: Nontuberculous mycobacterial (NTM) infections cause morbidity worldwide. They are difficult to diagnose in resource-limited regions, and most patients receive empiric treatment for tuberculosis (TB). Our objective here is to evaluate the potential impact of NTM diseases among patients treated presumptively for tuberculosis in Mali. METHODS: We re-evaluated sputum specimens among patients newly diagnosed with TB (naïve) and those previously treated for TB disease (chronic cases). Sputum microscopy, culture and Mycobacterium tuberculosis drug susceptibility testing were performed. Identification of strains was performed using molecular probes or sequencing of secA1 and/or 16S rRNA genes. RESULTS: Of 142 patients enrolled, 61 (43%) were clinically classified as chronic cases and 17 (12%) were infected with NTM. Eleven of the 142 (8%) patients had NTM disease alone (8 M. avium, 2 M. simiae and 1 M. palustre). All these 11 were from the chronic TB group, comprising 11/61 (18%) of that group and all were identified as candidates for second line treatment. The remaining 6/17 (35.30%) NTM infected patients had coinfection with M. tuberculosis and all 6 were from the TB treatment naïve group. These 6 were candidates for the standard first line treatment regimen of TB. M. avium was identified in 11 of the 142 (8%) patients, only 3/11 (27.27%) of whom were HIV positive. CONCLUSIONS: NTM infections should be considered a cause of morbidity in TB endemic environments especially when managing chronic TB cases to limit morbidity and provide appropriate treatment