Microscopic Observation Drug Susceptibility Assay Research Articles

Rapid detection of drug-resistant Mycobacterium tuberculosis by Modified MODS assay suitable for resource-poor settings.

Cross contamination and biosafety are concerns with the microscopic observation drug susceptibility assay. To address these issues, we modified the MODS technique in the current study. Two hundred and seventy-five samples were processed on LJ media and drug susceptibility was performed by the Indirect agar proportion method. A modified MODS test was done in tissue culture bottles. GenoType MTBDRplus assay was performed to detect the resistance and mutational pattern associated with the resistances. Sensitivity, specificity, positive predictive value, and negative predictive value for the detection of tuberculosis by modified MODS were 97.44%, 80.00%, 97.44%, and 80.00% respectively. The perfect agreement was seen between modified MODS and the Indirect agar proportion method for drug susceptibility testing of isoniazid (kappa = 0.923) and rifampicin (kappa = 1). The contamination rate, cost and TAT for modified MODS were less as compared to the solid media. In the case of MDR-TB isolates S531L (66.66%) was the most prevalent mutation in the rpoB gene followed by S315T2 mutation (58.33%) and T8C (41.66%) in katG and inhA gene respectively. In hetero-resistant strains, C-15T mutation (37.50%) was the most common followed by A-16G (12.50%) in the inhA gene. In INH mono-resistant strains only two mutations were observed i.e., S-315T1(50%) and C-15T (50%) in the katG and inhA genes respectively. Modified MODS proved to be cost-effective and user-friendly, with minimal risk to the handler and no cross-contamination between samples were observed. Hence, it can be used in low-income countries for early detection of tuberculosis and its resistance.

Evaluation of novel PCR-CTPP for simultaneous detection of Mycobacterium tuberculosis complex and identification of RpoB H526D point mutation

Background: Tuberculosis (TB) is a chronic and highly contagious disease caused of the acid-fast bacilli, Mycobacterium tuberculosis complex (MTC). Currently, it has been reported that rifampicin-mono resistant M. tuberculosis containing histidine to aspartate replacement at residue 526 (H526D) of the beta-subunit RNA polymerase enzyme (RpoB) increased the cell wall permeability and approximately eight times more susceptible to vancomycin. Objectives: To develop and evaluate the Polymerase chain reaction with confronting two-pair primers (PCR-CTPP) for simultaneous detection of MTC and identification of RpoB H526D mutation. Materials and methods: PCR-CTPP was implemented for TB diagnosis. Reaction and profile were optimized and applied for detection in a total of 308 clinical samples. Sensitivity and specificity of PCR-CTPP was calculated in comparison to the acid-fast bacilli (AFB) staining and standard culture method. In addition, microscopic observation drug susceptibility (MODS) assay was modified for vancomycin susceptibility determination in 15 clinical isolates. Results: Limit of detection of PCR-CTPP was approximately 2×103 bacilli and no cross-detection to other mycobacteria. PCR-CTPP was evaluated in 308 clinical samples. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of PCR-CTPP versus standard culture method were 90.95%, 79.31%, 91.78% and 77.53%, respectively. In addition, comparison of developed PCR-CTPP versus AFB staining were also represented. MODS was performed in fifteen samples, two multidrug-resistant (MDR) strains containing RpoB H526D were susceptible against vancomycin. Conclusion: The established PCR-CTPP is highly sensitive, specific for investigation of MTC and identification of RpoB H526D mutation. This method could be useful for TB diagnosis together with precision medicine application in vancomycin susceptibility determination in TB patients in the future.

In Vitro Antimycobacterial Activity of Human Lactoferrin-Derived Peptide, D-hLF 1-11, against Susceptible and Drug-Resistant Mycobacterium tuberculosis and Its Synergistic Effect with Rifampicin.

Tuberculosis is a highly contagious disease caused by the Mycobacterium tuberculosis complex (MTBC). Although TB is treatable, multidrug-resistant, extensively drug-resistant, and totally drug-resistant forms of M. tuberculosis have become a new life-threatening concern. New anti-TB drugs that are capable of curing these drug-resistant strains are urgently needed. The purpose of this study is to determine the antimycobacterial activity of D-enantiomer human lactoferricin 1-11 (D-hLF 1-11) against mycobacteria in vitro using a 3-(4,5-dimethylthiazol-2-yl)-2,5-dephenyltetrazolium bromide colorimetric assay, resazurin microplate assay, and microscopic observation drug susceptibility assay. Three previously described antimicrobial peptides, protegrin-1, AK 15-6, and melittin, with potent anti-TB activity, were included in this study. The findings suggest that D-hLF 1-11 can inhibit the growth of M. tuberculosis with a minimum inhibitory concentration of 100-200 µg/mL in susceptible, isoniazid (INH)-monoresistant, rifampicin (RF)-monoresistant, and MDR strains. The peptide can also inhibit some nontuberculous mycobacteria and other MTBC in similar concentrations. The antibiofilm activity of D-hLF 1-11 against the biofilm-forming M. abscessus was determined by crystal violet staining, and no significant difference is observed between the treated and untreated biofilm control. The checkerboard assay was subsequently carried out with M. tuberculosis H37Rv and the results indicate that D-hLF 1-11 displays an additive effect when combined with INH and a synergistic effect when combined with RF, with fractional inhibitory concentration indices of 0.730 and 0.312, respectively. The red blood cell hemolytic assay was initially applied for the toxicity determination of D-hLF 1-11, and negligible hemolysis (<1%) was observed, despite a concentration of up to 4 mg/mL being evaluated. Overall, D-hLF 1-11 has potential as a novel antimycobacterial agent for the future treatment of drug-sensitive and drug-resistant M. tuberculosis infections.

Evaluation of three alternatives cost-effective culture media for Mycobacterium tuberculosis detection and drug susceptibility determination using the microscopic observation drug susceptibility (MODS) assay

Tuberculosis phenotypic detection assays are commonly used in low-resource countries. Therefore, reliable detection methods are crucial for early diagnosis and treatment. The microscopic observation drug susceptibility (MODS) assay is a culture-based test to detect Mycobacterium tuberculosis and characterize drug resistance in 7–10 days directly from sputum. The use of MODS is limited by the availability of supplies necessary for preparing the enriched culture. In this study, we evaluated three dry culture media that are easier to produce and cheaper than the standard one used in MODS [1]: an unsterilized powder-based mixed (Boldú et al., 2007) [2], a sterile-lyophilized medium, and (Sengstake et al., 2017) [3] an irradiated powder-based mixed. Mycobacterial growth and drug susceptibility were evaluated for rifampin, isoniazid, and pyrazinamide (PZA). The alternative cultures were evaluated using 282 sputum samples with positive acid-fast smears. No significant differences were observed in the positivity test rates. The positivity time showed high correlations (Rho) of 0.925, 0.889, and 0.866 between each of the three alternative media and the standard. Susceptibility testing for MDR and PZA showed an excellent concordance of 1 compared to the reference test. These results demonstrate that dry culture media are appropriate and advantageous for use in MODS in low-resource settings.

A rapid and low-cost microscopic observation drug susceptibility assay for detecting TB and MDR-TB among individuals infected by HIV in South India

Background: The converging epidemics of HIV and tuberculosis (TB) pose one of the greatest public health challenges of our time. Rapid diagnosis of TB is essential in view of its infectious nature, high burden of cases, and emergence of drug resistance. Objective: The purpose of this present study was to evaluate the feasibility of implementing the microscopic observation drug susceptibility (MODS) assay, a novel assay for the diagnosis of TB and multi-drug-resistant tuberculosis (MDR-TB) directly from sputum specimens, in the Indian setting. Materials and Methods: This study involved a cross-sectional, blinded assessment of the MODS assay on 1036 suspected cases of pulmonary TB in HIV-positive and HIV-negative patients against the radiometric method, BD-BACTEC TB 460 system. Results: Overall, the sensitivity, specificity, positive predictive value, and negative predictive value of the MODS assay in detecting MTB among TB suspected patients were 89.1%, 99.1%, 94.2%, 95.8%, respectively. In addition, in the diagnosis of drug-resistant TB, the MODS assay was 84.2% sensitive for those specimens reporting MDR, 87% sensitivity for those specimens reporting INH mono-resistance, and 100% sensitive for specimens reporting RIF mono-resistance. The median time to detection of TB in the MODS assay versus BACTEC was 9 versus 21 days (P < 0.001). Conclusion: Costing 5 to 10 times lesser than the automated culture methods, the MODS assay has the potential clinical utility as a simple and rapid method. It could be effectively used as an alternative method for diagnosing TB and detection of MDR-TB in a timely and affordable way in resource-limited settings.

Direct Determination of Pyrazinamide (PZA) Susceptibility by Sputum Microscopic Observation Drug Susceptibility (MODS) Culture at Neutral pH: the MODS-PZA Assay.

Pyrazinamide (PZA) is considered the pivot drug in all tuberculosis treatment regimens due to its particular action on the persistent forms of Mycobacterium tuberculosis However, no drug susceptibility test (DST) is considered sufficiently reliable for routine application. Although molecular tests are endorsed, their application is limited to known PZA resistance associated mutations. Microbiological DSTs for PZA have been restricted by technical limitations, especially the necessity for an acidic pH. Here, for the first time, MODS culture at neutral pH was evaluated using high PZA concentrations (400 and 800 μg/ml) to determine PZA susceptibility directly from sputum samples. Sputum samples were cultured with PZA for up to 21 days at 37°C. Plate reading was performed at two time points: R1 (mean, 10 days) and R2 (mean, 13 days) for each PZA concentration. A consensus reference test, composed of MGIT-PZA, pncA sequencing, and the classic Wayne test, was used. A total of 182 samples were evaluated. The sensitivity and specificity for 400 μg/ml ranged from 76.9 to 89.7 and from 93.0 to 97.9%, respectively, and for 800 μg/ml ranged from 71.8 to 82.1 and from 95.8 to 98.6%, respectively. Compared to MGIT-PZA, our test showed a similar turnaround time (medians of 10 and 12 days for PZA-sensitive and -resistant isolates, respectively). In conclusion, MODS-PZA is presented as a fast, simple, and low-cost DST that could complement the MODS assay to evaluate resistance to the principal first-line antituberculosis drugs. Further optimization of test conditions would be useful in order to increase its performance.

Sources of Multidrug Resistance in Patients With Previous Isoniazid-Resistant Tuberculosis Identified Using Whole Genome Sequencing: A Longitudinal Cohort Study.

BackgroundMeta-analysis of patients with isoniazid-resistant tuberculosis (TB) given standard first-line anti-TB treatment indicated an increased risk of multidrug-resistant TB (MDR-TB) emerging (8%), compared to drug-sensitive TB (0.3%). Here we use whole genome sequencing (WGS) to investigate whether treatment of patients with preexisting isoniazid-resistant disease with first-line anti-TB therapy risks selecting for rifampicin resistance, and hence MDR-TB.MethodsPatients with isoniazid-resistant pulmonary TB were recruited and followed up for 24 months. Drug susceptibility testing was performed by microscopic observation drug susceptibility assay, mycobacterial growth indicator tube, and by WGS on isolates at first presentation and in the case of re-presentation. Where MDR-TB was diagnosed, WGS was used to determine the genomic relatedness between initial and subsequent isolates. De novo emergence of MDR-TB was assumed where the genomic distance was 5 or fewer single-nucleotide polymorphisms (SNPs), whereas reinfection with a different MDR-TB strain was assumed where the distance was 10 or more SNPs.ResultsTwo hundred thirty-nine patients with isoniazid-resistant pulmonary TB were recruited. Fourteen (14/239 [5.9%]) patients were diagnosed with a second episode of TB that was multidrug resistant. Six (6/239 [2.5%]) were identified as having evolved MDR-TB de novo and 6 as having been reinfected with a different strain. In 2 cases, the genomic distance was between 5 and 10 SNPs and therefore indeterminate.ConclusionsIn isoniazid-resistant TB, de novo emergence and reinfection of MDR-TB strains equally contributed to MDR development. Early diagnosis and optimal treatment of isoniazid-resistant TB are urgently needed to avert the de novo emergence of MDR-TB during treatment.

Microscopic Observation Drug Susceptibility (MODS) Assay: A Convenient Method for Determining Antibiogram of Clinical Isolates of Mycobacterium tuberculosis in Ghana.

(1) Background: Present methods for drug susceptibility tests (DST) rely on culture methods that are sophisticated and relatively faster, or a slow and cheaper option. These methods frustrate disease control; therefore, there is a need for methods that incorporate key functions of microscopy and culture, with reduced cost burden and sophistry. Thus, the purpose of this study was to identify which, among the most commonly used (in Ghana) methods, can conveniently be used at health centers located in rural areas for effective DST determination of Mycobacterium tuberculosis (MTB). (2) Methods: Mycobacterium tuberculosis isolates were tested for their susceptibility to streptomycin, isoniazid, rifampicin, ethambutol (SIRE), and pyrazinamide by microscopic observation drug susceptibility (MODS) and BACTEC MGIT 960 methods. Evaluations were based on shorter turnaround periods, rapidity, ease of use, cost, etc. A comparative analysis was statistically expressed as kappa values. (3) Results: Endpoints for drug susceptibilities by MODS averaged 13 days (7–32), whilst that for BACTEC MGIT 960 was 10 days with a further 12 days to detect resistance. Therefore, a turnaround period of 22 days was needed for DST by BACTEC MGIT 960, compared to 13 days for MODS. There were differences in correlation levels between the two methods, as determined by their kappa values. (4) Conclusion: The MODS assay was found to be less costly, more user-friendly, and still able to be conveniently used at health centers located in rural areas known to be endemic for TB, particularly in Ghana.

ASSESSMENT OF THE MICROSCOPIC– OBSERVATION- DRUG - SUSCEPTIBLITY ASSAY (MODS) AND GENEXPERT ASSAY FOR DIAGNOSIS OF PULMONARY TUBERCULOSIS

Background: Tuberculosis (TB) is considered the most common and highly contagious disease worldwide. Early and accurate diagnosis of TB with detection of multidrug-resistant Mycobacterium tuberculosis (M.TB) is of primary importance for both patient management and infection control. Objectives: To evaluate the performance of Microscopic-Observation Drug-Susceptibility (MODS) assay and GeneXpert MTB/RIF( GeneXpert ) assay in diagnosis of TB and detection of MDR-TB directly from sputum specimens in comparison to the convential diagnostic methods (direct Ziehl-Neelsen "Z-N" stained smear and culture on Lowenstein-Jenson media "LJ"). Patients and Methods: After approval of institutional ethical committee a total of 120 suspected pulmonary TB patients admitted to chest hospitals and dispensaries in Egypt (Giza, Cairo, Alexandria, Dakahlia, Suez, Beheira, Monufia, Qena). Their age was more than 16 years; all of them did not receive any anti tubercular drugs. Sputum samples were collected from each patient and subjected to direct ZN smear, culture and sensitivity of M.TB on LJ and Middlebrook 7H9 medium for MODS assay. Finally Genexpert M.TB/RIF assay was done directly for all sputum samples. Results: Infection rate among males was higher than female (81.7% & 18.7%). Also, it was observed that 56.7% of sputum samples gave positive direct Z-N smear, while 64.2% were positive for both LJ culture &MODS assay. On the other hand, by Genexpert, 68% of cases were positive. The sensitivity and specificity of the various diagnostic methods were studied in relation to LJ culture (gold standard). It was found that MODS assay was rapid that results were obtained in a median of 8 days ranged from (7– 21 days), while median time of LJ was 21dayes ranged from (21– 60). MODS had 100% sensitivity and specificity. Direct smear was less sensitive (88.3%), and GeneXpert was less specific (88.4%), but more rapid results were obtained in 2 hours. Finally, detection of MDR-TB using various methods for 77 isolated M.TB strains ranged from 16.9-19.5% to isoniazid (INH) and 9.8%-18.2% to rifampicin (RMP) by various diagnostic methods with statistically insignificant difference between them. Conclusion: MODS was an optimal alternative rapid diagnostic technique for TB and detection of M.TB and MDR-TB in resource limited settings, results were obtained in a median of 8 days. On the other hand, Gene Xpert was a novel integrated diagnostic PCR analysis in a single hand free step for rapid diagnosis of TB and detection of RMP resistance in clinical specimens in 2 hours but it was expensive.

ASSESSMENT OF THE MICROSCOPIC– OBSERVATION- DRUG - SUSCEPTIBLITY ASSAY (MODS) AND GENEXPERT ASSAY FOR DIAGNOSIS OF PULMONARY TUBERCULOSIS

Background: Tuberculosis (TB) is considered the most common and highly contagious disease worldwide. Early and accurate diagnosis of TB with detection of multidrug-resistant Mycobacterium tuberculosis (M.TB) is of primary importance for both patient management and infection control. Objectives: To evaluate the performance of Microscopic-Observation Drug-Susceptibility (MODS) assay and GeneXpert MTB/RIF( GeneXpert ) assay in diagnosis of TB and detection of MDR-TB directly from sputum specimens in comparison to the convential diagnostic methods (direct Ziehl-Neelsen stained smear and culture on Lowenstein-Jenson media LJ). Patients and Methods: After approval of institutional ethical committee a total of 120 suspected pulmonary TB patients admitted to chest hospitals and dispensaries in Egypt (Giza, Cairo, Alexandria, Dakahlia, Suez, Beheira, Monufia, Qena). Their age was more than 16 years; all of them did not receive any anti tubercular drugs. Sputum samples were collected from each patient and subjected to direct ZN smear, culture and sensitivity of M.TB on LJ and Middlebrook 7H9 medium for MODS assay. Finally Genexpert M.TB/RIF assay was done directly for all sputum samples. Results: Infection rate among males was higher than female (81.7% & 18.7%). Also, it was observed that 56.7% of sputum samples gave positive direct Z-N smear, while 64.2% were positive for both LJ culture &MODS assay. On the other hand, by Genexpert, 68% of cases were positive. The sensitivity and specificity of the various diagnostic methods were studied in relation to LJ culture (gold standard). It was found that MODS assay was rapid that results were obtained in a median of 8 days ranged from (7– 21 days), while median time of LJ was 21dayes ranged from (21– 60). MODS had 100% sensitivity and specificity. Direct smear was less sensitive (88.3%), and GeneXpert was less specific (88.4%), but more rapid results were obtained in 2 hours. Finally, detection of MDR-TB using various methods for 77 isolated M.TB strains ranged from 16.9-19.5% to isoniazid (INH) and 9.8%-18.2% to rifampicin (RMP) by various diagnostic methods with statistically insignificant difference between them. Conclusion: MODS was an optimal alternative rapid diagnostic technique for TB and detection of M.TB and MDR-TB in resource limited settings, results were obtained in a median of 8 days. On the other hand, Gene Xpert was a novel integrated diagnostic PCR analysis in a single hand free step for rapid diagnosis of TB and detection of RMP resistance in clinical specimens in 2 hours but it was expensive.

Protein analysis in skeletal muscle from gestational diabetes mellitus and their association with insulin signalling during pregnancy.

Mutations in pncA gene contributing to PZA resistance was not clearly elucidated in China. To reveal the correlated mutations of pncA gene on pyrazinamide (PZA) resistance. 148 Mycobacterium tuberculosis clinical isolates were included from multi-drug resistant tuberculosis suspects. The MGIT 960 test and microscopic observation drug susceptibility (MODS) assay were adopted for PZA phenotype drug susceptibility test. 120 isolates with consistent MGIT 960 and MODS results were selected for pncA gene sequencing. 68 samples (56.7%) were resistant to PZA while leaving 52 PZA susceptible samples. Out of the 68 PZA resistant isolates, 49 (72.1%) harbored mutations of pncA, and 4 (7.7%) of the 52 PZA susceptible samples harbored mutations of pncA as well. Compared to the phenotype drug resistant pattern of PZA, the mutations of pncA gene reached a sensitivity of 0.72 to report PZA resistance and a specificity of 0.92 to predict PZA susceptibility. Those mutations, Gln10Pro, Asp12Ala, Tyr41Stop, Gly97Asp, Val128Gly and FSC131(ins) exceeding 5% of the total PZA resistant isolates of each, might be helpful but not adequate in PZA molecular susceptibility test design and development.

PncA gene mutations in reporting pyrazinamide resistance among the MDR-TB suspects

Mutations in pncA gene contributing to PZA resistance was not clearly elucidated in China. To reveal the correlated mutations of pncA gene on pyrazinamide (PZA) resistance. 148 Mycobacterium tuberculosis clinical isolates were included from multi-drug resistant tuberculosis suspects. The MGIT 960 test and microscopic observation drug susceptibility (MODS) assay were adopted for PZA phenotype drug susceptibility test. 120 isolates with consistent MGIT 960 and MODS results were selected for pncA gene sequencing. 68 samples (56.7%) were resistant to PZA while leaving 52 PZA susceptible samples. Out of the 68 PZA resistant isolates, 49 (72.1%) harbored mutations of pncA, and 4 (7.7%) of the 52 PZA susceptible samples harbored mutations of pncA as well. Compared to the phenotype drug resistant pattern of PZA, the mutations of pncA gene reached a sensitivity of 0.72 to report PZA resistance and a specificity of 0.92 to predict PZA susceptibility. Those mutations, Gln10Pro, Asp12Ala, Tyr41Stop, Gly97Asp, Val128Gly and FSC131(ins) exceeding 5% of the total PZA resistant isolates of each, might be helpful but not adequate in PZA molecular susceptibility test design and development.

Rapid drug susceptibility testing and treatment outcomes for multidrug-resistant tuberculosis in Peru.

The detection of multidrug-resistant tuberculosis (MDR-TB) using rapid drug susceptibility testing (DST) has increased steadily in recent years in Peru, from 9216 tests in 2010 to 27 021 tests in 2015. Research examining the impact of rapid DST on treatment outcomes is required. To evaluate the association between rapid DST use (nitrate reductase assay, microscopic observation drug susceptibility assay [MODS] and GenoType® MTBDRplus) and treatment outcomes and mortality in MDR-TB patients in Peru. Retrospective cohort study of patients diagnosed with pulmonary MDR-TB between 2010 and 2013 (with treatment outcomes up to December 2015) using the electronic registry of the Peruvian National TB Programme. A total of 2671 MDR-TB patients were included; the median age was 27 years, 2.8% were co-infected with the human immunodeficiency virus. Use of rapid DST was associated with a 40% increase in the adjusted odds of treatment success (aOR 1.40, 95%CI 1.19-1.64) and a 54% reduction in mortality (aOR 0.46, 95%CI 0.33-0.64). Higher treatment success rates were driven by MODS and GenoType® MTBDRplus testing (aORs for unsuccessful outcomes respectively 0.68 and 0.66). The use of rapid DST (MODS and MTBDRplus) to diagnose MDR-TB was associated with a reduction in the odds of death and a substantial increase in the odds of treatment success.

Evaluation of Microscopic observation drug susceptibility (MODS) assay as a rapid, sensitive and inexpensive test for detection of tuberculosis and multidrug resistant tuberculosis

BackgroundMicroscopic observation drug susceptibility (MODS) assay has been suggested as a low cost method for rapid, accurate detection of tuberculosis (TB) and multidrug resistant tuberculosis (MDR-TB). MethodsA total of 2424 samples collected from 1063 eligible patients of suspected pulmonary or extrapulmonary TB were subjected to MODS assay. Performance of MODS was compared with culture and drug susceptibility testing (DST) by conventional solid Lowenstein–Jensen (LJ) media or liquid Mycobacteria Growth Indicator Tube (MGIT) culture. ResultsWhen compared to reference gold standard of positivity in either solid or liquid reference culture, the MODS assay had sensitivity, specificity, positive predictive value and negative predictive value of 91.3%, 98.2%, 96.0% and 95.9% respectively. MODS took a median time of 10.3 days to culture positivity as compared to 13.8 days using MGIT and 30.5 days using LJ culture. Culture and DST being concurrent in MODS, the median turnaround time for DST was the same as that for culture i.e. 10.3 days. The overall median turn around time for culture positivity and DST using manual MGIT and LJ medium was 23.6 days and 61.2 days respectively. The concordance between MODS culture and the reference susceptibility method was 97.7% for rifampicin, 95.6% for isoniazid, 98.5% for rifampicin and isoniazid. The cost of performing a single MODS assay was INR 200. ConclusionMODS is a rapid and sensitive, yet simple and inexpensive test that may be helpful to enhance diagnostic accuracy, and case detection of TB and MDR-TB in resource constrained settings.

A novel inexpensive electrochemical sensor for pyrazinoic acid as a potential tool for the identification of pyrazinamide-resistant Mycobacterium tuberculosis.

Tuberculosis (TB) is a significant cause of morbidity and mortality worldwide. The patient compliance with the long treatment regimens is essential for successful eradication. Pyrazinamide (PZA) shortens these regimens from 9 to 6 months, and therefore, improves treatment completion rates. Although PZA is a first-line medication for the treatment of TB, no simple or reliable assay to determine PZA resistance is yet available. In the presence of PZA, only susceptible Mycobacterium tuberculosis strains release pyrazinoic acid (POA). Therefore, the measurement and quantification of released POA is an indicator of PZA resistance. Two electrochemical sensors were constructed and tested with alternative working electrodes in conjunction with a portable potentiostat to measure the current produced when a potential difference of 2 V is applied to varying concentrations of POA in controlled solutions. The large (13.2 mm) electrochemical sensor was able to detect POA at a minimum concentration of 40 μM to a statistically significant level (P = 0.0190). Similar graphical trends were obtained when testing the electrochemical sensor in the supernatant of a negative microscopic observation drug susceptibility assay culture, irrespective of the presence of PZA. Inexpensive and reusable electrochemical sensors with a portable potentiostat are a promising tool for the detection of POA, a biomarker of PZA susceptible M. Tuberculosis.

Progress on Diagnosis of Tuberculous Meningitis.

Central nervous system (CNS) disease caused by Mycobacterium tuberculosis (MTB) is highly devastating. Tuberculous meningitis (TBM) is the most common form of CNS tuberculosis (TB). Rapid, sensitive, and affordable diagnostic tests are not available. Ziehl-Neelsen (ZN) stain has a very low sensitivity in cases of TBM, the sensitivity rates is of about 10-20%.The detection rate can be improved by taking large volume CSF samples (>6ml) and prolonged slide examination (30min). Culture of MTB from the CSF is slow and insufficiently sensitive. The sensitivity is different, which varies from 36% to 81.8%. The microscopic observation drug susceptibility (MODS) assay was recommended by the World Health Organization in 2011. The sensitivity is 65%, which is more sensitive and faster than CSF smear. Commercial PCR assays were found to be insensitive at detecting MTB in CSF samples. Many research provided the value of ADA on the TBM diagnosis. Interferon-gamma release assays (IGRAs) are not recommended for diagnosis of active TB disease. Imaging is essential in diagnosis and showing complications of CNS TB. Thwaites criteria and the Lancet consensus scoring system (LCSS) were developed to improve the diagnosis of TBM. Clinicians will continue to make judgment based on clinical examination, inflammatory CSF examinations, imaging studies, and scoring systems.

The value of microscopic-observation drug susceptibility assay in the diagnosis of tuberculosis and detection of multidrug resistance.

Inexpensive, rapid, and reliable tests for detecting the presence and drug susceptibility of Mycobacterium tuberculosis complex (MTBC) are urgently needed to control the transmission of tuberculosis. In this study, we aimed to assess the accuracy and speed of the microscopic-observation drug susceptibility (MODS) assay in the identification of MTBC and detection of multidrug resistance. Sputum samples from patients suspected to have tuberculosis were simultaneously tested with MODS and conventional culture [Löwenstein-Jensen (LJ) culture, BACTEC MGIT™ 960 (MGIT) system], and drug susceptibility testing (MGIT system) methods. A total of 331 sputum samples were analyzed. Sensitivity and specificity of MODS assay for detection of MTBC strains were 96% and 98.8%, respectively. MODS assay detected multidrug resistant MTBC isolates with 92.3% sensitivity and 96.6% specificity. Median time to culture positivity was similar for MGIT (8days) and MODS culture (8days), but was significantly longer with LJ culture (20days) (p<0.0001 for both comparisons). Median time to availability of the susceptibility results was significantly (p<0.0001) shorter with MODS assay (8days) than MGIT system (20days). In conclusion, MODS is an inexpensive and rapid test with good performance characteristics for direct diagnosis of tuberculosis and detection of multidrug resistance.

A suggested algorithm for detection of multi drug-resistant tuberculosis in Zimbabwe.

Rapid genotypic and phenotypic methods for multi-drug-resistant-tuberculosis (MDR-TB) detection are now widely available. Zimbabwe adopted the use of GeneXpert-MTB/RIF, microscopic-observation-drug-susceptibility-assay (MODS) and Mycobacteria-Growth-Indicator-Tube (MGIT) drug-susceptibility-testing (DST). Data is limited on the ideal combination of use of these methods in resource limited settings. Between August 2014 to July 2015, 211 sputa from MDR-TB suspects were tested with GeneXpert-MTB/RIF, MODS, manual-MGIT and Lowenstein-Jensen (LJ)-DST to determine diagnostic accuracy and turnaround-time (TAT), with LJ-DST as the gold standard. A performance score ranking table for diagnostic accuracy, TAT, costs, facilities and expertise requirements, was used to determine the most favourable tool. GeneXpert-MTB/RIF sensitivity was 96% (95%CI:80-100) and specificity was 95% (95%CI:90-97). MODS sensitivity was 88% (95%CI:68-97) and specificity was 97% (95%CI:87-100). Manual MGIT-DST had slightly lower sensitivity of 80% (95%CI:59-93). Median time to detection of MDR-TB was <1 day (IQR:0-0) for Xpert, 14 days (IQR:11-31) for MODS, 21 days (IQR:7-22) for MGIT-DST and 28 days (IQR:25-28) for LJ-DST. Operational costs for MODS, MGIT-DST, and GeneXpert-MTB/RIF were $21.20, $27.52 and $39.76 respectively. From a summation of scores including facility and expertise requirements per diagnostic technique, GeneXpert-MTB/RIF was the most favourable tool, followed by MODS and MGIT-DST. For best scale-up of MDR-TB diagnosis in Zimbabwe, GeneXpert-MTB/RIF can be used for rapid detection of TB in smear negative cases, RIF-susceptibility for early treatment initiation and probable MDR-TB. MODS can rapidly confirm probable MDR-TB detected by GeneXpert-MTB/RIF, manual-MGIT can provide early results for susceptibility to other antibiotics, with affordable costs, with LJ-DST confirming discordant DSTs.

Performance of microscopic observation drug susceptibility for the rapid diagnosis of tuberculosis and detection of drug resistance in Bamako, Mali

ObjectivesIn Mali early detection and treatment of multidrug-resistant tuberculosis (MDR-TB) are still challenging due to the cost, time and/or complexity associated with regular tests. Microscopic Observation Drug Susceptibility (MODS) is a low-cost assay validated by WHO in 2010. It is a liquid-culture-based assay to detect the ‘cording’ characteristic of Mycobacterium tuberculosis complex and to assess susceptibility to both isoniazid and rifampicin defining multidrug-resistant tuberculosis (MDR-TB). In this study we aimed to evaluate the performance of MODS as diagnostic tool compared with a validated method—Mycobacteria Growth Indicator Tube/Antimicrobial Susceptibility Testing/Streptomycin, Isoniazid, Rifampicin and Ethambutol (MGIT/AST/SIRE). Methods and ResultsBetween January 2010 and October 2015 we included 98 patients with suspected TB in an observational cohort study. The sensitivity and specificity of MODS assay for detecting TB were respectively 94.12% and 85.71% compared with the reference MGIT/7H11 culture, with a Cohen κ coefficient of 0.78 (95% CI 0.517–1.043). The median time to culture positivity for MODS assay and MGIT (plus interquartile range, IQR) was respectively 8 days (IQR 5–11) and 6 days (IQR 5–6). In detecting patients with MDR-TB, the sensitivity and specificity of MODS assay were respectively 100% and 95.92%. The positive predictive value and negative predictive value were, respectively, 66.7% and 100%. The median turnaround times for obtaining MDR-TB results using MODS assay and MGIT/AST/SIRE was respectively 9 days and 35 days. Hence, the MODS assay rapidly identifies MDR-TB in Mali compared with the MGIT/AST/SIRE. ConclusionAs an easy, simple, fast and affordable method, the MODS assay could significantly improve the management of TB.

Impact of Infection Control Measures to Control an Outbreak of Multidrug-Resistant Tuberculosis in a Human Immunodeficiency Virus Ward, Peru.

Multidrug-resistant tuberculosis (MDRTB) rates in a human immunodeficiency virus (HIV) care facility increased by the year 2000—56% of TB cases, eight times the national MDRTB rate. We reported the effect of tuberculosis infection control measures that were introduced in 2001 and that consisted of 1) building a respiratory isolation ward with mechanical ventilation, 2) triage segregation of patients, 3) relocation of waiting room to outdoors, 4) rapid sputum smear microscopy, and 5) culture/drug–susceptibility testing with the microscopic-observation drug-susceptibility assay. Records pertaining to patients attending the study site between 1997 and 2004 were reviewed. Six hundred and fifty five HIV/TB–coinfected patients (mean age 33 years, 79% male) who attended the service during the study period were included. After the intervention, MDRTB rates declined to 20% of TB cases by the year 2004 (P = 0.01). Extremely limited access to antiretroviral therapy and specific MDRTB therapy did not change during this period, and concurrently, national MDRTB prevalence increased, implying that the infection control measures caused the fall in MDRTB rates. The infection control measures were estimated to have cost US$91,031 while preventing 97 MDRTB cases, potentially saving US$1,430,026. Thus, this intervention significantly reduced MDRTB within an HIV care facility in this resource-constrained setting and should be cost-effective.