Detection of Nontuberculous Mycobacterial Species from Tissue Samples of Cattle and Buffaloes by PCR and PRA (PCR-RFLP)

Background: Nontuberculous mycobacteria are opportunistic pathogens and some of them may cause disease in humans and animals causing pulmonary infections, mastitis, lesions in respiratory tract and lymph nodes of cattle, due to which they are being recognized worldwide and also interfere with the diagnosis of bovine tuberculosis. Methods: The present study was conducted for detection of nontuberculous mycobacterial species (NTM) in tissue samples (with and without tubercle lesions) in cattle and buffaloes from postmortem hall GADVASU, Ludhiana. Polymerase Chain Reaction and PCR-RFLP which involved hsp65 gene amplification (439 bp) and restriction analysis of amplified product was performed on 30 tissue samples for detection of nontuberculous mycobacterial species. Result: Three out of 30 samples showed hsp65 gene amplification and 2 were identified as M. kansasii using restriction analysis technique and one could not be identified as the RFLP patterns was different from other known PCR-RFLP profiles. NTM such as M. kansasi may cause infection in animals and PRA (PCR-Restriction Fragment Length Polymorphism Analysis) technique was found to be a rapid tool for identification and differentiation of NTM upto species level.

Prevalence and molecular identification of Mycobacteria isolated from animals slaughtered at Sokoto modern abattoir, Sokoto State, Nigeria

This study investigated the molecular epidemiology of Mycobacteria isolated from animals slaughtered at Sokoto modern abattoir. During meat inspection, 104 suspected tuberculosis lesions were sampled from a total of 102,681 animals slaughtered between November 2016 and January 2018. These samples were subjected to Ziehl Neelsen staining, followed by culture on Lowenstein-Jensen media. Subsequently, polymerase chain reaction (PCR) and sequencing of the 65KDa heat shock protein (hsp65) gene were performed to identify and phylogenetically characterize the cultured organisms. Because sequencing of the hsp65 gene was unable to distinguish between Mycobacterium bovis (M. bovis) and M. tuberculosis, PCR was performed to amplify a genomic region-specific to M. bovis in order to differentiate them from M. tuberculosis. Results showed that, 14 samples yielded growth after culture. Furthermore, hsp65 was detected in 9 out of the 14 isolates screened, 5 of the amplicons were successfully sequenced. Similarity search using NCBI BLAST tool showed the five sequences to share highest identities with Mycobacterium novocastrense (95.99%), M. canettii (94.54%), and M. tuberculosis/M. bovis (100%). Two out of the 5 isolates were confirmed to be M. bovis after PCR amplification using M. bovis specific primers. Phylogenetic tree further confirmed the identity of these isolates by placing them close to species of their kind. Further studies should be conducted to establish the transmission dynamics of the zoonotic Mycobacteria between animals and their owners, to facilitate control and eradication of tuberculosis.

Genetic Diversity of Non-Tuberculous Mycobacteria among Symptomatic TB negative Patients in Kenya

Non-Tuberculous Mycobacteria (NTM) transmission to humans occurs through inhalation of dust particles or vaporized water containing NTM leading to pulmonary manifestations. NTM infections are often misdiagnosed for tuberculosis (TB) due to their similar clinical and radiological manifestations. We, therefore, performed a species-level identification of NTM in symptomatic TB negative patients through sequencing of the hsp65 gene. We conducted a cross-sectional study at the National Tuberculosis Reference Laboratory in the period between January to November 2020. One hundred and sixty-six mycobacterial culture-positive samples that tested negative for TB using capilia underwent Polymerase Chain Reaction targeting the hsp65 gene. Isolates showing a band with gel electrophoresis at 441 bp position were sequenced using Sanger technology. Geneious software was used to analyze the obtained sequences, and the National Center for Biotechnology Information gene database identified NTM species for each isolate. A phylogenetic tree was constructed from the DNA sequences and evolutionary distances computed using the general time-reversible method. Pearson chi-square was used to determine the association between NTM infection and participants characteristics. Our study identified 43 different NTM species. The dominant NTM belonged to Mycobacterium avium complex 37 (31%). Slow-growing NTM were the majority at 86 (71%) while rapid-growing NTM were 36 (29%). A significant association (p<0.05) was observed for regions and age, while patient type had a week likelihood of NTM infection. Our study characterized the diversity of NTM in Kenya for the first time and showed a high diversity of NTM species.

Peritoneal dialysis-associated peritonitis caused by Mycobacteroides massiliense: the first case and review of the literature

Background Peritoneal dialysis (PD)-associated peritonitis caused by nontuberculous Mycobacterium is rare; however, the number of cases has increased over the past decades. Mycobacteroides massiliense is a subspecies of the Mycobacteroides abscessus complex. It has different clinical characteristics compared to the other subspecies of the complex. Previous case reports of PD-associated peritonitis caused by Mycobacteroides abscessus complex have not distinguished the subspecies in detail. Case presentation A 40-year-old man presented with an exit-site and tunnel infection refractory to antibiotic therapy. Peritonitis occurred after simultaneous catheter removal and reinsertion. The Mycobacteroides abscessus complex was detected in the culture of the dialysis effluent. Removal of the PD catheter combined with antibiotics, including macrolides, resulted in a good clinical course. Further analysis of multiplex PCR and the hsp65 gene sequence identified the bacterium as Mycobacteroides massiliense. Conclusions The Mycobacteroides abscessus complex is classified into three subspecies; Mycobacteroides abscessus, Mycobacteroides massiliense, and Mycobacteroides bolletii. These have different characteristics, particularly antibiotic susceptibility. Therefore, clear identification of the subspecies of the Mycobacteroides abscessus complex is necessary for definitive treatment.

Isolation and Molecular Identification of Nontuberculous Mycobacterium from Different Species of Fish in Karbala Province, Iraq

This study was designed for isolation and molecular identification of Nontuberculous Mycobacterium (NTM) from fish during the period between October and December 2017 from Karbla province-Iraq. This study included 200 fresh fish samples from four different species including Spondyliosoma cantharus, Liza abu, Carassius carassius and Cyprinuscarpio .Three samples of each fish were taken including gills, muscles and all internal organs. The samples were processed by decontamination, concentration of 4% sodium hydroxide, and 0.1 ml of sediment was streaking on Löwenstein Johnson (LJ) media; then the bacterial cultures were incubated at 28-30 °C for 3days up to 4 weeks and suspected colonies were stained with acid fast stain to confirm the presence of Mycobacterium. Further identification, biochemical tests were carried out to confirm the diagnosis of isolates, PCR was done using 16s RNA gene for all isolates, hsp65 gene was used in unidentified NTM spp and to confirm the others. Results revealed that out of 200 fish samples, 19 isolates 9.5% were identified as NTM belonged to Rapid Growth Mycobacterium (RGM). of the total isolates, 18.26 % was investigated from Liza abu (Kishni, Abu khraiza). NTM (RGM) isolates on spp level identified six spp of these isolates. M. porcinum was 26.32% which was followed by M. fortuitum of 21.05%, others included M. neworleansense and M. mucogenicum 10.5% of each, M. cosmeticum and M. pallens 5.26% of each. The distribution of NTM spp in the fish organs, nine out of 47.37% NTM isolate were recovered from gills followed by muscles 36.84 %, while 15.79% from internal organs. These results were the first study concerning isolation of these spp of NTM from fish in Iraq, and some spp are not reported in other studies. This study concluded that the fish is an importance source or reservoir for NTM, especially the pathogenic spp

Immunotherapeutic Activities of a DNA Plasmid Carrying the Mycobacterial hsp65 Gene (DNAhsp65)

DNA vaccines have become relevant subject matter, and efforts for their development have been increasing due to their potential as technology platforms applicable for prophylactic and therapeutic approaches for infectious diseases and for cancer treatment, allergies, and autoimmune diseases. This review aimed to summarize current knowledge about the plasmid DNA vaccine carrying the mycobacterial hsp65 gene (DNAhsp65), which demonstrates immunomodulatory and immunoregulatory properties of both the innate and adaptive immune systems. The possible mechanisms associated with the modulation and regulatory role of DNAhsp65 in the control of various conditions is also discussed.

Meningitis caused by Mycobacterium fortuitum mimicking tuberculous meningitis in an immunocompetent patient: a case report and literature review.

Background: Nontuberculous mycobacteria (NTM) are a large group of microorganisms that mainly affect persons who have immune deficiency. Mycobacterium fortuitum is one of them, which usually cause soft tissue or pulmonary disease. Meningitis caused by M. fortuitum is extremely rare and is often confused with tuberculous meningitis (TBM) due to its low prevalence. Case presentation: A 35-year-old woman was referred to our hospital with a history of headache, followed by fever, chills, cough, and vomiting. The results of mycobacterial culture and TSPOT. TB of her cerebrospinal fluid were positive. Therefore, she was diagnosed with tuberculous meningitis and was given anti-tuberculous chemotherapy. During the observation, the CSF results were not significantly improved. Finally, hsp65 gene PCR revealed that the real pathogen was M. fortuitum . The treatment therapy was changed and the patient recovered after one-year combined chemotherapy. Conclusions: In this case we described a patient with M. fortuitum meningitis who was immunocompetent with no trauma or surgical history. The wrong diagnosis with TBM was made due to positive results of both blood and CSF TSPOT. TB . The pathological evidence and microbiological analysis of hsp65 gene PCR suggested the real pathogen to be M. fortuitum . The diagnostic accuracy of blood and CSF TSPOT. TB in case of TBM is moderate. Infections of NTM cannot be excluded when the patient does not respond to anti-tuberculous therapy and it is of great significance to do molecular identification for clinical isolates of mycobacterium.

Meningitis caused by Mycobacterium fortuitum mimicking tuberculous meningitis in an immunocompetent patient: a case report and literature review.

Background: Nontuberculous mycobacteria (NTM) are a large group of microorganisms that mainly affect persons who have immune deficiency. Mycobacterium fortuitum is one of them, which usually cause soft tissue or pulmonary disease. Meningitis caused by M. fortuitum is extremely rare and is often confused with tuberculous meningitis (TBM) due to its low prevalence. Case presentation: A 35-year-old woman was referred to our hospital with a history of headache, followed by fever, chills, cough, and vomiting. The results of mycobacterial culture and TSPOT. TB of her cerebrospinal fluid were positive. Therefore, she was diagnosed with tuberculous meningitis and was given anti-tuberculous chemotherapy. During the observation, the CSF results were not significantly improved. Finally, hsp65 gene PCR revealed that the real pathogen was M. fortuitum . The treatment therapy was changed and the patient recovered after one-year combined chemotherapy. Conclusions: In this case we described a patient with M. fortuitum meningitis who was immunocompetent with no trauma or surgical history. The wrong diagnosis with TBM was made due to positive results of both blood and CSF TSPOT. TB . The pathological evidence and microbiological analysis of hsp65 gene PCR suggested the real pathogen to be M. fortuitum . The diagnostic accuracy of blood and CSF TSPOT. TB in case of TBM is moderate. Infections of NTM cannot be excluded when the patient does not respond to anti-tuberculous therapy and it is of great significance to do molecular identification for clinical isolates of mycobacterium.

Meningitis caused by Mycobacterium fortuitum mimicking tuberculous meningitis in an immunocompetent patient: a case report and literature review.

Background: Nontuberculous mycobacteria (NTM) are a large group of microorganisms that mainly affect persons who have immune deficiency. Mycobacterium fortuitum is one of them, which usually cause soft tissue or pulmonary disease. Meningitis caused by M. fortuitum is extremely rare and is often confused with tuberculous meningitis (TBM) due to its low prevalence. Case presentation: A 35-year-old woman was referred to our hospital with a history of headache, followed by fever, chills, cough, and vomiting. The results of mycobacterial culture and TSPOT. TB of her cerebrospinal fluid were positive. Therefore, she was diagnosed with tuberculous meningitis and was given anti-tuberculous chemotherapy. During the observation, the CSF results were not significantly improved. Finally, hsp65 gene PCR revealed that the real pathogen was M. fortuitum. The treatment therapy was changed and the patient recovered after one-year combined chemotherapy. Conclusions: In this case we described a patient with M. fortuitum meningitis who was immunocompetent with no trauma or surgical history. The wrong diagnosis with TBM was made due to positive results of both blood and CSF TSPOT. TB . The pathological evidence and microbiological analysis of hsp65 gene PCR suggested the real pathogen to be M. fortuitum . The diagnostic accuracy of blood and CSF TSPOT. TB in case of TBM is moderate. Infections of NTM cannot be excluded when the patient does not respond to anti-tuberculous therapy and it is of great significance to do molecular identification for clinical isolates of mycobacterium.

Identification of Non-tuberculous Mycobacteria by Partial Gene Sequencing and Matrix Assisted Laser Desorption Ionisation Time of Flight at a Tertiary Care Hospital in Telangana, India

Introduction: Non-tuberculous Mycobacteria (NTM) pulmonary disease is often unrecognised or misdiagnosed as Mycobacterium tuberculosis (MTB), Multi-Drug Resistant Tuberculosis (MDRTB), because of similar clinical presentation in counties with high burden of Tuberculosis (TB) including India. In India due to lack of awareness among clinicians and lack of laboratory facilities to diagnose these infections, its prevalence is largely unknown. Aim: To evaluate the efficacy of identification of NTM species by Matrix Assisted Laser Desorption Ionisation Time of Flight (MALDI-TOF) and Heat Shock Protein (hSP65) gene sequencing and to determine their Antimicrobial Susceptibility Testing (AST). Materials and Methods: All the clinical specimens from pulmonary and extra-pulmonary TB suspects at Nizam’s Institute of Medical Sciences, Hyderabad, Telangana, India, over a period of one year i.e., from June 2017 to May 2018 were included in the study. The specimens were subjected to microscopy, culture and GeneXpert. The NTMs isolated in the culture were further characterised genotypically by MALDI-TOF and hsp65 gene sequencing. The identified NTM isolates were subjected to AST. All the methods were followed as per the standard protocols. Data was analysed using SPSS 25. Results: A total of 1085 samples were processed out of which Mycobacteria was detected in 201 cases (18.5%). Among the culture positives, MTB complex was detected in 146 cases (13.5%) and NTM in 55 (5.06%). Mycobacterium abscessus was the predominant isolate. The most common co-morbidities were bronchiectasis and Chronic Obstructive Pulmonary Disease (COPD). Linezolid, clarithromycin, moxifloxacin and amikacin showed high sensitivity. Conclusion: Molecular assay helps in rapid identification which can lead to targeted therapy and can thus combat antimicrobial resistance. The MALDI-TOF and hsp65 gene sequencing also offers quick results at a low cost and is easy to perform hence it can be considered as an alternate diagnostic tool for identification.