Detection Of Multidrug-resistant Tuberculosis Research Articles

Early detection of multi-drug resistance and common mutations in Mycobacterium tuberculosis isolates from Delhi using GenoType MTBDRplus assay

Purpose: There is scarcity of prevalence data of multi-drug–resistant tuberculosis (MDR-TB) data and common mutations responsible in North India. This study aimed to detect MDR-TB among MDR-TB suspects from Delhi and mutation patterns using GenoType MTBDRplus assay. Materials and Methods: All MDR suspects in five districts of New Delhi were referred to the laboratory from 1st October 2011 to 31st December 2012 as per criterion defined by Programmatic Management of Drug Resistant Tuberculosis (PMDT). GenoType MTBDRplus assay was performed on 2182 samples or cultures and mutations in the rpoB gene for rifampicin (RIF) and katG and inhA genes for isoniazid (INH) were analyzed. Results: A total of 366 (16.8%) MDR-TB cases were diagnosed. MDR rate was found to be 32%, 16.6% and 10.2% during criterion A, B and C respectively. The most common mutation detected for RIF was S531L (59.0%) and for INH was S315T1 (88.3%). Mutations S531L and S315T1 occurred significantly higher in MDR strains as compared to RIF mono-resistant and INH mono-resistant strains, respectively. Average laboratory turn-around time (TAT) for dispatch of result to districts for test conducted on samples was 4.4 days. Conclusion: GenoType MTBDRplus is a useful assay for rapid detection of MDR-TB. The common mutations for RIF and INH were similar to those seen in other regions. However, mutations determining MDR strains and mono-resistant strains differed significantly for both RIF and INH.

Rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis isolates using high resolution melting curve analysis

Aims and objectivesEarly detection of multidrug-resistant tuberculosis (MDR-TB) is essential to prevent its transmission in the community and to initiate an effective anti-TB treatment regimen. The objective of this study is to evaluate molecular technique high resolution melting curve (HRM) assay to rapidly detect resistance-conferring mutations in rpoB and katG genes. MethodsHRM analysis was used to screen 95 Mycobacterium tuberculosis (MTB) clinical isolates including 20 rifampin resistant (RIF-R), 21 isoniazid resistant (INH-R), and 54 fully susceptible (S) isolates determined by proportion method of drug susceptibility testing. 19 MTB isolates with known drug susceptibility genotypes were used as control strains for initial validation of the assay. Polymerase chain reaction (PCR) amplicons from rpoB and katG genes were sequenced to investigate the frequency and type of mutations and to confirm HRM results. ResultsAll RIF-S and INH-S isolates generated wild-type HRM curves and were accurately identified as susceptible by this method. Similarly 19 out of 20 RIF-R and 18 out of 21 INH-R isolates correctly exhibited mutant-type HRM curves. These results were similar to those obtained by direct sequencing. However, 1 RIF-R and 3 INH-R isolates were falsely identified as susceptible by HRM assay. These strains were confirmed as having no mutation in their target regions by sequencing. The main mutations involved in RIF and INH resistance were found at codons rpoB531 (60% of RIF-R isolates) and katG315 (85.7% of INH-R isolates), respectively. ConclusionsHRM was found to be a reliable, rapid and low-cost method to characterize drug susceptibility of clinical TB isolates in resource-limited settings.

Diagnosis and Treatment of Multidrug-Resistant Tuberculosis

Despite global efforts to control tuberculosis (TB), multidrug-resistant TB (MDR-TB) is still a serious problem worldwide. The diagnosis of MDR-TB is based on mycobacterial culture followed by drug susceptibility testing, with results available in weeks to months. This requirement calls for rapid direct tests, especially genotypic tests, in which specimens are amplified directly for the detection of MDR-TB. The treatment of MDR-TB is challenging because of the high toxicity of second-line drugs and the longer treatment duration required compared to drug-susceptible TB. The selection of drugs in MDR-TB is based on the treatment history, drug susceptibility results, and TB drug resistance patterns in each region. Recent World Health Organization guidelines recommend the use of at least four second-line drugs (i.e., a newer fluoroquinolone, an injectable agent, prothionamide, and cycloserine or para-aminosalicylic acid) in addition to pyrazinamide. Kanamycin is the initial choice of an injectable drug, and newer fluoroquinolones include levofloxacin and moxifloxacin. For extensively drug-resistant TB, group 5 drugs such as linezolid and clofazimine need to be included. New drugs such as delamanid and bedaquiline have recently been approved for treating MDR-TB and other agents with novel mechanisms of action that can be given for shorter durations (6-12 months) for MDR-TB are under investigation. (Korean J Med 2015;88:509-517)

Isolation of Mycobacterium tuberculosis from sputum of tribal, non-tribal pulmonary tuberculosis patients of Andaman & Nicobar islands by conventional culture method and assessment of first line anti-tuberculosis drug susceptibility patterns

BackgroundDrug resistance surveys have not yet conducted in these Islands and as such no data exists on drug resistance currently. Aimsthe present study was initiated with the objective of isolation and assessment of Drug resistance patterns of Mycobacterium tuberculosis isolates from sputum specimens collected from different categories of Tribal, Non-Tribal pulmonary tuberculosis patients treated under DOTS and Non-DOTS program by conventional culture and Proportion sensitivity (PST) method to detect patients with Multidrug resistant strains. MethodsThe investigation was hospital based laboratory surveillance study carried out for a period of 3 years at the selected hospitals of Andaman district (TB ward GB Pant Hospital at Port Blair, CHC Bamboflat at Port Blair and CHC Rangat at Rangat) and Nicobar district (CHC Nancowry at Nancowry groups of Islands), among the new cases and re-treatment cases of tuberculosis patient under DOTS program and Non-DOTS patients attended selected hospitals of Andaman & Nicobar districts chosen for the study. Results83 culture positive isolates obtained (74 identified as M. tuberculosis) from the sputum specimen of 162 cases of tuberculosis patient by conventional culture method. 60 M. tuberculosis isolates subjected to drug susceptibility test using PST method, 7 patients (11.67%) found to be Multidrug resistant tuberculosis (MDR-TB), resistant patterns were S + H + R + E = 1(Cat II-DOTS),H + r = 3(Cat-I DOTS = 1, Cat II-DOTS-1,Non-DOTS = 1), Rifampicin resistant alone = 2 (Non-DOTS = 1, Cat II-DOTS = 1) and R + E = 1(Cat I-DOTS). ConclusionsLaboratory finding suggested that nine MDR-TB strains detected in DOTS and Non-DOTS among 60 M. tuberculosis isolates were selected for drug susceptibility testing but two isolates detected as MDR-TB from patients was already on Second line drugs treatment were not included in the MDR-TB detection criteria. Hence 7 patients (11.67%) declared to be Multidrug resistant tuberculosis (MDR-TB). 2 MDR-TB strains with resistant patterns H + r = 1(Cat II-DOTS), Rifampicin resistant alone = 1(Non-DOTS) detected from 12 isolates of Tribal patients from Nicobar district and 5 MDR-TB strains with resistant patterns S + H + R + E = 1 (Cat II-DOTS), H + r = 2(Cat I-DOTS = 1,Non-DOTS = 1), Rifampicin resistant alone = 1 (Cat II-DOTS) and R + E = 1(Cat I-DOTS) detected from 48 isolates of Non-Tribal patients from Andaman district. To assess the MDR-TB burden in the islands, systematic drug resistant surveillance study needs to be conducted.

Evaluation of sensitivity and specificity of GenoType MTBDRplus line probe assay in rapid diagnosis of tuberculous pleural effusion

BackgroundPakistan has an annual tuberculosis (TB) incidence rate of 233 per 100,000 population and ranks sixth among the high TB burden countries worldwide. Tuberculous pleural effusion (TPE) occurs in up to 25% of TB patients. Owing to the pauci-bacillary nature of the pleural fluid, the diagnosis of TPE is a challenge. ObjectiveThe aim of the present study is to evaluate the performance of MTBDRplus line probe assay in terms of its sensitivity and specificity for the rapid TB diagnosis in TPE and frequency of multidrug-resistant TB (MDR-TB) in TPE through the detection of rifampicin (Rif) and isoniazid (INH) resistance. MethodsThis study is an ongoing prospective cross-sectional study being performed at Aga Khan University Hospital. Patients aged 18years or more with suspected TPE were asked to participate. In addition to a preformed questionnaire which gathered information regarding clinical history and previous laboratory investigations, already tapped pleural effusion samples are tested for pleural fluid composition, TB smear and culture, MTBDRplus line probe assay and ADA analysis. ResultsSo far, 22 patients with a mean age of 49.81±21.75 (range: 18–86; IQR: 29.25, 68.00) years have been enrolled. Almost two-thirds are males (n=15, 68.2%). The majority of the patient population belongs to Karachi (n=18, 81.8%), followed by Quetta (n=2, 9.1%), Faisalabad (n=1, 4.5%) and Sukkur (n=1, 4.5%). Half of the patients had a history of TB contact (n=11, 50.0%), whereas only two (9.1%) had a past history of TB. Fifty percent (n=11) had a right-sided pleural effusion. Effusion size was moderate in 36.4% (n=8), mild in 27.3% (n=6) and massive in 22.5% (n=5). Pleural fluid MTBDRplus positively detected TB in 9.1% (n=2) of cases. INH resistance was detected in one of these two cases. Pleural fluid culture was positive in two cases. Pleural fluid AFB smear was negative in all cases. ConclusionGiven the study is still under way, it is hoped that the results of this study may investigate the relevance, sensitivity and specificity of MTBDRplus line probe assay as a tool in the rapid detection of MTB in TPE and the early detection of MDR-TB through the detection of Rif and INH resistance. It may also provide an estimate of prevalence of drug resistant strains in TPE, which would be useful for guiding empirical anti-tuberculous therapy in TPE.

Molecular characterization of multi drug-resistant tuberculosis isolated from Baghdad, Iraq

BackgroundTuberculosis (TB) remains a major cause of mortality and morbidity all over the world. Multi drug-resistant tuberculosis (MDR-TB) is an important cause for mortality among TB patients, and management of MDR-TB remains a major challenge for national TB programs. Early detection of MDR-TB provides better treatment outcomes and reduces the transmission of MDR. Nucleic acid amplification test (NAAT) like Line Probe Assays have been recently endorsed for use in low-income settings and can be used to screen smear-positive sputum specimens for diagnosing of resistance to rifampicin and isoniazid in 1–2days. ObjectiveOverall objective of the study was to evaluate performances of Line Probe Assay GenoType® MTBDRplus (Hain Life science, GmbH, Nehren, Germany) to move forward its introduction into routine diagnostics. Secondary objective was to determine the type of mutations in rpoB, katG and inhA genes from culture specimens isolated from Iraqi selected MDR-TB patients. Materials and methodsThe DNA was extracted by CTAB method from 51 clinical isolates which were previously characterized as MDR strains in reference laboratory/center of Chest and Respiratory Diseases obtained from patients living in Baghdad/Iraq (2010–2011). GenoType® MTBDRplus ver. 2.0 (HainLifescience, GmbH, Nehren, Germany) was performed at the Supra-national TB Reference Laboratory in Milan (Italy) as well as interpretation of mutations involved in drug resistance to rifampin and Isoniazide according to manufacturer’s instructions. Results and conclusionLine Probe Assays were performed on 51 MDR of Mycobacterium tuberculosis strains; 6 strains were excluded from the analysis due to un-interpretable results; 5 strains were identified as sensitive to both rifampicin (RIF) and isoniazid (INH) by MTBDR plus assay. The most common genetic mutation conferring RIF resistance was S531L of rpoB gene which was detected in 33 (82.5%) resistant strains. Other mutations in this gene were D516Vand H526Y which were detected in a single strain (2.5%) for each. Also, there were 5 (12.5%) RIF-resistant strains with mutations that could not be identified specifically in this assay.Isoniazid (INH) resistance due to katG mutation S315T1 was found in 17 (42.5%) strains of INH-resistant M. tuberculosis. The second most common site of mutation was in the upstream promoter sequence of inhA, which was found in 15 (37.5%) strains, 14 (35%) strains of which carried a C→T transition at the −15 position, while single strains (2.5%) carried a T→A mutation at the −8 position. The result showed 8 (20%) strains missing a band on the wild-type region of these strains, but no hybridization with mutation probes on this gene suggested resistance due to mutations other than those included in this assay.Regardless the type of mutations detected, GenoType® MTBDR plus ver. 2.0 showed an overall accuracy of 95.5% (C.I. 95% 89.9–98.1) in detecting MDR strains. Despite the relatively low number of MDR strains tested, they show very common mutations patterns being the majority mutated at the codon 531 and 315 of rpoB and katG gene. In such setting, GenoType® MTBDR plus ver. 2.0 could be a great tool to rapidly screen for MDR-TB and has the potential to substantially reduce the turnaround time of drug sensitivity test (DST) results.

Implementación de un sistema de telediagnóstico de tuberculosis y determinación de multidrogorresistencia basada en el método MODS en Trujillo, Perú

To implement a system for remote diagnosis of tuberculosis and multidrug resistance (MDR) using the Microscopic-Observation Drug Susceptibility Assay (MODS) method in the Mycobacteria Laboratory, Trujillo Center of Excellence in Tuberculosis (CENEX-Trujillo). The system included a variant of an algorithm for recognition of Mycobacterium tuberculosis recently reported from digital images of MODS cultures of sputum samples. The recognition algorithm was optimized using a retraining statistical model based on digital images of MODS cultures from CENEX-Trujillo. Images of 50 positive MODS cultures of patients with suspected multidrug-resistant tuberculosis were obtained between January and October 2012 in the CENEX-Trujillo. The sensitivity and specificity to recognize strings of tuberculosis were 92.04% and 94.93% respectively using objects. The sensitivity and specificity to determine a positive tuberculosis field were 95.4% and 98.07% respectively using pictures. The results demonstrated the feasibility of the implementation of telediagnostics in remote locations, which may contribute to the early detection of multidrug-resistant tuberculosis by MODS method.

Line Probe Assay as a Rapid Tool for Detection of MDRTB

Aims: To evaluate genotypic Line Probe Assay (LPA) for rapid detection of Multidrug Resistant Tuberculosis (MDRTB) directly from sputum samples in comparison with Drug Susceptibility Testing (DST) on phenotypic MBBacT liquid media. Study Design: Data analysis from 86 Mycobacterium tuberculosis (MTB) strains was done using SPSS version 17. Place and Duration of Study: Department of Microbiology, JSS Medical College, Mysore, Karnataka, between January 2011 to January 2012. Methodology: MDRTB rate detected by LPA assay from 92 samples by noting the mutations in hot spot region of rpoB gene, katG and inhA regulatory region and compared with DST on MBBacT liquid media. Research Article Annual Research & Review in Biology, 4(1): 246-257, 2014 247 Results: Out of 86 MTB isolates, resistant rates for Rifampicin (RIF) and Isoniazid (INH) were 41.8%, 39.53% by LPA and 45.34%, 55.81% by MBBacT. LPA assay showed sensitivity and specificity as 92.35%, 100% for RIF resistance detection and 70.83%, 100% for INH resistance detection, 94.74%, 100% for MDRTB detection compared to conventional DST results. Conclusion: This study showed that LPA has high detection rate for RIF resistance. However to improve the detection of INH resistance in MTB strains additional probes are to be included in LPA. LPA has good sensitivity and specificity for MDRTB detection with turnaround time of less than 48 hours.

A Pilot Study on the Detection of Multidrug Resistant Tuberculosis in Hospital Based Population of Chennai, India

Background: Community-acquired pneumonia (CAP) is associated with high mortality. Drug resistance is common in countries where the alternative treatments are limited and available drugs are misused. In resource limited countries like Ethiopia; it is wise to determine antimicrobial susceptibility pattern of common bacterial pathogens of CAP. The objective of our study was to determine antimicrobial susceptibility pattern of common bacterial pathogens of CAP among adult patients visiting Arba Minch Hospital. Methods: A cross sectional study conducted at Arba Minch Hospital, Southern Ethiopia from February to May 2013. Sputum specimens were collected; microbiological investigations and antimicrobial susceptibility testing were performed using standard procedures. Data was processed and analyzed with SPSS version16.0. Results: Out of 170 cases, only 73 (42.9%) were culture positive. Majority of tested bacterial isolates (>86%) were sensitive to Ceftriaxone and Ciprofloxacin. Most S. pneumoniae isolates (60%) were resistant to Oxacillin. Most of S. aureus and gram negative bacterial isolates were resistance to Tetracycline (100%), Penicillin (83.3%), Ampicillin (50-100%), Doxycycline (50-100%), and Trimethoprim-sulfamethoxazole (83.3-100%). Multidrug resistance (MDR) was observed to most (60.3%) bacterial isolates. Conclusion: Antimicrobial resistance including MDR was observed to a number of commonly used antibiotics, such as trimethoprim- sulfamethoxazole, penicillin groups and doxycycline. Hence, periodic monitoring of drug resistant pattern is essential for better management of CAP.

Multidrug-resistant Tuberculosis Patients in Bhutan, August 2011 to July 2012

This study aimed to describe the factors associated with multidrug-resistant tuberculosis (MDR-TB) in Bhutan. The study covered all MDR-TB patients admitted to Gidhakom Hospital, Thimphu from August 2011 to July 2012. Data were collected from MDR-TB registers, laboratory registers and inpatient records as well as from interview with patients about demographic characteristics, history of previous anti-TB treatment, compliance and contact history. There were total 19 MDR-TB patients. Majority of the patients were males (63%). Median age was 25 years for males and 30 years for females. Of the 19 cases, 47% and 37% had contacted with TB case and MDR-TB case in the same house respectively. About 74% reported previous history of anti-TB treatment. Among those with previous treatment, 79% did not comply with the directly observed treatment short-course (DOTS). It was found that 20% of new cases and 50% of previously treated persons were resistant to four first-line drugs. In conclusion, previous anti-TB treatment, non-compliance to DOTS and contact with MDR-TB were observed in majority of the cases. We highlighted the importance of early detection of MDR-TB, providing health education on prevention of disease transmission and strengthening DOTS policy. Investigation on household contacts should be given priority to identify and control the spread of TB.

A simple, rapid and economic method for detecting multidrug-resistant tuberculosis

ObjectiveTo evaluate multiplex allele specific polymerase chain reaction as a rapid molecular tool for detecting multidrug-resistant tuberculosis. MethodsBased on drug susceptibility testing, 103 isolates were multidrug-resistant tuberculosis and 45 isolates were sensitive to isonicotinylhydrazine and rifampin. Primers were designed to target five mutations hotspots that confer resistance to the first-line drugs isoniazid and rifampin, and multiplex allele specific polymerase chain reaction was performed. Whole-genome sequencing confirmed drug resistance mutations identified by multiplex allele specific polymerase chain reaction. ResultsDNA sequencing revealed that 68.9% of multidrug-resistant strains have point mutations at codon 315 of the katG gene, 19.8% within the mabA-inhA promoter, and 98.0% at three hotspots within rpoB. Multiplex allele specific polymerase chain reaction detected each of these five mutations, yielding 82.3% sensitivity and 100% specificity for isoniazid resistance, and 97.9% sensitivity and 100% specificity for rifampin resistance as compared to drug susceptibility testing. ConclusionsThe results show that multiplex allele specific polymerase chain reaction is an inexpensive and practical method for rapid detection of multidrug-resistant tuberculosis in developing countries.

Comparative Evaluation of GenoType MTBDRplus Line Probe Assay with Solid Culture Method in Early Diagnosis of Multidrug Resistant Tuberculosis (MDR-TB) at a Tertiary Care Centre in India

BackgroundThe objectives of the study were to compare the performance of line probe assay (GenoType MTBDRplus) with solid culture method for an early diagnosis of multidrug resistant tuberculosis (MDR-TB), and to study the mutation patterns associated with rpoB, katG and inhA genes at a tertiary care centre in north India.MethodsIn this cross-sectional study, 269 previously treated sputum-smear acid-fast bacilli (AFB) positive MDR-TB suspects were enrolled from January to September 2012 at the All India Institute of Medical Sciences hospital, New Delhi. Line probe assay (LPA) was performed directly on the sputum specimens and the results were compared with that of conventional drug susceptibility testing (DST) on solid media [Lowenstein Jensen (LJ) method].ResultsDST results by LPA and LJ methods were compared in 242 MDR-TB suspects. The LPA detected rifampicin (RIF) resistance in 70 of 71 cases, isoniazid (INH) resistance in 86 of 93 cases, and MDR-TB in 66 of 68 cases as compared to the conventional method. Overall (rifampicin, isoniazid and MDR-TB) concordance of the LPA with the conventional DST was 96%. Sensitivity and specificity were 98% and 99% respectively for detection of RIF resistance; 92% and 99% respectively for detection of INH resistance; 97% and 100% respectively for detection of MDR-TB. Frequencies of katG gene, inhA gene and combined katG and inhA gene mutations conferring all INH resistance were 72/87 (83%), 10/87 (11%) and 5/87 (6%) respectively. The turnaround time of the LPA test was 48 hours.ConclusionThe LPA test provides an early diagnosis of monoresistance to isoniazid and rifampicin and is highly sensitive and specific for an early diagnosis of MDR-TB. Based on these findings, it is concluded that the LPA test can be useful in early diagnosis of drug resistant TB in high TB burden countries.

Slide drug susceptibility test for the detection of multidrug-resistant tuberculosis in Bangladesh

The present study attempted to comparatively assess and establish a suitable detection method of multidrug-resistant tuberculosis (MDR-TB) from previously treated TB cases in Bangladesh. Of 130 Zeihl-Neelsen smear-positive fresh sputum specimens, 112 samples were found to contain viable bacilli as visualized under the light-emitting diode fluorescence microscope after fluorescein di-acetate staining, and 109 positive cases were detected through Löwenstein-Jensen culture. The samples were further tested to survey the drug resistance both by slide drug susceptibility test (DST) and by conventional DST: 94 MDR-TB cases were detected within 10days through the slide DST, whereas 82 cases were observed through the conventional DST, requiring about 3months. Because the rapidity, sensitivity and accuracy of the slide DST method were found to be comparatively satisfactory when compared to the conventional DST method; we recommend the slide DST method as the standard diagnostic tool in perspective of Bangladesh for the detection of MDR-TB.

Screening and Rapid Molecular Diagnosis of Tuberculosis in Prisons in Russia and Eastern Europe: A Cost-Effectiveness Analysis

Prisons of the former Soviet Union (FSU) have high rates of multidrug-resistant tuberculosis (MDR-TB) and are thought to drive general population tuberculosis (TB) epidemics. Effective prison case detection, though employing more expensive technologies, may reduce long-term treatment costs and slow MDR-TB transmission. We developed a dynamic transmission model of TB and drug resistance matched to the epidemiology and costs in FSU prisons. We evaluated eight strategies for TB screening and diagnosis involving, alone or in combination, self-referral, symptom screening, mass miniature radiography (MMR), and sputum PCR with probes for rifampin resistance (Xpert MTB/RIF). Over a 10-y horizon, we projected costs, quality-adjusted life years (QALYs), and TB and MDR-TB prevalence. Using sputum PCR as an annual primary screening tool among the general prison population most effectively reduced overall TB prevalence (from 2.78% to 2.31%) and MDR-TB prevalence (from 0.74% to 0.63%), and cost US$543/QALY for additional QALYs gained compared to MMR screening with sputum PCR reserved for rapid detection of MDR-TB. Adding sputum PCR to the currently used strategy of annual MMR screening was cost-saving over 10 y compared to MMR screening alone, but produced only a modest reduction in MDR-TB prevalence (from 0.74% to 0.69%) and had minimal effect on overall TB prevalence (from 2.78% to 2.74%). Strategies based on symptom screening alone were less effective and more expensive than MMR-based strategies. Study limitations included scarce primary TB time-series data in FSU prisons and uncertainties regarding screening test characteristics. In prisons of the FSU, annual screening of the general inmate population with sputum PCR most effectively reduces TB and MDR-TB prevalence, doing so cost-effectively. If this approach is not feasible, the current strategy of annual MMR is both more effective and less expensive than strategies using self-referral or symptom screening alone, and the addition of sputum PCR for rapid MDR-TB detection may be cost-saving over time.

Comparative evaluation of the microplate nitrate reductase assay and the rezasurin microtitre assay for the rapid detection of multidrug resistant Mycobacterium tuberculosis clinical isolates

The microplate nitrate reductase assay (MNRA) and the rezasurin microtitre assay (REMA) were used for the susceptibility testing of 73 clinical isolates and the results were compared with those that were obtained using the Bactec 460 TB and Bactec MGIT 960 systems. The REMA and the MNRA were performed in 96-well plates. For the REMA, the concentrations of isoniazid (INH) and rifampicin (RIF) ranged from 1.0-0.01 µg/mL and 2.0-0.03 µg/mL, respectively. For the MNRA, the INH concentration was between 1.0-0.03 µg/mL and the RIF concentration was between 2.0-0.06 µg/mL. For the MNRA, the sensitivity, specificity, positive predictive value, negative predictive value and INH/RIF agreement were 100/95.6, 97.6/100, 96.8/100, 100/98 and 98.6/98.6, respectively, and for the REMA, they were 100/91.3, 90.4/100, 88.5/100, 100/96.1 and 94.5/97.2, respectively. Our data suggest that these two rapid, low-cost methods may be inexpensive, alternative assays for the rapid detection of multidrug resistant tuberculosis in low-income countries.

Use of multiplex allele-specific polymerase chain reaction (MAS-PCR) to detect multidrug-resistant tuberculosis in Panama.

The frequency of individual genetic mutations conferring drug resistance (DR) to Mycobacterium tuberculosis has not been studied previously in Central America, the place of origin of many immigrants to the United States. The current gold standard for detecting multidrug-resistant tuberculosis (MDR-TB) is phenotypic drug susceptibility testing (DST), which is resource-intensive and slow, leading to increased MDR-TB transmission in the community. We evaluated multiplex allele-specific polymerase chain reaction (MAS-PCR) as a rapid molecular tool to detect MDR-TB in Panama. Based on DST, 67 MDR-TB and 31 drug-sensitive clinical isolates were identified and cultured from an archived collection. Primers were designed to target five mutation hotspots that confer resistance to the first-line drugs isoniazid and rifampin, and MAS-PCR was performed. Whole-genome sequencing confirmed DR mutations identified by MAS-PCR, and provided frequencies of genetic mutations. DNA sequencing revealed 70.1% of MDR strains to have point mutations at codon 315 of the katG gene, 19.4% within mabA-inhA promoter, and 98.5% at three hotspots within rpoB. MAS-PCR detected each of these mutations, yielding 82.8% sensitivity and 100% specificity for isoniazid resistance, and 98.4% sensitivity and 100% specificity for rifampin resistance relative to DST. The frequency of individual DR mutations among MDR strains in Panama parallels that of other TB-endemic countries. The performance of MAS-PCR suggests that it may be a relatively inexpensive and technically feasible method for rapid detection of MDR-TB in developing countries.

Complete atrioventricular block due to overdose of pregabalin

Pregabalin, a synthetic derivate of the inhibitory neurotransmitter γ-aminobutyric acid, shows antiepileptic, analgesic, anticonvulsant, anxiolytic, and sleep-modulating activities. The major advantage of pregabalin is its relative reliability, easy use, high tolerance, and lack of negative interaction with other drugs. A 65-year-old woman with medical histories of diabetes mellitus, lumbar spondylosis, diabetic nephropathy, chronic renal failure, and anemia of chronic disease was admitted with the complaint of dizziness and syncope. She had been taking pregabalin 300 mg daily for 8 months. Electrocardiogram revealed complete atrioventricular (AV) block and right bundle-brunch block with a heart rate of 39 per minute. Her creatinine was 1.8 mg/dL, and creatinine clearance was 50 mL/min. Pregabalin treatment was discontinued. Four days later, the complete AV block resolved spontaneously to Mobitz type II block and to sinus rhythm with right bundle-brunch block on the seventh day. To our knowledge, this is the first case of complete AV block associated with pregabalin. We believe that AV block occurred as a result of pregabalin's effect on L-type Ca++ channels in the heart. Pregabalin's different effects on electrocardiogram and on the heart in different individuals may have an association with the patterns of distribution of the L-type calcium channels in myocardium.

Prospective evaluation of simply modified MODS assay: an effective tool for TB diagnosis and detection of MDR-TB

Background and settingThailand is one of the highest tuberculosis (TB)-burdened countries. Chiang Rai, the northernmost province of Thailand has high tuberculosis and human immunodeficiency virus (HIV) prevalence and the laboratory workload for TB culture and drug susceptibility testing is increasing.ObjectivesTo evaluate the simply modified microscopic-observation drug-susceptibility assay (MODS) in the setting of a developing country.MethodsIn this cross-sectional diagnostic study, a total of 202 sputum samples of clinically diagnosed TB patients were used to test the performance of MODS assay in reference to gold standard BACTEC™ MGIT™ 960 liquid culture system and Ogawa solid culture. Sputum samples were collected from clinically diagnosed TB patients. Culture growth rate and time to culture positivity were compared among three methods. Performance of modified MODS assay was evaluated for detection of mycobacterium drug resistance in reference to MGIT antimicrobial susceptibility test (AST).ResultMedian time to culture positivity by MODS, solid, and liquid culture were 12, 30, and 6 days respectively. Compared to the drug susceptibility test (DST) result of reference liquid culture, the sensitivity and specificity of MODS for detection of multidrug-resistant tuberculosis (MDR-TB) was 85.7% and 97.5% respectively. MODS assay has a positive predicative value of 80% and negative predictive value of 96.5% for isoniazid resistance, 70% and 100% for rifampicin resistance, and 66.7% and 99.1% for MDR-TB.ConclusionMODS is a highly effective screening test for detection of MDR-TB.

Evaluation of the GenoType® MTBDRplus assay and identification of a rare mutation for improving MDR-TB detection

To assess the new GenoType® MTBDRplus assay for the rapid detection of multidrug-resistant tuberculosis (MDR-TB) in comparison with DNA sequencing to identify drug resistance mutation profiles in China. Using MTBDRplus, drug susceptibility testing (DST) and DNA sequencing, 237 Mycobacterium tuberculosis strains were tested. The sensitivity of MTBDRplus was 75.0% (126/168) for isoniazid (INH) resistant strains, and 93.5% (157/168) for rifampicin (RMP) resistant strains. It correlated well with sequencing, with 94.9% and 99.6% agreement for each strain category and 100% specificity for all categories. The two most common rpoB mutations were S531L (53.6%, 90/168) and D516G (17.3%, 29/168) in RMP-resistant strains. INH resistance was dominated by the katG 315 locus (S to T, N, R, I) mutation (73.7%, 124/168), and a rare katG mutation, S315N (6.5%, 11/168), not covered by MTBDRplus was identified. The mutation combination inhA-15/inhA-8 and katG315 (34 strains) was characteristically displayed in MDR-TB strains (23.5%), but not in INH-monoresistant strains. Although Genotype MTBDRplus is a rapid and reliable molecular test for detecting MDR-TB, a significant proportion of strains in China contain a rare katG S315N mutation that would be missed by the assay. Further improvements may be achieved by incorporating this mutation into the assay to increase sensitivity in detecting INH resistance in China.

Multidrug-resistant tuberculosis in Port-au-Prince, Haiti

To determine the prevalence of multidrug-resistant tuberculosis (MDR-TB) among patients with new smear-positive pulmonary TB in Port-au-Prince, Haiti. Sputum samples were cultured from 1 006 patients newly diagnosed with TB in 2008. The core region of the rpoB gene that is associated with resistance to rifampin was sequenced. All isolates with rpoB mutations were sent to the New York State reference laboratory for conventional drug susceptibility testing (DST). All isolates were also tested with the GenoType MTBDRplus line-probe assay. Mycobacterium tuberculosis was isolated from 906 patients. Twenty-six (2.9%) of the isolates had missense mutations or deletions in rpoB and were resistant to rifampin by DST. All 26 were also resistant to isoniazid and classified as MDR-TB. Forty-six control isolates without rpoB mutations were found to be rifampin sensitive by DST. The GenoType MTBDRplus line-probe assay correctly identified 26 MDR-TB strains. It misclassified one pansusceptible isolate as rifampin resistant. This study shows an MDR-TB prevalence of 2.9% in newly diagnosed TB patients in Haiti and suggests that rpoB sequencing and hybridization assays are good screening tools for early detection of MDR-TB.