Growth Of Mycobacteria Research Articles

Molecular Approaches for Detection of Rifampin-Resistant Mycobacterium tuberculosis and Its Associated Risk Factors among General Population

GeneXpert MTB/RIF is a molecular technique for the diagnosis of tuberculosis. This method not only detects M. tuberculosis but also identifies the absence or presence of different drug resistant mutations in DNA fragments like rifampicin resistance. In this study, about 200 septum samples were collected from tuberculosis suspected people of Isfandyar Bukhari District Head Quarter Hospital Attock for screening and determination of Mycobacterium tuberculosis and multi-drug-resistant tuberculosis. The samples were screened for the presence of M. tuberculosis using Fluorescence Microscopy staining followed by GeneXpert MTB/RIF assay. The results were further confirmed by Line Probe Assay and Mycobacterium Growth Indicator Tube 960 techniques. In a total of 200 samples, 45(22.5%) were found positive, 155 (77.5%) were classified negative on culture while 38(19%) were positive and 162(81%) were negative on FM, 33(16.5%) were positive and 167(83.5%) were negative on ZN staining microscopy and 47(23.5%) were noted positive and 153 (76.5%) were negative on GeneXpert. The overall sensitivity of GeneXpert was 07% higher than that of smear microscopy and 4.2 % higher than culture.Different risk factors reported for TB are; malnutrition (23.40%), health care workers (14.89%), immunosuppression (12.76%), unimmunized with Bacille Calmette-Guérin (BCG) vaccine and contact with infected persons (10.63%), diabetes mellitus (8.51%), poverty and foreign-born children are (4.2%). It is concluded that the most common risk factor for TB was malnutrition (23.40%) and the foreign-born children (4.2%) were least affected. GeneXpert assay for identification of tuberculosis was found more sensitive than fluorescence microscopy and for isolation of MTB and rifampicin resistance, GeneXpert RIF assay can efficiently be usedwhich is simple, robust and efficient with minimum handling technique.

Identification of novel inhibitors for mycobacterial polyketide synthase 13 via in silico drug screening assisted by the parallel compound screening with genetic algorithm-based programs.

Identifying small compounds capable of inhibiting Mycobacterium tuberculosis polyketide synthase 13 (Pks13), in charge of final step of mycolic acid biosynthesis, could lead to the development of a novel antituberculosis drug. This study screened for lead compounds capable of targeting M. tuberculosis Pks13 from a chemical library comprising 154,118 compounds through multiple in silico docking simulations. The parallel compound screening (PCS), conducted via two genetic algorithm-based programs was applied in the screening strategy. Out of seven experimentally validated compounds, four compounds showed inhibitory effects on the growth of the model mycobacteria (Mycobacterium smegmatis). Subsequent docking simulation of analogs of the promising leads with the assistance of PCS resulted in the identification of three additional compounds with potent antimycobacterial effects (compounds A1, A2, and A5). Further, molecular dynamics simulation predicted stable interaction between M. tuberculosis Pks13 active site and compound A2, which showed potent antimycobacterial activity comparable to that of isoniazid. The present study demonstrated the efficacy of in silico structure-based drug screening through PCS in antituberculosis drug discovery.

Assessment of Antimycobacterial Activity of Newly Synthesized Pyrimidine Derivatives Against Mycobacterium tuberculosis

Background. The current trend of growing antibiotic resistance among pathogenic microorganisms remains one of the urgent and significant problems of mankind. The constant spread of resistant strains of microorganisms requires the development of innovative methods and the search for medicinal compounds with a highly effective mechanism of action. One of these multi-resistant pathogens that are difficult to eradicate is the causative agent of tuberculosis — Mycobacterium tuberculosis. The aim is to study the effect of newly synthesized pyrimidine derivatives on the growth of Mycobacterium tuberculosis culture, as well as on the structural changes in cells.Material and methods. In order to assess the effect of a number of pyrimidine derivatives on the growth of Mycobacterium tuberculosis culture, 6 samples of 5-(arylmethylene) hexahydropyrimidine-2,4,6-triones (TAG1 — TAG6), 7 samples of 5-hetarylmethylidene-2,4,6-triones (TAG7 — TAG13), and 2 new samples of 3-(2-Benzyloxy-2-oxoethyl)quinazoline-4(3H)-one and 3-[2-(1-Naphthyl)-2-oxoethyl]quinazoline-4(3H)-one were screened under the laboratory ciphers VMA-13-03 and VMA-13-04 in the course of the study. M.tuberculosis H37RV strain was used as a test culture; it was provided by the bacteriological laboratory of the Regional Infectious Clinical Hospital named after A. M. Nichoga. A 4-week culture of M.tuberculosis, synchronized by cold (+4°C) for 72 hours, was used to prepare a suspension of mycobacteria. The number of mycobacteria in the suspension was determined using the McFarland 0.5 turbidity standard. 0.2 ml of M.tuberculosis working suspension was added to each tube of a series of successive dilutions of the studied substances, including the control. The study was carried out in 4 series of replicates. The minimum bactericidal concentration of the compounds, at which no colony growth was detected, as well as the minimum inhibitory concentration, at which mycobacterium growth was delayed by 50% compared to the control, were determined. Smears were prepared from the sediment for staining using theZiehl-Neelsen method to determine the presence of acid-resistant and non-acid-resistant forms of mycobacteria, as well as to study the effect of pyrimidines and a comparison drug on structural changes in M.tuberculosis cells.Results. In the course of the study, the TAG4, TAG6, and TAG8 compounds were found to have the closest antibacterial activity to the comparison drug isoniazid, according to the indicator of mycobacteria growth retardation. The greatest bactericidal activity against M.tuberculosis was observed in TAG4, TAG7, and VMA–13–04. The remaining compounds have shown minimal inhibitory effect on the growth of M.tuberculosis. Microscopic studies have shown that under the influence of TAG3, TAG4, TAG7, TAG12, VMA-13-03, and VMA-13-04, the main structural components of M.tuberculosis cells undergo fragmentation and morphological changes compared to mycobacterium cells without exposure.Conclusion. As a result, it was found that all the studied compounds possess antimycobacterial activity. Compounds under the laboratory ciphers TAG1, TAG4, TAG7, and TAG13 were comparable to isoniazid by the nature of the inhibitory effect on the growth of M.tuberculosis, and the TAG3 compound even slightly exceeded the effect of the comparison drug. Compounds under the laboratory codes VMA-13-03, and VMA-13-04 had the least pronounced anti-tuberculosis effect. Compounds under the laboratory codes TAG5, TAG6, TAG11, and TAG12 showed the least antimycobacterial activity.

Potential Efficacy of β-Amyrin Targeting Mycobacterial Universal Stress Protein by In Vitro and In Silico Approach.

The emergence of drug resistance and the limited number of approved antitubercular drugs prompted identification and development of new antitubercular compounds to cure Tuberculosis (TB). In this work, an attempt was made to identify potential natural compounds that target mycobacterial proteins. Three plant extracts (A. aspera, C. gigantea and C. procera) were investigated. The ethyl acetate fraction of the aerial part of A. aspera and the flower ash of C. gigantea were found to be effective against M. tuberculosis H37Rv. Furthermore, the GC-MS analysis of the plant fractions confirmed the presence of active compounds in the extracts. The Mycobacterium target proteins, i.e., available PDB dataset proteins and proteins classified in virulence, detoxification, and adaptation, were investigated. A total of ten target proteins were shortlisted for further study, identified as follows: BpoC, RipA, MazF4, RipD, TB15.3, VapC15, VapC20, VapC21, TB31.7, and MazF9. Molecular docking studies showed that β-amyrin interacted with most of these proteins and its highest binding affinity was observed with Mycobacterium Rv1636 (TB15.3) protein. The stability of the protein-ligand complex was assessed by molecular dynamic simulation, which confirmed that β-amyrin most firmly interacted with Rv1636 protein. Rv1636 is a universal stress protein, which regulates Mycobacterium growth in different stress conditions and, thus, targeting Rv1636 makes M. tuberculosis vulnerable to host-derived stress conditions.

Comparison of TrueNat polymerase chain reaction and mycobacterium growth indicator tube culture in the diagnosis of pulmonary and extrapulmonary tuberculosis

Comparison of TrueNat polymerase chain reaction and mycobacterium growth indicator tube culture in the diagnosis of pulmonary and extrapulmonary tuberculosis

Resistance Profile of Mycobacterial Strains Causing Cluster Pulmonary Infection in Bayelsa State, Nigeria

Aim: The aim of this study was to profile the anti-microbial resistant of Mycobacterial strains causing cluster pulmonary infections in Bayelsa State, Nigeria.
 Study Design: Cross sectional facility based.
 Place and Duration: This study was carried out in all the in tuberculosis centers in all the eight local government area in Bayelsa State, Nigeria between January 2019 to February 2021.
 Materials and Methods: A total of 102 tuberculosis patients who had been previously diagnosed of Tuberculosis (both new and old cases) participated in the study. Two sputum samples were collected from each subject in a wide mouth screw cape container and falcon tube container respectively. The first was used for GenXpert technique while the second was used for culture on Lowenstein Jensen solid Media. The visible and confirmed Mycobacterium growth was subjected to Line Probe Assay (MTBDRplus assay) and genetic sequencing. The DNA of the isolates where extracted using Genolyse method. The extracted DNA was used to perform a gene mutation profiling using MTBRplus Assay. The 16Sr RNA sequencing was done on the amplified genes using the BigDye terminator kit on a 3510 AB1 sequencer.
 Results: Out of the 102 sputum samples analyzed a total of 91(89.2%) were GeneXpert positive. Drug resistant profiling by GeneXpert shows 8(7.8) strain mutation at the rpoB gene only while the resistant profiling of the isolated on Lowenstein Jensen solid media had 14(13.7) strain with mutation on genes responsible for first and second line drug resistant. Two non-tuberculosis Mycobacterium species which are Mycobacteriode abscessus subsp. abscessus st and Mycobacterium Kansasii strain FDAARGOS_1534 where isolated among Tuberculosis patients, both are multidrug-resistant strains. The Mycobacterium tuberculosis strains in circulation causing cluster TB infection in Yenagoa especially and other parts of Bayelsa State, Nigeria are MG003 and R2092 strains, but the most predominant strain that is traced to cluster TB infection is MG003. The extraction of MTB gene from a pure culture of a Lowenstein Jensen selected media reduced the possibility of contamination and enhanced the reliability of gene sequencing and Bioinformatics analysis which includes comparing strains of MTB with the once in the National center for Biotechnology information (BLAST) data base.
 Conclusion: The resistance profiling of MTB reveals that most strains were resistant to rifampicin. This means that there were more mutation on the rifampicin activation gene (rpoB) than any other gene. Most cases of multi drug resistance is associated with rifampicin resistance while cases of extensive drug resistance is not common.

Tuberculosis Molecular Bacterial Load Assay Reveals Early Delayed Bacterial Killing in Patients With Relapse.

Bacterial killing in patients with tuberculosis (TB) relapse was compared to that in patients achieving cure, measured by TB molecular bacterial load assay (TB-MBLA) or mycobacteria growth indicator tube (MGIT) time to positivity (TTP). TB-MBLA in 4 relapsed patients was significantly different compared to 132 cured patients after 2 weeks of treatment; MGIT TTP showed a significant difference from week 8.

Comparative study of nutrient media for the isolation of mycobacteria

Laboratory diagnostics is of decisive importance in the detection of animal tuberculosis. The proposed nutrient media for isolating of the pathogen are rich in variety. The practical significance of frequently used culture media and their modifications have been studied. Samples of Petragnani, Gerold, Evglevsky, Mordovsky, Pavlovsky, Levenshtein-Jensen, Finn-II and some of their modifications for the primary isolation and subculturing of mycobacteria were studied. Also, in a comparative plan, the Levenshtein-Jensen and Finn-II media were studied in various versions: Levenshtein-Jensen, prescribed and frozen; Levenshtein-Jensen, dry and frozen; Finna-II in the author's version and four modifications, AF-I (glycocol instead of monosodium glutamate), AF-II- (with potato extract), AF-III - (without glycocol) and MF variant (with M. flei extract). For sowing, biomaterial from sick animals, also artificially infected, was used. The studies made it possible to establish the effectiveness of the Levenshtein-Jensen and Finn-II media for the primary isolation of the pathogen. In terms of growth properties, the dry Levenshtein-Jensen medium is superior to the nitive one, but is significantly inferior to Finn's native medium in terms of properties that inhibit foreign microflora. The ability to accelerate the growth of mycobacteria with hydrocarbons was determined by the addition of n-alkanes with a wide range of carbohydrates - from decane to nonadecane (from C10 to C22). A total of 30 different options were tested: by adding liquid paraffin; alcohol extract of M. flei; wax solutions; propolis on hexane; bovine serum and others. The use of n-alkanes as a growth stimulator in the amount of 2-3 drops per 10 ml of nutrient medium (1 drop per 4 ml of medium) made it possible to reduce the cultivation time on 15-20 days and increase the bacterial mass in about 2 times. The reliability of growth stimulation of M. bovis (P<0.05) by n-alkanes on egg media according to the c2 criterion was also confirmed by the results of variation statistics. Weak growth properties in relation to M. avium and a slight stimulation of the growth of M. tuberculosis were revealed.

Species diversity among the genus <i>Streptomyces</i> members isolated from clinical material

Recently, both in Russia and around the world, the number of cases detecting acid-fast microbial members from the order Actinomycetales while developing human bacterial infections has been increased. The most important pathogens in this bacterial order are the members from the families Mycobacteriaceae, Nocardiaceae, Gordoniaceae, Tsukamurellaceae, Promicromonosporaceae, Brevibacteriaceae, Streptomycetaceae. This work is devoted to analyzing prevalence and species diversity of representatives of the family Streptomycetaceae from the genus Streptomyces isolated from clinical material upon examining for tuberculosis. There were examined 865 cultures of clinical material samples while examining for tuberculosis, in which signs of growth of contaminating microflora were revealed, as well as 316 cultures of clinical material samples obtained during examination for tuberculosis, in which signs of growth of non-tuberculosis mycobacteria (NTM) were detected. The material was collected from January 2016 to January 2019. Samples with signs of growth of contaminating microflora allowed to identify 1,093 strains, samples with signs of growth of NTM 352 strains. Among them, the number of representatives of the genus Streptomyces comprised 39 strains. All Streptomyces strains were isolated from sputum. Variety of isolated Streptomyces: S. phaeochromogenes (13 strains), S. albus (1 train), S. avidinii (1 strain), S. badius (2 strains), S. chartreusis (2 strains), S. griseus (1 strain), S. hirsutus (2 strains), S. lavendulae (3 strains), S. violaceoruber (10 strains). Species identification was not possible for 4 strains. Analyzing the data obtained, it is possible to draw a conclusion about the moderate distribution of representatives of the genus Streptomyces in pattern of contaminating microflora while examining for tuberculosis. In pattern structure of isolated microorganisms, Streptomyces accounted for 3.3%. Among the microflora isolated from culture with signs of NTM growth, Streptomyces was presented by single strains. However, it should be noted that Streptomyces was the dominant group among acid-resistant actinomycetes in pattern of contaminating microflora and accounted for 38.3%. Taking into account the fact that a significant proportion of them were isolated as microbial associations, it can be concluded that Streptomyces turned out to be classical contaminants in this case. Nevertheless, we believe that the isolation of Streptomyces in association with clinically significant NTMs can be considered as an unfavorable factor due to the fact that antibiotic resistance genes are widespread among Streptomyces and may be transmitted to other types of microorganisms from the group of acid-resistant actinomycetes, including mycobacteria. Thus, the clinical material examining for tuberculosis is an interesting research object from which various representatives of acid-fast actinomycetes, including Streptomyces, can be isolated.

Characterization of Non-tuberculous Mycobacteria by Line Probe Assay Isolated from Pulmonary Tuberculosis Suspected Patients at Rajasthan

Non-tuberculous Mycobacteria (NTM) are emerging as an important opportunistic pathogen. Since clinical presentation of NTM infection is similar to tuberculosis (TB), patients present as suspected TB or drug resistant TB. Presently in the National Tuberculosis Elimination Programme (NTEP ) NTM are not being speciated, but there is an urgent need to characterize the NTM so that appropriate treatment can be given as many species are multi-drug resistant. The purpose of the present study was to use Line Probe Assay (LPA) i.e. GenoType Mycobacterium CM/AS assay to characterize NTM for rapid early reporting and to know the pattern of NTM at Rajasthan. Sputum samples from 5000 TB and Multi Drug Resistant TB (MDRTB) suspect patients were processed and cultured on Mycobacterium Growth Indicator Tube (MGIT). Culture isolates found positive for mycobacteria in Ziehl Neelsen (ZN) staining and negative by MPT64 antigen test were then subjected for GenoType Mycobacterium CM/AS Among sputum samples from 5000 patients 1520 (30.4%) patient samples were positive for mycobacteria, among these 1488 (97.9%) were Mycobacterium tuberculosis (MTB) and 32 (2.1%) were NTM, among them 56.2% were Mycobacterium intracellulare, 21.8% Mycobacterium abscessus, 9.3% Mycobacterium fortuitum, 1% Mycobacterium simiae and 9.3% isolates showed invalid results. Incidence of NTM was very low (2.1%) among them M. intracellulare and M. abscessus were the most commonly isolated species, GenoType Mycobacterium CM/AS assay was found to be easy, rapid and reliable test giving valid results in 91% cases in 3-5 days of getting growth.

ENDOMETRIAL TUBERCULOSIS REVEALED BY POST-MENOPAUSAL METRORRHAGIA REPORT CASE

Because genital tuberculosis is frequently asymptomatic, the disease may not manifest itself for years after the initial infection. Clinical symptoms usually appear 10 to 15 years after the initial infection. We report a case of 47 years old women with a history of right breast cancer who was suffering from post menauposicmetrorrhagia, ultrasound revealed thickening of the endometrium, and the biopsy revealed endometrial tuberculosis, Genital tuberculosis is uncommon in postmenopausal women and accounts for 1% of postmenopausal metrorrhagia. In 60-70 percent of cases, the endometrium is affected. It is difficult to explain the diseases low prevalence in this age group. The majority of authors believe that the endometrial atrophy and decreased vascularity associated with this hypoestrogenic phase provide an unfavorable environment for Mycobacterium growth. The standard treatment regimen is currently well codified and is based on daily administration of Isoniazid and Rifampicin for 6 months, followed by Pyrazinamide and Ethambutol for the first 2 months. Throughout the treatment, clinical and paraclinical monitoring is performed on a regular basis.

Tapping into the antitubercular potential of 2,5-dimethylpyrroles: A structure-activity relationship interrogation

An exploration of the chemical space around a 2,5-dimethylpyrrole scaffold of antitubercular hit compound 1 has led to the identification of new derivatives active against Mycobacterium tuberculosis and multidrug-resistant clinical isolates. Analogues incorporating a cyclohexanemethyl group on the methyleneamine side chain at C3 of the pyrrole core, including 5n and 5q, exhibited potent inhibitory effects against the M. tuberculosis strains, substantiating the essentiality of the moiety to their antimycobacterial activity. In addition, selected derivatives showed promising cytotoxicity profiles against human pulmonary fibroblasts and/or murine macrophages, proved to be effective in inhibiting the growth of intracellular mycobacteria, and elicited either bactericidal effects, or bacteriostatic activity comparable to 1. Computational studies revealed that the new compounds bind to the putative target, MmpL3, in a manner similar to that of known inhibitors BM212 and SQ109.

Construction of EF-G knockdown strain of Mycobacterium smegmatis and drug resistance analysis

As the only translational factor that plays a critical role in two translational processes (elongation and ribosome regeneration), GTPase elongation factor G (EF-G) is a potential target for antimicrobial agents. Both Mycobacterium smegmatis and Mycobacterium tuberculosis have two EF-G homologous coding genes, MsmEFG1 (MSMEG_1400) and MsmEFG2 (MSMEG_6535), fusA1 (Rv0684) and fusA2 (Rv0120c), respectively. MsmEFG1 (MSMEG_1400) and fusA1 (Rv0684) were identified as essential genes for bacterial growth by gene mutation library and bioinformatic analysis. To investigate the biological function and characteristics of EF-G in mycobacterium, two induced EF-G knockdown strains (Msm-ΔEFG1(KD) and Msm-ΔEFG2(KD)) from Mycobacterium smegmatis were constructed by clustered regularly interspaced short palindromic repeats interference (CRISPRi) technique. EF-G2 knockdown had no effect on bacterial growth, while EF-G1 knockdown significantly retarded the growth of mycobacterium, weakened the film-forming ability, changed the colony morphology, and increased the length of mycobacterium. It was speculated that EF-G might be involved in the division of bacteria. Minimal inhibitory concentration assay showed that inhibition of EF-G1 expression enhanced the sensitivity of mycobacterium to rifampicin, isoniazid, erythromycin, fucidic acid, capreomycin and other antibacterial agents, suggesting that EF-G1 might be a potential target for screening anti-tuberculosis drugs in the future.

Direct Attachment with Erythrocytes Augments Extracellular Growth of Pathogenic Mycobacteria.

ABSTRACTPathogenic intracellular mycobacteria, such as Mycobacterium tuberculosis and Mycobacterium avium, which cause lung diseases, can grow in macrophages. Extracellular mycobacteria have been reported in the lungs, blood, and sputum of patients, indicating the involvement of these pathogens in disease progression. Erythrocytes are involved in the symptoms associated with pulmonary mycobacterial diseases, such as bloody sputum and hemoptysis; however, little attention has been paid to the role of erythrocytes in mycobacterial diseases. Herein, we found that Mycobacterium avium subsp. hominissuis (MAH) and Mycobacterium intracellulare colocalized with erythrocytes at the sites of lung infection, inside capillaries and necrotic areas of granulomas, using histopathological examinations. Electron microscopy showed that MAH adhered and entered human erythrocytes when they were cocultured in vitro. MAH adhered to erythrocytes through complement receptor 1 and cell-surface sialo-glycoproteins. Importantly, MAH grew vigorously without causing any pronounced damage to erythrocytes. This erythrocyte-mediated enhancement of MAH growth occurred extracellularly depending on its direct attachment to erythrocytes. In contrast, MAH failed to multiply inside erythrocytes. Similarly, erythrocytes augmented the growth of other pathogenic mycobacteria, such as M. intracellulare and M. tuberculosis. THP-1 cell-derived human macrophages preferentially phagocytosed erythrocytes that were attached to mycobacteria (compared to bacteria alone), suggesting that erythrocyte-attached mycobacteria are an efficient infectious source for macrophages. Our findings provide new insights into the pathogenesis of mycobacterial diseases and offer an alternative and useful strategy for treating mycobacterial disease.IMPORTANCE Pathogenic mycobacteria, such as Mycobacterium tuberculosis, Mycobacterium avium subsp. hominissuis (MAH), and Mycobacterium intracellulare, cause pulmonary infections as intracellular parasites of lung macrophages and epithelial cells. Here, using histopathological examinations we found that MAH and M. intracellulare colocalized with erythrocytes in lung infection sites. Subsequent studies demonstrated that direct interaction with erythrocytes enhances the extracellular proliferation of mycobacteria based on the following results: 1. MAH adhered and invaded human erythrocytes upon coculture in vitro; 2. MAH adhered to erythrocytes through complement receptor 1 and cell-surface sialo-glycoproteins; 3. MAH rapidly proliferated when directly attached to erythrocytes but not within them; 4. other mycobacteria, such as M. intracellulare and M. tuberculosis, also proliferated in the same way as MAH. The finding that pathogenic mycobacteria grow extracellularly in an erythrocyte-dependent manner is of considerable clinical importance for understanding disease progression and latent infection.

Diagnostic utility of chest computerized tomography in the diagnosis of recurrence among sputum scarce and sputum negative previously treated pulmonary tuberculosis suspects.

Objective:The objective was to study the sensitivity, specificity, and diagnostic accuracy of various computed tomography (CT) chest findings in diagnosing recurrence among pulmonary tuberculosis (PTB) suspects.Materials and Methods:A prospective observational study was conducted in a tertiary care hospital in New Delhi. A total of 130 suspects with a past history of treatment for PTB, who presented with any of the symptoms suggestive of recurrence were included. Sputum-positive, HIV-positive patients, pregnant females, and patients aged <18 years were excluded. Patients underwent CT chest followed by bronchoalveolar lavage (BAL).Results:A total of 62 patients were there in the final analysis. The median age of the patients with recurrent PTB was 27.5 years. Cough was the universal symptom in all these patients (>90%). Hemoptysis was the predominant symptom among patients with chronic pulmonary aspergillosis (66.6%). Necrotic mediastinal lymph nodes had good diagnostic accuracy of 88.71% with area under the curve of 0.806, P < 0.001 in diagnosing recurrent TB. BAL GeneXpert and mycobacteria growth indicator tube had good sensitivity (83.33% and 84.62%, respectively), specificity (100% for both), and excellent diagnostic accuracy (95.16% and 96.36%, respectively) for diagnosing recurrence in sputum negative and sputum scarce patient, (P < 0.001) when compared with composite reference standard. For culture-positive cases, BAL GeneXpert MTB/RIF had 100% sensitivity and 97.73% specificity in diagnosing recurrent PTB patients.Conclusion:The presence of mediastinal necrotic lymph node is the most accurate CT finding that can differentiate recurrent TB from post-TB sequelae. No other single chest CT scan finding had reliable diagnostic accuracy in comparison to microbiological tools in diagnosing recurrence among sputum negative or scarce previously treated PTB suspects.

ANTITUBERCULOSIS EFFECTIVENESS OF ENDOPHYTIC ANDALAS MUTANT BACTERIA PRODUCS INDUCED BY UV LIGHT WILL BE CONDUCTED

Indonesia is the 3rd largest country in the world in cases of tuberculosis (TB) infection. Infections caused by the bacteria Mycobacterium Tuberculosis have claimed 100,000 lives per year. This death rate is expected to continue to increase due to resistance to Antituberculosis Drugs (OAT). One strategy to overcome the increasing number of resistance of M. tuberculosis bacteria to OAT is to find new and better antituberculosis active compounds. This study aims to determine the potential of Andalas Endophytic Bacteria mutated by UV light on antituberculosis activity. This research is descriptive research. The research was conducted at the Research Laboratory of the Faculty of Mathematics and Natural Sciences, Padang State University. The mutation process is carried out as a form of increasing the production of active compounds. Natural anti microbial compounds can be obtained through the fermentation process. The active compounds from the fermentation products were extracted using methanol and aquadest as solvents. Testing against M. tuberculosis using the Mycobacterium Growth Indicator Tube (MGIT) 960 System as a medium for sensitivity testing. The fermented supernatant with absolute concentration and extract of each solvent with a concentration of 75% were mixed into the MGIT 960 tube. The results showed that the length of time UV irradiation could reduce the number of endophytic bacterial cells of Andalas isolate ATB 10-6. UV light also causes changes in the morphology of mutant bacterial colonies. UV irradiation can produce mutant isolates that have better antifungal activity, where of the 8 mutant bacteria produced, 7 mutants have better antifungal activity than the wild type, and the growth of mutant bacteria is faster than the wild type.

Viable Staining for Detecting Mycobacterium Pulmonary Tuberculosis in Vulnerable Population

Background: Diagnosis of pulmonary tuberculosis (PTB) is the major concern of proper diagnosis and for effective treatment of PTB. Despite the recent advancement in diagnostic methods, the smear microscopy remains the gold standard for the diagnosis of PTB in high burden countries. Design: This study, therefore, aimed at assessing the diagnostic performance of fluorescein diacetate (FDA) and Ziehl–Neelsen (ZN) staining techniques in the diagnosis of PTB for vulnerable group of population HIV, smoker, health care workers, and malnutrition people and confirmed with Mycobacterium Growth Indicator Tube (MGIT) liquid culture to check the viability. Samples were collected from Intermediate Reference Laboratory, Karnataka, from November 2019 to March 2020. A total of 350 samples were collected and these were stained using ZN method as per National Tuberculosis Elimination Program norms and FDA method followed by liquid culture using the MGIT 960 to check for the viability of the bacilli and correlate with the results of the FDA viability staining method. Results: The study demonstrates that the sensitivity and specificity of FDA viable fluorescent staining to that of liquid MGIT culture were 98.6% and 100%, respectively, while those of ZN staining were 55.8% and 100%, respectively. Conclusion: Hence, we concluded that the FDA staining technique is a more sensitive test for checking the viability of AFB among PTB suspect, as compared to the conventional ZN-stained smear observation.

Chemical susceptibility testing of non-tuberculous mycobacterium strains and other aquatic bacteria: Results of a study for the development of a more sensitive and simple method for the detection of NTM in environmental samples

The methods employed to detect non-tuberculous mycobacteria on environmental samples are essentially those classically used in clinical microbiology, which envisage a decontamination step to reduce the overgrowth of non-mycobacterial organisms before plating them on the culture medium. The aim of this study was to propose alternative culture techniques to improve non-tuberculous mycobacteria detection in environmental samples. We used artificially contaminated samples to compare the membrane filter washing procedure against direct plating of membrane filters on culture media in relation to M.chimaera and M.chelonae recovery efficiency. Moreover, we compared the efficacy of NTM Elite agar in inhibiting the growth of aquatic bacteria with that of cetylpyridinium chloride and N-acetyl-L-cysteine sodium hydroxide decontamination treatments. The washing procedure yielded a low release of both mycobacterium strains (6.6% for Mycobacterium chimaera and 7.5% for Mycobacterium chelonae) from the membrane filters; on the contrary, direct plating of membrane filters led to a 100% cell recovery. Water sample pretreatment with N-acetyl-L-cysteine sodium hydroxide (1%), despite achieving complete suppression of non-acid fast bacilli, caused a reduction in mycobacteria growth. Decontamination with cetylpyridinium chloride (0.005%) was found to be ineffective against Methylobacterium spp. and Burkholderia multivorans. NTM Elite agar was ineffective against B. multivorans, but it inhibited the growth of all other aquatic bacteria.Our results indicate that NTM Elite agar provides a valid alternative method of recovering non-tuberculous mycobacteria from environmental samples. It does not involve a decontamination step and provides greater recovery efficiency by skipping the washing step and directly plating the filters on the media.

Sphingosine-1-phosphate receptors 2 and 3 reprogram resting human macrophages into M1 phenotype following mycobacteria infection

Mycobacteria tuberculosis (M.tb) the causative agent for tuberculosis has been accredited for a high rate of morbidity and mortality worldwide. The rise in MDR and XDR cases has further created new obstacles in achieving the “End TB Strategy”, which is aimed for 2035. In this article, we have demonstrated the potential of sphingosine-1-phosphate (S1P) analogs in providing an anti-mycobacterial effector response by altering macrophage polarity into M1. Among S1PR1 and S1PR3 analogs, S1PR2 analogs proficiently favor selective polarization of infected human macrophages into M1 phenotypes, marked by increased expression of M1 markers and decreased M2 markers. Furthermore, S1PR1-3 analogs treated macrophages were also able to decrease the secretion of anti-inflammatory cytokine IL-10 and can induce NO secretion in infected macrophages. Lastly, only S1PR2-3 analogs were able to restrict the growth of mycobacteria in human macrophages. Taken together our study reflects the potential of S1PR2-3 analogs in providing host defenses following mycobacterial infection by favoring M1 macrophage polarization.

Antimycobacterial and anti-inflammatory activities of fractions and substances from Erythrina verna Vell focusing on dual severe TB treatment approach.

Tuberculosis remains a major health problem worldwide. Drug-resistant and hypervirulent Mycobacterium tuberculosis (Mtb) strains can lead to a hyperinflammatory response and necrotic pathology in hyper-reactive individuals that require adjunctive treatment. Plant-derived substances have been investigated for TB treatment, among which flavonoids stand out. We evaluate the anti-Mtb, anti-inflammatory and cytotoxicity activities of fractions and substances 1, 2 and 3 isolated from Erythrina verna through a bioassay guided fractionation. Seven fractions (1, 3-5 and 7-9) obtained from dichloromethane E. verna extract inhibited NO production (IC50 ≤ 15 μg/mL) with none or poor cytotoxic effect, while the fractions 4 and 5 notably reduced TNF-a production. Fractions 4, 6 and 9 suppressed Mycobacterium growth with MIC50 ≤ 20 μg/mL. Fraction 4 was the most potent due to dual biological activities. Erythratidinone and alpinumisoflavone inhibited the growth of Mtb H37Rv and hypervirulent strain in bacterial cultures (MIC50 ≤ 20 μg/mL), with erythratidinone standing out in reducing intracellular growth of Mtb H37Rv (5.8 ± 1.1 μg/mL). Alpinumisoflavone and erythratidinone were capable of inhibiting NO and TNF-α production besides showing significant inhibitory effects against Mycobacterium tuberculosis strains with low toxicity in macrophages. Both substances are promising for further studies focusing on an anti-TB dual treatment approach.