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).

Performance of GeneXpert MTB/RIF for diagnosing tuberculosis among symptomatic household contacts of index patients in South Africa

Background We describe the performance of GeneXpert MTB/RIF (Xpert) for diagnosing tuberculosis (TB) among symptomatic household contacts (HHCs) of rifampicin-resistant and drug-sensitive index cases. Methods We conducted a cross-sectional study among HHCs of recently diagnosed (<2 weeks) smear-positive and Xpert-positive index cases in the Bojanala District, South Africa. HHCs were screened for TB symptoms; persons with ≥1 TB symptom provided one sputum for smear microscopy, Xpert and MGIT culture. Diagnostic test performance of Xpert was determined using MGIT as reference standard. Results From August 2013 to July 2015, 619 HHCs from 216 index cases were enrolled: 60.6% were female, median age was 22 years (inter-quartile range: 9, 40) and 126 (20.4%) self-reported/tested HIV-positive. 54.3% (336/619) of contacts had ≥1 TB symptom (cough, fever, night sweats, weight loss), of which 297/336 (88.4%) provided a sputum; 289 (97.3%) had complete testing and 271 were included in the analysis. In total, 42 (6.8%) of 619 HHCs had microbiologically-confirmed TB. MGIT identified 33 HHCs as positive for Mycobacterium tuberculosis (MTB); of these, 7 were positive on Xpert resulting in a sensitivity of 21.2% (95% confidence interval [CI]: 9.0, 38.9), specificity 98.3% (95% CI: 95.6, 99.5), positive predictive value (PPV) 63.6% (95% CI: 30.8, 89.1), negative predictive value (NPV) 90.0 (95% CI: 85.7, 93.4). Conclusions Among symptomatic HHCs investigated for TB, Xpert performed sub-optimally compared to MGIT culture. The poor performance of Xpert for diagnosing TB suggests that a more sensitive test, such a Xpert Ultra or culture, may be needed to improve yield of contact investigation, where feasible.

Presence of Infection by Mycobacterium avium subsp. paratuberculosis in the Blood of Patients with Crohn’s Disease and Control Subjects Shown by Multiple Laboratory Culture and Antibody Methods

Mycobacterium avium subspecies paratuberculosis (MAP) has long been suspected to be involved in the etiology of Crohn’s disease (CD). An obligate intracellular pathogen, MAP persists and influences host macrophages. The primary goals of this study were to test new rapid culture methods for MAP in human subjects and to assess the degree of viable culturable MAP bacteremia in CD patients compared to controls. A secondary goal was to compare the efficacy of three culture methods plus a phage assay and four antibody assays performed in separate laboratories, to detect MAP from the parallel samples. Culture and serological MAP testing was performed blind on whole blood samples obtained from 201 subjects including 61 CD patients (two of the patients with CD had concurrent ulcerative colitis (UC)) and 140 non-CD controls (14 patients in this group had UC only). Viable MAP bacteremia was detected in a significant number of study subjects across all groups. This included Pozzato culture (124/201 or 62% of all subjects, 35/61 or 57% of CD patients), Phage assay (113/201 or 56% of all subjects, 28/61 or 46% of CD patients), TiKa culture (64/201 or 32% of all subjects, 22/61 or 36% of CD patients) and MGIT culture (36/201 or 18% of all subjects, 15/61 or 25% of CD patients). A link between MAP detection and CD was observed with MGIT culture and one of the antibody methods (Hsp65) confirming previous studies. Other detection methods showed no association between any of the groups tested. Nine subjects with a positive Phage assay (4/9) or MAP culture (5/9) were again positive with the Phage assay one year later. This study highlights viable MAP bacteremia is widespread in the study population including CD patients, those with other autoimmune conditions and asymptomatic healthy subjects.

Presence Of Infection By Mycobacterium Avium Subsp. Paratuberculosis In The Blood Of Patients With Crohn’s Disease And Control Subjects Shown By Multiple Laboratory Culture And Antibody Methods

Mycobacterium avium subspecies paratuberculosis (MAP) has long been suspected to be involved in the etiology of Crohn’s disease (CD). An obligate intracellular pathogen, MAP persists and influences host macrophages. The primary goals of this study were to assess the degree of viable culturable MAP bacteremia in humans, definite identification of the organisms cultured and to assess if CD patients have a significantly higher rate of MAP infection compared to controls. A secondary goal was to compare the efficacy of three culture methods plus a phage assay and four antibody assays performed in separate laboratories, to detect MAP from the parallel samples. Culture and serological MAP testing was performed blind on whole blood samples obtained from 201 subjects including 61 CD patients, two patients with CD and concurrent ulcerative colitis (UC), 14 patients with UC only and 140 non-CD controls. Viable MAP bacteremia was detected in a significant number of study subjects across all groups. This included Pozzato culture (124/201 or 62% of all subjects, 35/61 or 57% of CD patients), Phage assay (113/201 or 56% of all subjects, 28/61 or 46% of CD patients), TiKa culture (64/201 or 32% of all subjects, 22/61 or 36% of CD patients) and MGIT culture (36/201 or 18% of all subjects, 15/61 or 25% of CD patients). A link between MAP detection and CD was observed with MGIT culture and one of the antibody methods (Hsp65) confirming previous studies. Other detection methods showed no association between any of the groups tested. Nine subjects with positive Phage assay (8/9) or MAP culture (1/9) were again positive with the Phage assay one year later. This study highlights viable MAP bacteremia is widespread in the study population including CD patients, those with other autoimmune conditions and asymptomatic healthy subjects.

The MPB64 immunochromatography assay: an analysis of doubtful results

Culture remains the gold standard for tuberculosis (TB) diagnosis, and the mycobacteria growth indicator tube (MGIT), endorsed by the World Health Organization (WHO), is widely used. Further identification of a positive culture is done with the help of an immunochromatography assay, which often shows faint bands that are difficult to interpret. We analysed 125 BACTEC MGIT culture positive results, of which 11/16 (68.7%) of the doubtful assays, analysed by MGIT™ TBc Identification test (TBcId), were positive for Mycobacterium tuberculosis complex (MTBC), the remaining being non-tuberculous mycobacteria as determined by an in-house duplex polymerase chain reaction and line probe assay. Guidelines on faint or doubtful bands in immunochromatography assays are important so as not to overlook true-positive cases of TB.

IP-MS Analysis of ESX-5 and ESX-1 Substrates Enables Mycobacterial Species Identification

Pulmonary disease resulting from non-tuberculous mycobacteria (NTM) infection has emerged as an increasingly prevalent clinical entity in the past two to three decades, but there are no standardized, commercial assays available for the molecular diagnosis of NTM infections from clinical samples. Herein we discuss the development of an assay that employs immunoprecipitation coupled with mass spectrometry (IP-MS) to rapidly and accurately discriminate prevalent slow-growing mycobacterial species (i.e., M. avium and M. intracellulare, M. kansasii, M. gordonae, M. marinum and M. tuberculosis) during early growth in mycobacterial growth indicator tube (MGIT) cultures. This IP-MS assay employs antibodies specific for conserved tryptic peptides of M. tuberculosis EsxN (AQAASLEAEHQAIVR) and CFP-10 (TQIDQVESTAGSLQGQWR) to capture and identify specific mycobacterial species and allows species-specific mycobacterium identification at the first sign of MGIT culture positivity.

Accuracy of Xpert Ultra in Diagnosis of Pulmonary Tuberculosis among Children in Uganda: a Substudy from the SHINE Trial

ABSTRACT Childhood tuberculosis (TB) presents significant diagnostic challenges associated with paucibacillary disease and requires a more sensitive test. We evaluated the diagnostic accuracy of Xpert MTB/RIF Ultra (Ultra) compared to other microbiological tests using respiratory samples from Ugandan children in the SHINE trial. SHINE is a randomized trial evaluating shorter treatment in 1,204 children with minimal TB disease in Africa and India. Among 352 samples and one cervical lymph node fine needle aspirate, one sample was randomly selected per patient and tested with the Xpert MTB/RIF assay (Xpert) and with Lowenstein-Jensen medium (LJ) and liquid mycobacterial growth indicator tube (MGIT) cultures. We selected only uncontaminated stored sample pellets for Ultra testing. We estimated the sensitivity of Xpert and Ultra against culture and a composite microbiological reference standard (any positive result). Of 398 children, 353 (89%) had culture, Xpert, and Ultra results. The median age was 2.8 years (interquartile range [IQR], 1.3 to 5.3); 8.5% (30/353) were HIV infected, and 54.4% (192/353) were male. Of the 353, 31 (9%) were positive by LJ and/or MGIT culture, 36 (10%) by Ultra, and 16 (5%) by Xpert. Sensitivities (95% confidence intervals [CI]) were 58% (39 to 65% [18/31]) for Ultra and 45% (27 to 64% [14/31]) for Xpert against any culture-positive result, with false positives of <1% and 5.5% for Xpert and Ultra. Against a composite microbiological reference, sensitivities were 72% (58 to 84% [36/50]) for Ultra and 32% (20 to 47% [16/50]) for Xpert. However, there were 17 samples that were positive only with Ultra (majority trace). Among children screened for minimal TB in Uganda, Ultra has higher sensitivity than Xpert. This represents an important advance for a condition which has posed a diagnostic challenge for decades.

Diagnostic value of polymerase chain reaction targeting insertion sequence IS1081 for the diagnosis of pediatric tuberculosis

Background: Aim of this study was to evaluate the efficacy of PCR targeting IS1081in diagnosis of pediatric tuberculosis and compare the results with MGIT culture.Methods: This prospective study was conducted in the department of pediatrics, S.N. medical college, Agra. 100 subjects (28 pulmonary 72 extra pulmonary) were registered in study. The specimens obtained from these cases were subjected to Ziehl–Neelsen staining (ZN), MGIT 960 TB culture and PCR targeting insertion sequence IS1081. Sensitivity, specificity, PPV and NPV of PCR were calculated in pulmonary and extra pulmonary specimens. The results of PCR IS1081 were compared to MGIT culture.Results: Microscopy with ZN staining was positive in 12 (12%) samples. MGIT culture was positive in 44% samples with maximum positivity in sputum (70%). PCR IS1081 has shown 93.3% sensitivity in pulmonary tuberculosis, while PCR IS1081 has shown 93.1% sensitivity in extra pulmonary tuberculosis. In diagnosis of childhood tuberculosis PCR IS1081 was found to be statistically significant (p value <0.05) as compared with MGIT culture. Result was statistically significant (p value <0.05) in CSF samples only.Conclusions: The study concluded that the PCR targeting sequence IS1081 technique is the most sensitive technique for a quick identification of MTB in pulmonary and extra pulmonary tuberculosis.

Comparison of Diagnostic Yield of Tuberculosis Loop-Mediated Isothermal Amplification Assay With Cartridge-Based Nucleic Acid Amplification Test, Acid-Fast Bacilli Microscopy, and Mycobacteria Growth Indicator Tube Culture in Children With Pulmonary Tuberculosis

Background Cartridge-based nucleic acid amplification test (CB-NAAT) has been recommended for diagnosis of tuberculosis (TB) in children, but its wide use is limited by high cost and the need for well-equipped laboratories. This study was conducted in children with pulmonary TB to compare the diagnostic yield of TB-LAMP (loop-mediated isothermal amplification test) with CB-NAAT and other conventional methods. Methods Patients ≤ 14 years of age diagnosed with probable pulmonary TB were included in the study. Induced sputum/gastric aspirate was obtained and subjected to acid-fast bacilli (AFB) microscopy, mycobacteria growth indicator tube (MGIT) culture, CB-NAAT, and TB-LAMP. The TB-LAMP assay was performed using 2 different primers, IS6110 and mpb64, for detection of Mycobacterium tuberculosis (MTB). TB-LAMP assays were compared to other assays using appropriate statistical tests. Results One hundred fourteen subjects were recruited in the study. AFB microscopy, MGIT culture, CB-NAAT, TB-LAMP IS6110, and TB-LAMP mpb64 showed positivity of 32 (28.1%), 59 (51.7%), 66 (57.9%), 75 (65.8%), and 81 (71%), respectively. TB-LAMP IS6110 showed significantly higher MTB detection in comparison to AFB microscopy and MGIT culture (P = .0001 and P = .03, respectively), and showed no significant difference in MTB detection in comparison with CB-NAAT (P = .219). TB-LAMP mpb64 showed significantly higher MTB detection as compared to AFB microscopy, MGIT culture, and CB-NAAT (P = .0001, P = .003, and P = .037, respectively). TB-LAMP mpb64 and IS6110 showed sensitivity of 94.9% (95% confidence interval [CI], 85.9%–98.9%) and 89.8% (95% CI, 79.7%–96.2%), respectively, in reference to MGIT culture. The degree of agreement between TB-LAMP (mpb64 and IS6110) with CB-NAAT showed κ values of 0.718 and 0.834, respectively. Conclusions TB-LAMP assay can be a useful alternative test in diagnosis of pulmonary TB in children.