Improved detection of Mycobacterium tuberculosis and M. bovis in African wildlife samples using cationic peptide decontamination and mycobacterial culture supplementation

In South Africa, mycobacterial culture is regarded as the gold standard for the detection of Mycobacterium tuberculosis complex (MTBC) infection in wildlife even though it is regarded as “imperfect.” We compared a novel decontamination and mycobacterial culture technique (TiKa) to the conventional mycobacterium growth indicator tube (MGIT) system using known amounts of bacilli and clinical samples from MTBC-infected African buffaloes ( Syncerus caffer), white rhinoceros ( Ceratotherium simum), and African elephants ( Loxodonta africana). Use of the TiKa-KiC decontamination agent on samples spiked with 10,000 to 10 colony forming units (cfu) of M. bovis (SB0121) and M. tuberculosis (H37Rv) had no effect on isolate recovery in culture. In contrast, decontamination with MGIT MycoPrep resulted in no growth of M. bovis samples at concentrations < 1,000 cfu and M. tuberculosis samples < 100 cfu. Subsequently, we used the TiKa system with stored clinical samples (various lymphatic tissues) collected from wildlife and paucibacillary bronchoalveolar lavage fluid, trunk washes, and endotracheal tube washes from 3 species with known MTBC infections. Overall, MTBC recovery by culture was improved significantly ( p < 0.01) by using TiKa compared to conventional MGIT, with 54 of 57 positive specimens versus 25 of 57 positive specimens, respectively. The TiKa mycobacterial growth system appears to significantly enhance the recovery of MTBC members from tissue and paucibacillary respiratory samples collected from African buffaloes, African elephants, and white rhinoceros. Moreover, the TiKa system may improve success of MTBC culture from various sample types previously deemed unculturable from other species.

Comparison of Line Probe Assay (LPA) and Mycobacterium Growth Indicator Tubes (MGIT) Assay for Second-line TB Drug Susceptibility Testing

BACKGROUND: Tuberculosis (TB) infection is one of the most prominent health issues in the world, including in Indonesia. TB is evolving into multidrug-resistant tuberculosis (MDR-TB) and requiring second-line TB drugs. Mycobacterium growth indicator tube (MGIT) is the gold standard for susceptibility testing of second-line TB drugs. Alternatively, line probe assay (LPA), which detects genes resistant to second-line TB drugs, takes a shorter time to run. This study aims to compare MGIT and LPA's ability to detect TB resistance to second-line TB drugs and observe mutation patterns of genes encoding second-line TB drugs.METHODS: This was an observational analytic study, using cross-sectional method. The data were acquired from the MDR-TB clinic’s medical records at the Dr. Hasan Sadikin Hospital from September to December 2019. LPA and MGIT test were conducted at the Health Laboratory Hall of West Java Province, then tested using Kolmogorov-Smirnov and chi-square statistic.RESULTS: From 121 subjects, 113 people were not resistant to the second-line TB drugs, which was examined using both LPA and MGIT (93.4%), p=0.991. Mutations were found in gyrA and rrs gene. There was no significant difference between the proportion of subjects resistant to the second-line of TB drugs tested using LPA and MGIT.CONCLUSION: LPA is an alternative method to MGIT because it requires a shorter time and reduces the risk of exposure that will improve MDR-TB patients management.KEYWORDS: line probe assay (LPA), multidrug-resistant TB, mycobacterium growth indicator tube (MGIT), second-line TB drugs

Direct detection of resistance to fluoroquinolones/SLIDs in sputum specimen by GenoType MTBDRsl v.2.0 assay A study from Eastern Uttar Pradesh, India

Background According to World Health Organization (WHO), drug-resistant tuberculosis (DR-TB) is a major contributor to antimicrobial resistance globally and continues to be a public health threat. Annually, about half a million people fall ill with DR-TB globally. The gradual increase in resistance to fluoroquinolones (FQs) and second-line injectable drugs (SLIDs), poses a serious threat to effective TB control and adequate patient management. Therefore, WHO suggests the use of GenoType MTBDRsl v.2.0 assay for detection of multiple mutations associated with FQs and SLIDs. Hence, the study was conducted to determine the prevalence of resistance to FQs and SLIDs by comparing direct GenoType MTBDRsl v.2.0 assay with phenotypic drug susceptibility testing (DST). Methods The study was conducted on 1320 smear positive sputum samples from a total of 2536 RR-TB, confirmed by GeneXpert MTB/RIF. The smear positive specimens were decontaminated, and DNA extraction was performed. Furthermore, the extracted DNA was used for GenoType MTBDRsl v.2.0 assay. While 20% of the decontaminated specimens were inoculated in Mycobacterium growth indicator tube (MGIT) for drug susceptibility testing (DST). Results Out of 1320 smear positive sputum samples, 1178 were identified as Mycobacterium tuberculosis complex (MTBC) and remaining were negative by GenoType MTBDRsl v.2.0 assay. Of the 1178 MTBC positive, 26.6% were sensitive to both FQs and SLIDs, whereas 57.3% were only FQs resistant and 15.9% were resistant to both FQs and SLIDs. Further DST of 225 isolates by liquid culture showed that 17% were sensitive to both FQs and SLIDs, 61.3% were only FQs resistant and 21.3% were resistant to both. The specificity for FQs and SLIDs was 92.31% and 100% whereas sensitivity was 100% respectively by GenoType MTBDRsl v.2.0 assay in direct sputum samples. Conclusions Our study clearly suggests that GenoType MTBDRsl v.2.0 assay is a reliable test for the rapid detection of resistance to second-line drugs after confirmation by GeneXpert MTB/RIF assay for RR-TB. Though, high rate FQ (ofloxacin) resistance was seen in our setting, moxifloxacin could be used as treatment option owing to very low resistance.

The threat of persistent bacteria and fungi contamination in tuberculosis sputum cultures

Background: Tuberculosis (TB) sputum culture contaminants make it difficult to obtain pure TB isolates.We aimed to study and identify persistent TB sputum culture contaminants post the standard laboratory pre-culture sample decontamination techniques. Methods: This was a longitudinal study of TB sputum culture contamination for a cohort of TB patients on standard treatment at: baseline, during TB treatment and post TB treatment. Sputum samples were decontaminated with 1.5%NaOH and neutralized using 6.8 Phosphate buffer solution.Sputum was then inoculated into MGIT (mycobactrial growth indicator tube) supplemented with 0.8ml PANTA. A drop of each positive MGIT culture was sub cultured onto blood agar and incu- bated for 48 hours at 35 -37OC.Any growth was identified using growth characteristics and colony morphology. Results: From October 2017 through May 2019;we collected 8645 sputum samples of which 8624(99.8%) were eligible and inoculated into MGIT where 2444(28.3%)samples were TB culture positive and 255(10.4%)were positive for contam- inants:237 none-tuberculosis bacteria, 12 fungi and 6 mixed(none-tuberculous bacteria+fungi).There was no statistically significant difference between none tuberculosis bacteria and fungi in the treatment (OR=1.4,95%CI:0.26–7.47,p=0.690) and the post treatment TB phases(OR=2.02,95%CI:0.38–10.79,p=0.411)Vs baseline. Conclusion: None-tuberculous bacteria and fungi dominate the plethora of TB sputum culture contamination and persist beyond the standard laboratory pre-culture decontamination algorithm. Keywords: Bacteria; Fungi; Inoculation; PANTA (Polymyxin B; Amphotericin B; Nalidixic acid; Trimethoprim; Azlocillin).

Comparative study of different methods of microscopy followed by a culture method in the detection of mycobacterium tuberculosis in clinically and radiologically suspected cases of pulmonary tuberculosis

To compare the sensitivity of 2 microscopic methods for the diagnosis of Mycobacterium tuberculosis (M.tb) along with culture and drug susceptibility testing to first line drugs.: The cross-sectional study comprises 200 suspected cases of pulmonary tuberculosis both clinically and radiologically in KIMS, Bangalore over a period of 2 years. Samples (sputum/BAL fluid) were collected, processed and stained by Ziehl Neelson (ZN) and Fluorescent methods. Culture and drug susceptibilty testing was done for Streptomycin, Isoniazid, Rifampicin and Ethambutol by Mycobacterium growth indicator tube (MGIT) method after decontamination.Fischer’s test : 1. Out of 200 samples: 1.120 were male and 80 were female; 2. 18 were positive by Ziehl Neelson, 21 by Fluorescent and 28 by culture; 3. Majority of the patients belonged to age group 41-50 years (23%); 4. InMGIT, 26 were M.tb and 2 were Non-tubercular mycobacteria; 5. Out of 26 M.tb isolates, 4 were resistant to streptomycin, 6 to isoniazid, 2 to rifampicin and 9 to ethambutol.1. The sensitivity of Fluorescent staining (64.28%) is higher than that of Ziehl-Neelson (51.7%); 2. In MGIT, 26 were M.tb and 2 were Non tubercular mycobacteria; 3. 2 were Multi-drug resistant- tuberculosis (MDR-TB) This study made us aware of the need for prompt detection, identification and appropriate treatment of Tuberculosis due to the rising incidence of MDR-TB.

Using Bronchoalveolar Lavage Fluid for Active Pulmonary Tuberculosis Laboratory Diagnosis

Objective: To evaluate the diagnostic efficiency of bronchoalveolar lavage fluid (BALF) for MycobacteriumTuberculosis (MTB) infection using laboratory methods. Methods: A retrospective study was conducted in patients diagnosed with active pulmonary tuberculosis (APTB) and lacking sputum quality/quantity. BALF collected during the operation processes of Electric bronchoscopy were tested using Ziehl-Neelsen staining acid-fast bacilli smear microscopy (Z-N-AFB-SM), GeneXpert MTB/RIF（Xpert），loop-mediated isothermal amplification (LAMP), or culturing with BACTEC™ Mycobacterial Growth Indicator Tube™ 960 (MGIT). Chi-square test was used for statistic analysis. Results: 331 suspected APTB patients were enrolled in this study. 224 of them were sputum-scarce. 89 were sputum-sufficient andnegative in both Z-N-AFB-SM and MGIT 960 testing. Of the sputum sufficient patients, BALF-testing confirmed APTB diagnosis in 20.2% (18/89) via Z-N-AFB-SM, and 53.0% (35/89) via MGIT. The total positive rates of BALF testing via four aforementioned methods were 18.2% (57/313), 66.4% (168/253), 61.0% (83/136) and 48.2% (140/290) respectively. The positive rate of MTB discovered in BALF collected by well-trained respiratory physicians are significantly higher than those collected by anesthetists (χ2=22.48, P<0.01). Total adverse events incidence of BAL was 1.9% (6/313). Conclusion: BALF has a similar sensitivity and specificity for APTB laboratory diagnosis. It can be used as a complementary diagnostic method for APTB when sputum availability is poor. The proficiency of BALF collection is an important factor affecting the detection results.

Tale of compounding oddities

We present a case of a 59-year-old man, who on being evaluated for abdominal pain and headache, was found to have a pancreatic head mass and inflammatory hypophysitis. Xpert MTB/Rif of the pancreatic mass biopsy showed the presence of tuberculosis (TB) with a very low load, and rifampicin resistance was detected with absence of probes A and B. Pyrosequencing (a novel genotypic test for TB) of the Xpert MTB/Rif isolate detected a single, rare, high-confidence mutation (S512T) in the rpoB region (rifampicin resistance determining region in the MTB genome). The TB mycobacteria growth indicator tube (TBMGIT) phenotypic drug susceptibility test (DST), however, showed rifampicin susceptibility. Incidentally, he was unable to tolerate rifampicin and responded well to a non-rifampicin-based regimen. We discuss a possible hypothesis of the Xpert-DST discordance in accordance with a recent literature review on phenotypic DST methods. We also discuss the utility of pyrosequencing in clinical practice for the diagnosis of TB and its resistance patterns.

Epidemiological cutoff values for a 96-well broth microdilution plate for high-throughput research antibiotic susceptibility testing of M. tuberculosis

Drug susceptibility testing of M. tuberculosis is rooted in a binary susceptible/resistant paradigm. There are considerable advantages in measuring the minimum inhibitory concentrations (MICs) of a panel of drugs for an isolate, including quantifying the magnitude of effect conferred by genetic variants and being able to identify isolates with elevated MICs that can still be treated with standard therapy. It is necessary, however, to measure the epidemiological cutoff values (ECOFF/ECVs) to permit comparison with qualitative data. Here we present ECOFF/ECVs for 13 anti-TB compounds, including bedaquiline and delamanid, derived from 20,637 clinical isolates collected by 14 laboratories based in 11 countries on five continents. Each isolate was incubated for 14 days on a dry 96-well broth microdilution plate and then read. Resistance to the majority of the drugs due to prior exposure is expected and the MIC distributions for many of the compounds are complex and therefore a phenotypically wild-type population could not be defined. Since a majority of samples also underwent genetic sequencing, we defined a genotypically wild-type population and measured the MIC of the 99th percentile by direct measurement and via fitting a Gaussian using interval regression. The proposed ECOFF/ECV values were then validated by comparing to the MIC distributions of high-confidence genetic variants that confer resistance and to qualitative drug susceptibility tests obtained via Mycobacterial Growth Indicator Tube and the Microscopic-Observation Drug-Susceptibility assay.

On the Consequences of Poorly Defined Breakpoints for Rifampin Susceptibility Testing of Mycobacterium tuberculosis Complex

ABSTRACT In a recent report of a systematic review of critical concentrations (CCs), the World Health Organization (WHO) lowered the rifampin (RIF) CC for antimicrobial susceptibility testing (AST) of the Mycobacterium tuberculosis complex using Middlebrook 7H10 medium and the Bactec Mycobacterial Growth Indicator Tube (MGIT) 960 system from 1 to 0.5 μg/ml. The previous RIF CC for 7H10 had been in use for over half a century. Because it had served as the de facto reference standard, it contributed to the endorsement of inappropriately high CCs for other AST methods, including the U.S. Food and Drug Administration (FDA)-approved MGIT system. Moreover, this resulted in confusion about the interpretation of seven borderline resistance mutations in rpoB (i.e., L430P, D435Y, H445L, H445N, H445S, L452P, and I491F). In this issue of the Journal of Clinical Microbiology, Shea et al. (J Clin Microbiol 59:e01885-20, 2021, https://doi.org/10.1128/JCM.01885-20) provide evidence that the CC endorsed by the Clinical and Laboratory Standards Institute for the Sensititre MYCOTB system, which is not FDA approved but is CE-IVD marked in the European Union, is likely also too high. These findings underscore the importance of calibrating AST methods against a rigorously defined reference standard, as recently proposed by the European Committee on Antimicrobial Susceptibility Testing, as well as the value of routine next-generation sequencing for investigating discordant AST results.