Identification Of Mycobacterium Species Research Articles

Evaluation of nucleotide MALDI-TOF-MS for the identification of Mycobacterium species.

The accurate identification of the Mycobacterium tuberculosis complex (MTBC) and different nontuberculous mycobacteria (NTM) species is crucial for the timely diagnosis of NTM infections and for reducing poor prognoses. Nucleotide matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been extensively used for microbial identification with high accuracy and throughput. However, its efficacy for Mycobacterium species identification has been less studied. The objective of this study was to evaluate the performance of nucleotide MALDI-TOF-MS for Mycobacterium species identification. A total of 933 clinical Mycobacterium isolates were preliminarily identified as NTM by the MPB64 test. These isolates were identified by nucleotide MALDI-TOF-MS and Sanger sequencing. The performance of nucleotide MALDI-TOF MS for identifying various Mycobacterium species was analyzed based on Sanger sequencing as the gold standard. The total correct detection rate of all 933 clinical Mycobacterium isolates using nucleotide MALDI-TOF-MS was 91.64% (855/933), and mixed infections were detected in 18.65% (174/933) of the samples. The correct detection rates for Mycobacterium intracellulare, Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium avium, MTBC, Mycobacterium gordonae, and Mycobacterium massiliense were 99.32% (585/589), 100% (86/86), 98.46% (64/65), 94.59% (35/37), 100.00% (34/34), 95.65% (22/23), and 100% (19/19), respectively. For the identification of the MTBC, M. intracellulare, M.abscessus, M. kansasii, M. avium, M. gordonae, and M. massiliense, nucleotide MALDI-TOF-MS and Sanger sequencing results were in good agreement (k > 0.7). In conclusion, nucleotide MALDI-TOF-MS is a promising approach for identifying MTBC and the most common clinical NTM species.

Issues of morphological diagnosis and pathogenesis of tuberculosis

Tuberculosis remains a serious global problem of our time. The epidemiological situation regarding tuberculosis in the Russian Federation and in Moscow is quite favorable, however, many manifestations of diseases and morphological changes in tuberculosis require a serious approach to the timely diagnosis of the disease, especially intravital morphological verification of the process. The article outlines the main aspects of the pathogenesis of tuberculosis, including deep immunosuppression associated with HIV. A typical microscopic picture of tuberculosis inflammation is described with an algorithm for identifying the causative agent of tuberculosis in histological sections and cytological preparations, and species identification of mycobacteria in material from paraffin blocks. Attention is paid to the morphology of HIV-associated tuberculosis, which is characterized by the loss of signs of granulomatous inflammation in condition of deep immune suppression. The need for differential diagnosis of tuberculosis with many infectious and non-infectious granulomatous-necrotic processes is noted, which requires the morphologist to compare the cellular composition of granulomas, study perifocal tissue reactions and features of vascular damage, correctly assess the activity of inflammatory changes, etc. Attention is drawn to the features of the morphological diagnostic search in cases combined pathology of tuberculosis with other infectious diseases, incl. with Covid 19. Changes are difficult to analyze due to the combination of morphological manifestations of several diseases, each of which may have atypical microscopic manifestations, as well as the varying activity of several simultaneously occurring infectious processes.

The Homologous Gene of Chromosomal Virulence D (chvD) Presents High Resolution as a Novel Biomarker in Mycobacterium Species Identification.

To evaluate the resolution of chromosomal virulence D (chvD) as a novel marker for mycobacterial species identification. A segment of chvD (652 bp) was amplified by PCR from 63 mycobacterial reference strains, 163 nontuberculous mycobacterial clinical isolates, and 16 M. tuberculosis complex (MTBC) clinical isolates. A phylogenetic tree based on the reference strains was constructed by the neighbor-joining and IQ-tree methods. Comparative sequence analysis of the homologous chvD gene efficiently differentiated the species within the genus Mycobacterium. Slowly growing Mycobacterium (SGM) and rapidly growing Mycobacterium (RGM) were separated in the phylogenetic tree based on the chvD gene. The sequence discrepancies were obvious between M. kansasii and M. gastri, M. chelonae and M. abscessus, and M. avium and M. intracellulare, none of which could be achieved by 16S ribosomal RNA (rRNA) homologous gene alignment. Furthermore, chvD manifested larger intraspecies diversity among members of M. intracellulare subspecies. A total of 174 of the 179 (97.21%) clinical isolates, consisting of 12 mycobacterial species, were identified correctly by chvD blast. Four M. abscessus subsp. abscessus were identified as M. abscessus subsp. bolletii by chvD. MTBC isolates were indistinguishable, because they showed 99.84%-100% homology. Homologous chvD is a promising gene marker for identifying mycobacterial species, and could be used for highly accurate species identification among mycobacteria.

Molecular Identification of Mycobacterium species Based on Genotype MTBC in Goats Slaughtered at Kano Abattoir, Kano State, Nigeria

Molecular Identification of Mycobacterium species Based on Genotype MTBC in Goats Slaughtered at Kano Abattoir, Kano State, Nigeria

Molecular Characterization of Mycobacterium species Isolates from Patients with Pulmonary Tuberculosis in Sabah, Malaysia

Tuberculosis (TB) is one of the deadliest diseases worldwide, caused by members of Mycobacterium tuberculosis complex (MTBC), commonly by Mycobacterium tuberculosis (Mtb) and Mycobacterium bovis. In Malaysia, Sabah is one of the states of public health concern with the highest TB cases. Clinical presentations of TB and non-tuberculous mycobacteria (NTM) lung disease are similar, and mycobacteria appear to be identical under standard diagnosis with sputum smear microscopy, causing difficulty to diagnose TB. Identification of Mycobacterium species is essential for effective management of mycobacterial diseases treatment and their control strategy. Thus, this study aimed to identify the Mycobacterium species from suspected TB patients in Sabah using molecular methods. Sputum samples (n=595) were screened with GeneXpert MTB/RIF (Xpert), and positive TB samples (n=67) were processed and cultured in BACTEC MGIT. Forty-five isolates were successfully recovered in MGIT and characterisation of the mycobacterial isolates using PCR and/or sequencing with rpoB, RD9, hsp65, and 16S rRNA genes confirmed the presence of Mtb in 41 samples, and four non-mycobacteria, i.e. Microbacterium laevaniformans, Streptomyces sp., Streptomyces misionensis and Gordonia sp. These non-mycobacteria isolates showed negative results when tested directly with Xpert. In conclusion, Mtb is the predominant species of MTBC circulating in Sabah. The presence of non-mycobacteria in this study was due to bacterial contamination in MGIT, not bacterial cross-reactivity in Xpert, implying the high sensitivity and specificity of Xpert for diagnosis of TB.

New design of multilocus sequence analysis of rpoB, ssrA, tuf, atpE, ku, and dnaK for identification of Mycobacterium species.

Differentiating Mycobacterium tuberculosis (MTB) from nontuberculous mycobacteria (NTM) is very important in the treatment process of patients. According to the American Thoracic Society guideline (ATS), NTM clinical isolates should be identified at the species level proper treatment and patient management. This study aimed to identify NTM clinical isolates by evaluationg rpoB, ssrA, tuf, atpE, ku, and dnaK genes, and use multilocus sequence analysis (MLSA) to concatenate the six genes. Ninety-six Mycobacterium isolates, including 86 NTM and 10 MTB isolates, from all the patients referred to the certain TB Reference Centres were included. All isolates were evaluated by PCR amplification of rpoB, ssrA, tuf, ku, atpE, and dnaK genes and MLSA. Out of 96 isolates, 91 (94.8%), 87 (90.6%), 72 (75%), 84 (87.5%) and 79 (82.3%) were differentiated to the species level by rpoB, tuf, ssrA, dnaK and atpE genes, respectively. The ku gene was able to identify 69 (80.2%) isolates of the 86 NTM isolates to the species level. We could identify 100% of the isolates to the species level by MLSA. None of the PCR targets used in this study were able to completely differentiate all species. The MLSA technique used to concatenate the six genes could increase the identification of clinical Mycobacterium isolates and all 16 species were well-differentiated.

Evaluation of the MeltPro Myco Assay for the Identification of Non-Tuberculous Mycobacteria.

PurposeThe purpose of this study was to evaluate a new commercial kit for Mycobacterium species identification and compare its results with that of the commonly used GenoType Mycobacterium CM assay. In addition, we were committed to identifying the main frequent species of nontuberculous mycobacteria (NTM) in Fujian.MethodsA total of 261 clinical strains, collected at the Center for Disease Control and Prevention of Fujian Province, China, were preliminarily identified as NTM based on p-nitrobenzoic acid (PNB) growth test. The genomic DNA of all 261 strains was extracted and subjected to species identification using MeltPro Myco assay and GenoType Mycobacterium CM assay. The results of the latter were used as a control to calculate the positive agreement, negative agreement, agreement and the total agreement of the former. For samples with inconsistent detection results, sequencing was performed for verification.ResultsCompared to GenoType Mycobacterium CM assay, the total agreement of MeltPro Myco assay was 96.55% (252/261 strains). Both the positive and negative agreement of MeltPro Myco assay in identifying M. intracellulare, M. abscessus, M. kansasii, M. fortuitum, M. avium, M. gordonae, M. scrofulaceum, and mixed infections were higher than 99.00%, but the positive agreement of M. chelonae was relatively low at only 33.33%. In Fujian, the predominant strain of NTM was M. intracellulare (64.36%, 130/202 strains), followed by M. abscessus (19.31%, 39/202 strains), M. kansasii (4.46%, 9/202 strains), and M. fortuitum (3.96%, 8/202 strains).ConclusionThe reliability of MeltPro Myco assay for identifying mycobacterium species was strongly demonstrated in this study, which greatly supports its usage for the clinical identification of mycobacteria. The present study also showed that the distribution of mycobacteria in Fujian, China, was significantly different from that in other regions and provided important data for future epidemiological study of NTM.

Utility of urine lipoarabinomannan (LAM) in diagnosing mycobacteria infection among hospitalized HIV-positive patients

ObjectivesCross-reactivity with nontuberculous mycobacteria (NTM) species might limit the use of urine lipoarabinomannan (LAM) test to diagnose tuberculosis (TB) in people living with HIV (PLWH). This study aimed to investigate the utility of the LAM test among hospitalized HIV-positive patients. MethodsThis prospective study enrolled HIV-positive inpatients with any TB symptom or seriously ill patients with advanced immunodeficiency. Urine samples were tested using the Alere Determine LAM Ag, and participants were categorized as confirmed TB, confirmed NTM infection, unclassified mycobacteria infection, and no mycobacteria infection based on microbiologic reference standards. ResultsA total of 382 participants were included. The prevalence of confirmed TB and NTM infection was 5.24% (20 of 382) and 4.45% (17 of 382), respectively. The sensitivity and specificity of the urine LAM for TB diagnosis were 65.00% (95% confidence interval [CI] 40.78-84.61) and 89.36% (95% CI 85.68-92.36), respectively. The LAM test for NTM yielded a sensitivity of 58.82% (95% CI 32.92-81.56) and specificity of 88.61% (95% CI 84.87-91.70). Notably, the negative predictive values of the urine LAM for TB and NTM were 97.85% (95% CI 95.63-99.13) and 97.85% (95% CI 95.63-99.13), respectively. ConclusionsCross-reactivity with NTM cause high false-positive LAM for TB diagnosis in PLWH. The correct identification of mycobacteria species is crucial for deciding treatment strategies.

Use of MALDI-TOF MS (Bruker Daltonics) for identification of Mycobacterium species isolated directly from liquid medium

ObjectivesThe aim of this study was to describe the evaluation of the use of MALDI-TOF MS for the identification of non-tuberculous mycobacteria (NTM) and Mycobacterium tuberculosis directly from liquid MGIT cultures from January 2017 to December 2017. Material/methodsA total of 155 isolates (mainly respiratory) were analyzed by MALDI-TOF MS (Bruker Daltonics) directly from MGIT liquid medium with a previous extraction procedure. ResultsMALDI-TOF MS generated acceptable scores for 152 isolates (98.06%). Fifty isolates were identified as M. tuberculosis complex and the remaining 105 as NTM (M. abscessus subsp. abscessus, M. avium, M. celatum, M chelonae, M. chimaera, M. fortuitum, M. gordonae, M. intracellulare, M. kansasii, M. lentiflavum, M. mageritense, M. mucogenicum and M. xenopi). ConclusionsThese results indicate that MALDI-TOF MS can be useful to identify mycobacteria directly from MGIT cultures and is an accurate, rapid and cost-effective system to be used as a routine method.

Detection and identification of Mycobacterium species

Background: Successful diagnosis and effective treatment for mycobacterial infections are mainly depending on a rapid and sensitive identification method. Objective: To detect and identify the Mycobacterium species. Methodology: PCR and LCD-microarry techniques were compared with the classical methods of Ziehl-Neelsen staining (ZN) and culturing. Two primers based on two conservative regions within the mycobacterium 16S rRNA gene were designed and amplified a DNA fragment of about 1350 bp for both complex of Mycobacterium tuberculosis (MTB) and non-tuberculous mycobacteria (NTM). Results: Regarding to the standard method of culture, 57 positive individuals were identified out of 100 urine samples. The PCR showed 96.30 % sensitivity and 96.70% specificity, while ZN gave Se = 67.50 % and Sp = 100 %. The LCD-microarray analysis exhibited 100 % sensitivity and specificity. One species of MTB was determined as M. tuberculosis and positively represented by 12.3% (n=7). Five species of NTM were determined and represented as M. kansasii 36.8 % (n=21), M. celatum 21 % (n=12), M. gordonae 12.2% (n=7), M. chelonae 10.5 % (n=6), and M. phlei 7% (n=4). Conclusion: The results recommended utilizing the simple and rapid PCR method for early mycobacteria detection. Also, the fast LCD-microarray protocol is very beneficial for identification and differentiation between MTB and of NTM species.

Evaluation of Transbronchial Needle Aspiration Samples from Sarcoidosis Patients for the Presence of Mycobacterium

Background: Tuberculosis (TB) and sarcoidosis are two chronic, systemic, and granulomatous diseases that have similar pulmonary and extrapulmonary manifestations. Given the similarities between the two diseases, it is suggested that the Mycobacterium genome may play a role in sarcoidosis. Identification of Mycobacterium species isolated from sarcoidosis patients optimizes the treatment process of patients. The present study investigates biopsy tissue samples from patients with sarcoidosis for the presence of Mycobacterium in the tissues. Methods: This descriptive cross-sectional study was performed on all sarcoidosis patients referred to Masih Daneshvari Hospital during 2017–2018 (401 patients). All patients who were outpatients or hospitalized in the bronchoscopy ward of Masih Daneshvari Hospital underwent transbronchial needle aspiration (TBNA). The cause of the TBNA procedure in patients was the presence of bilateral hilar lymphadenopathy in patients. Out of 401 patients with sarcoidosis, five patients showed positive smear or culture for Mycobacterium and were excluded from the study. Finally, 396 patients were included in the study. Results: The majority of participants in the present study were female (56.1%) with a mean age of 48.2 years and a mean ACE index of 73.4. Hypertension was found in16.2% of patients with sarcoidosis. 16.4% of patients had hyperlipidemia and 4.3% of patients had a history of ischemic heart disease. Redness of the eyes was seen in 13.1% of patients and 12.4% of the patients had diabetes. Also, 43.4% of the patients had skin lesions. Out of a total of 396 patients with sarcoidosis in the present study, 3.5% of the lymph and lung tissues of sarcoidosis patients (who were not infected with Mycobacterium tuberculosis by clinical signs and culture) contained amplifiable MTB DNA. Conclusion: In the present study and many previous related studies, the Mycobacterium genome has been observed in lymph node samples of mediastinal nodes and lung tissue of sarcoidosis patients. One of the strengths of the present study, which can make the results very reliable, is the high sample size in this study. It is suggested that similar studies with the use of control groups in large samples, investigate the presence of the Mycobacterium genome and its type in sarcoidosis patients.

Rapid identification of Mycobacterium species isolated from clinical specimens of city Jahrom by real time PCR

Diagnosis and identification of mycobacterial infections is often a challenge that engages different medical fields. Due to the time-consuming diagnostic procedures for most mycobacteria and the lack of rapid differentiation between Mycobacterium tuberculosis and NTMs species, incorrect diagnosis of non-tuberculosis leads to the use of TB treatment protocol and prolonged hospitalization. So, the aim of this study was to rapid identification of Mycobacterium tuberculosis strains from non-Mycobacterial tuberculosis by the multiplex Real time PCR method to prevent misdiagnosis in the treatment procedures.In this study, 82 sputum specimens were collected from patients suspected to tuberculosis referred to Peymanieh Hospital and health centers of Jahrom, as well as those treated with TB drugs referred to Jahrom Tuberculosis Center in 2015. Sediment was collected from the clinical samples by Petroff's method, DNA extraction was done with commercial DNA kit. 10 pairs primer; 23srRNA, grvB, ITS 152 bp, ITS172bp 164 bp, ITS, dnaJ, katG, rpoB, ITS 185 bp, ITS 185 bp were used. Multiplex real time PCR was performed in one run and in three different reactions and their melting curve was evaluated for both species and drug resistance.Among 82 suspected tuberculosis patients referred to Jahrom Tuberculosis Center, 26 (31.7% of total samples) were Mycobacterium positive, of which 13 (15.85%) were Mycobacterium tuberculosis, 7 (53.58%) Mycobacterium kansasii, 1 specimen (1.21%) were Mycobacterium gordonae and 5 specimens (6.09%) were uncommon Mycobacterium tuberculosis. No MDR was found in Mycobacterium tuberculosis specimens and no non-tuberculosis resistant specimens were found in Mycobacterium tuberculosis specimens.According to the results of this study, the prevalence of Mycobacterium tuberculosis and non-tuberculosis in Jahrom city was different from other parts of the country. Also, this procedure is convenient, easy and quick to detect common mycobacterial strains to prevent ectopic treatments and is also a good way to follow up treatment in treated cases.

Use of MALDI-TOF MS (Bruker Daltonics) for identification of Mycobacterium species isolated directly from liquid medium

ObjectivesThe aim of this study was to describe the evaluation of the use of MALDI-TOF MS for the identification of non-tuberculous mycobacteria (NTM) and Mycobacterium tuberculosis directly from liquid MGIT cultures from January 2017 to December 2017. Material/methodsA total of 155 isolates (mainly respiratory) were analyzed by MALDI-TOF MS (Bruker Daltonics) directly from MGIT liquid medium with a previous extraction procedure. ResultsMALDI-TOF MS generated acceptable scores for 152 isolates (98.06%). Fifty isolates were identified as M. tuberculosis complex and the remaining 105 as NTM (M. abscessus subsp. abscessus, M. avium, M. celatum, M chelonae, M. chimaera, M. fortuitum, M. gordonae, M. intracellulare, M. kansasii, M. lentiflavum, M. mageritense, M. mucogenicum and M. xenopi). ConclusionsThese results indicate that MALDI-TOF MS can be useful to identify mycobacteria directly from MGIT cultures and is an accurate, rapid and cost-effective system to be used as a routine method.

Identification of mycobacteria species through mass spectrometry (MALDI-TOF)

Mycobacterial diseases are very important both clinically and epidemiologically. Mycobacterium tuberculosis complex (MTBc) infections confer higher morbidity and mortality rate than non-tuberculous mycobacteria (NTM) infections. Traditional species identification techniques are based on phenotypic characteristics which take a long time by laborious processes and in occasions are no conclusive. Currently, most used techniques are based on molecular methods, which are accurate but are expensive and complex. Matrix Assisted Laser Desorption/Ionization Time-of-Flight mass spectrometry (MALDI-TOF MS) is a simple, cheap and fast identification method based on comparing protein spectra with a reference database. To assess the performance of MALDI-TOF MS in the identification of MTBc and NTM, compared with molecular methods. For that purpose, 28 isolates of 9 different species were analyzed through MALDI-TOF MS. 78.5% (22/28) of isolates were correctly identified, 100% (9/9) of rapidly growers NTM, 60% (9/15) of slow growing NTM and 100% (4/4) of MTBc. Every unidentified isolate (6/6) corresponded to M. avium/intracellulare complex. MALDI-TOF MS is fast, simple and cheaper than molecular methods and also has adequate accuracy.

Identification of mycobacteria species by molecular methods.

In this study, mycobacteria, which were previously identified as Mycobacterium tuberculosis complex (MTC), and mycobacteria other than tuberculosis (MOTT) with cord factor and the p-nitro-alpha-acetyl-amino-beta-hydroxypropiophenone (NAP) test were reanalysed using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis method in order to confirm the identification, and at the same time, species accepted as MOTT were identified. Although the results of the NAP test were obtained within 3-5 days, the PCR-RFLP results were obtained in 1 day. Ten species identified as MTC with the NAP test and cord factor were confirmed with the PCR-RFLP method. Fourteen species accepted as MOTT were identified as Mycobacterium species with the evaluation of the bands observed after the restriction of PCR product with the PCR-RFLP method. These were as follows: three species Mycobacterium intracellulare type I, two species Mycobacterium phlei, two species Mycobacterium kansasii, one species Mycobacterium fortuitum type I, one species Mycobacterium gordonae type I, one species Mycobacterium abscessus type I, one species Mycobacterium scrofulaceum, one species Mycobacterium szulgai type I, one species Mycobacterium avium type II, and one species Mycobacterium terrae. Hence, the results of both the cord factor and the NAP test were confirmed with the molecular method, and at the same time, mycobacteria species identification was made by determining the fastest, easiest, and the most accurate result-giving method. Because PCR-RFLP is a very rapid method that provides exact identification of mycobacteria species, it can be performed in routine procedures.

Establishment of a multiplex PCR for rapid identification of Mycobacterium species

Objective To establish and evaluate a multiplex PCR method for rapid identification of Mycobacterium species in order to provide an approach for rapid diagnosis of pathogens causing tuberculosis. Methods Four genes including 16S rRNA, Rv0577, RD9 and mtbk_20680 were selected to establish the multiplex PCR method. Specific primers were designed and the reaction system and conditions were optimized. The sensitivity and specificity of the multiplex PCR method were evaluated through identifying Mycobacterium africanum (M.africanum), Mycobacterium bovis (M.bovis), M. bovis BCG, seven common non-tuberculosis Mycobacterium (NTM), seven reference species of common respiratory bacteria and 93 clinical strains of Mycobacterium tuberculosis (MTB) isolated from patients with tuberculosis in Gansu Province of China. Results The fragments of 16S rRNA, Rv0577, RD9 and mtbk_20680 genes were 543 bp, 786 bp, 369 bp and 231 bp in length, respectively. MTB strains of the Beijing genotype were positive for all of the four genes, while the non-Beijing genotype strains were negative for mtbk_20680. M. africanum, M. bovis and M. bovis BCG strains were negative for 16S rRNA and Rv0577. NTM strains only carried 16S rRNA and none of four genes were detected in the seven species of respiratory bacteria. Among the 93 clinical MTB strains, 80 (86.02%) belonged to the Beijing genotype and the other 13 (13.98%) were non-Beijing genotype strains. The specificity of the multiplex PCR method was 100%. Conclusions The established multiplex PCR method could accurately distinguish Mycobacterium, Mycobacterium tuberculosis complex (MTBC), NTM, MTB, and Beijing and non-Beijing genotype MTB with high sensitivity and specificity. Key words: Multiplex PCR; Mycobacterium; Mycobacterium tuberculosis; Beijing genotype

Discovery of a novel Mycobacterium asiaticum PRA-hsp65 pattern

Twenty-one pulmonary sputum samples from nine Brazilian patients were analyzed by the PRA-hsp65 method for identification of Mycobacterium species and the results were compared by sequencing. We reported a mutation at the position 381, that generates a suppression cutting site in the BstEII enzyme, thus leading to a new PRA-hsp65 pattern for M. asiaticum identification.

Non Tuberculosis Mycobacterium, E. coli and E. faecalis from Biofilm in Drinking Water Distribution Systems from Selected Sites of Addis Ababa

Background:The decline in microbial quality of drinking water may be attributed to many factors among which the presence of biofilm within the distribution system is the major cause of contamination. Drinking water distribution systems provide an oligotrophic environment, for post-treatment recovery and regrowth of microorganisms including the opportunistic Nontuberculosis Mycobacterium (NTM).Objective:The aim was to look for opportunistic non tuberculosis mycobacterium and indicator organisms of fecal contamination from biofilm in drinking water distribution pipeline from selected sites of Addis Ababa.Materials and Methods:A total of 40 biofilm samples were collected from two sub-cities of Addis Ababa. Biofilm samples were taken from the inner surfaces of the get valve and water meter. For the detection ofE. coliandE. faecalis,diluted biofilm samples were filtered, then it was incubated on respective culture media. For non-tuberculosis mycobacterium, the homogenized biofilm sediment was processed using the standard SD bio line method, whereby, The processed sediment was inoculated to appropriate solid and liquid culture media. The DNA extraction was conducted by chemical lysis followed by PCR amplification, from the grown colonies on LJ media (Löwenstein–Jensen). The identification of Mycobacterium species was performed by reverse hybridization using a membrane strip and an enzymatic color reaction.Results:From the total biofilm samples, 14 out of 40 (35%) were positive for mycobacteria species.M. gordoneawas the most prevalent specie of Mycobacterium, whereby 8/14 (57.1%) of the isolates were from this species followed byM. fortuitum1/14 (7.14%). About (35.7%) 5/14 of the genus Mycobaterium were unidentified species. Indicator organisms of fecal contamination (E. coliandE. faecalis)were found in 3/40(7.5%) and 6/40(15%) respectively. There was no statistically significant association between nontuberculosis mycobacterium and the indicator organisms atpvalue of 0.01.Conclusion:The study has highlighted that the occurrence of NTM in drinking water distribution in a significant proportion.M. gordonaewas found to be the most dominant species of nontuberculosis mycobacterium found in the distribution line biofilm samples.

Nontuberculous mycobacteria pulmonary disease: A retrospective analysis

To analyze the clinical characteristics and drug resistance in patients with non-tuberculous mycobacteria (NTM) pulmonary disease in Changsha Central Hospital of Hunan Province in recent three years. Methods: The clinical data of 153 patients with NTM pulmonary disease, who were diagnosed in Changsha Central Hospital of Hunan Province from February 2014 to May 2017, were retrospectively analyzed. According to the concentration of drug sensitivity test, the patients were divided into a low concentration group and a high concentration group. The status of drug sensitivity and drug resistance were examined. Results: Among 153 patients, 79 patients (51.63%) were male, 74 patients (48.37%) were female. The mean ages were (60.27±19.46) years. The NTM pulmonary disease mainly occurred in the individuals with bronchiectasis, and the course of disease was long (mean 7.8 years). The clinical symptoms were not specific and mostly misdiagnosed as pulmonary tuberculosis (92.81%). Mycobacterium avium-intracellulare (56.21%) and mycobacterium chelonae-abscess (20.92%) were the majority. The drug-resistance rate of the first-line and second-line anti-tuberculosis drugs was high. The majority was resistant to more than eight drugs, 38.56% patients in the low concentration group were resistant to total drugs, and 25.49% patients in the high concentration group were resistant to total drugs. Conclusion: The NTM pulmonary disease is easily misdiagnosed, and the drug resistance rate is high. Identification of mycobacterium species and detection of drug sensitivity play an important role in clinical diagnosis and treatment.

Evaluation of the performance of MALDI-TOF MS and DNA sequence analysis in the identification of mycobacteria species

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is an alternative way of identifying mycobacteria via the analysis of biomolecules. It is being increasingly used in routine microbiology practice since it permits early, rapid, and cost-effective identification of pathogens of clinical importance. In this study, we aimed to evaluate the efficacy of phenotypic identification of mycobacteria by the MALDI-TOF MS MBT Mycobacteria Library (ML) 4.0 (Bruker, Daltonics) compared to standard sequence analysis. A total of 155 Mycobacterium clinical and external quality control isolates, comprising nontuberculous mycobacteria (NTM) (n = 95) and the Mycobacterium tuberculosis complex (MTC) (n = 60), were included in the study. Identification by MBT ML4.0 was correctly performed in 100% of MTC and in 91% of NTM isolates. All of the MTC isolates were correctly differentiated from NTM isolates. Based on our results, MBT ML4.0 may be used reliably to identify both NTM and MTC.