Discordancia entre pruebas de susceptibilidad genotípicas y fenotípicas para tuberculosis en Cali, Colombia: un reto en la práctica clínica

Introducción: La detección de patrones de resistencia de Mycobacterium tuberculosis se basa en pruebas de susceptibilidad fenotípicas y genotípicas. Los resultados discordantes entre ellas son un desafío clínico para el manejo de pacientes con tuberculosis resistente a fármacos. Objetivo: Evaluar la concordancia entre pruebas fenotípicas y moleculares en pacientes con tuberculosis resistente a fármacos atendidos en una institución de Cali, Colombia. Materiales y Métodos: Se realizó un estudio transversal en el que se obtuvo el perfil de sensibilidad fenotípico de cultivos de micobacterias y la susceptibilidad genotípica con las pruebas moleculares Xpert-MTB/ RIF® o Genotype-MDRTBplus ®. Se evaluó el porcentaje de resistencia y porcentaje de acuerdo entre los resultados de las pruebas fenotípicas y genotípicas. Se estimó un coeficiente de kappa de Cohen (κ) para cada tipo de resistencia según la prueba utilizada. Resultados: Se incluyeron 30 casos con resultados de pruebas genotípicas y fenotípicas. Las pruebas fenotípicas detectaron resistencia a fármacos de primera línea en 29/30 casos, mientras que las moleculares detectaron la resistencia en todos los casos evaluados. El porcentaje de resistencia a rifampicina detectado entre la prueba fenotípica y Genotype-MDRTBplus ® &e 61,5% (acuerdo global 41,1%, κ = 0,40, p = 0,96), mientras que el porcentaje de resistencia detectado con Xpert-MTB/RIF® fue 100% (acuerdo global 81,82%, κ: 0,00, p < 0,001) para este mismo medicamento. El porcentaje de resistencia a isoniacida detectado entre la prueba fenotípica y Genotype-MDRTBplus ® fue 94,4% (acuerdo global 89,47%, κ: -0,055 p = 0,59). Conclusiones: La discordancia entre los resultados de las pruebas genotípicas y fenotípicas es posible, por lo que es importante usar e interpretar ambos tipos de pruebas de manera complementaria en el diagnóstico de la resistencia a fármacos de primera línea en la infección por M. tuberculosis.

Background: The detection of Mycobacterium tuberculosis resistance patterns is based on phenotypic and genotypic susceptibility tests. The discordant results between them are a clinical challenge for the management of patients with drug-resistant tuberculosis. Aim: To evaluate the concordance between phenotypic and molecular tests in patients with drug-resistant tuberculosis treated in an institution in Cali, Colombia. Methods: A cross-sectional study was conducted. A phenotypic sensitivity profile was obtained from mycobacterial cultures. The genotypic susceptibility was obtained with Xpert-MTB/ RIF® or Genotype-MDRTBplus ®. The percentage of resistance and percentage of agreement between the results of the phenotypic and genotypic tests were evaluated. A Cohen's kappa coefficient (κ) was estimated for each type of resistance according to the test used. Results: A total of 30 cases with both genotypic and phenotypic testing were included. The phenotypic tests detected resistance to first-line drugs in 29/30 cases, while the molecular tests detected resistance in all the cases evaluated. The percentage of resistance detected between Genotype-MDRTBplus® and the phenotypic test for rifampicin was 61.5% (overall agreement 41.1%, κ = 0.40, p = 0.96), while the percentage of resistance detected with XpertMTB/RIF® was 100% (overall agreement 81.82%, κ: 0.00, p < 0.001) for this same drug. Resistance to isoniazid detected by both types of tests was 94.4% (overall agreement 89.47%, κ: -0.055 p = 0.59). Conclusions: Discordance between the results of genotypic and phenotypic tests is possible, so it is important to use and interpret both types of tests in a complementary way in the diagnosis of resistance to first-line drugs in M. tuberculosis infection.

A rare case of smear positive tuberculous arthritis

TB arthritis is a very rare extrapulmonary presentation in an immunocompetent host. It is usually the result of direct hematogenous spread from the primary focus. Our patient presented with pain and swelling of the right knee for 6 months. The blood investigations and CT chest revealed findings consistent with active tuberculosis. Synovial fluid was positive for acid-fast bacilli (AFB) which is a very rare finding. Cartridge-based nucleic acid amplification test (CBNAAT) revealed Mycobacterium tuberculosis and sensitivity to rifampicin. Establishing the diagnosis of Mycobacterium tuberculosis beyond doubt is very important, and early initiation of antitubercular treatment (ATT) is important as delay in treatment may lead to irreversible damage to the joint and restriction of joint mobility.

Drug-Resistant Mycobacterium tuberculosis Isolates from New and Previously Treated TB Patients in China, 2017-2019

Abstract INTRODUCTION: Mycobacterium tuberculosis (MTB) is a causative agent of tuberculosis (TB) that causes death worldwide. METHODS: MTB was subjected to phenotypic drug-susceptibility tests (DST), and drug-resistant genes were sequenced. RESULTS: Previously treated patients were more likely to have positive smear results and exhibit drug resistance. New patients were more likely to be mono SM-resistant and less likely to be INH- and RIF-resistant. The most common mutations were katG (S315T), rpoB (S450L), rpsL (K43R), and embB (M306V). CONCLUSIONS: The proportion of mono-SM-resistant TB among new patients was higher.

Research advances in M. Tuberculosis polyketide synthase 13 inhibitors / 药学学报

Polyketide synthase 13 (Pks13) performs a critical role in the final assembly step of mycolic acid synthesis in Mycobacterium tuberculosis. The inhibition of Pks13 can influence the biosynthesis of mycolic acid, which leads to Mycobacterium tuberculosis cell death. Researchers have discovered Pks13 inhibitors with five chemical scaffolds as antituberculosis agents. Herein, we summarize recent advances in the study of Pks13 inhibitors including the process of discovery, the mechanism of action and structure-activity relationships.

Interrelation of Ca2+ and PE_PGRS proteins during Mycobacterium tuberculosis pathogenesis

In today’s era tuberculosis is a major threat to human population. The lethality of this disease is caused by very efficientlythrived bacteria Mycobacterium tuberculosis (M. tuberculosis). Ca2? plays crucial role in maintenance of cellular homeostasis. Bacilli survival in human alveolar macrophages majorly depends on disruption in Ca2? signaling. Bacilli sustainability in phagosome lies in the interruption of phagolysosomal fusion, which is possible because of low intracellularCa2? concentration. Bacilli contain various Ca2? binding proteins which help in regulation of Ca2? signaling for its ownbenefit. For the survival of pathogen, it requires alteration in normal Ca2? concentration in healthy cell. In this review weaim to find the various Ca2? binding domains which are present in several Ca2? binding proteins of M. tuberculosis andvariety of roles played by Ca2? to survive bacilli within host cell. This manuscript emphasizes the Ca2? binding domainspresent in PE_PGRS group of gene family and their functionality in M. tuberculosis survival and pathogenesis.

Análisis de una serie de casos de tuberculosis en pacientes pediátricos atendidos en un hospital de tercer nivel / A case-series analysis of tuberculosis in pediatric patients treated in a tertiary level hospital

Resumen: Introducción: La tuberculosis (TB) continúa siendo un reto ya que las formas graves se presentan con mayor frecuencia en los menores de 5 años y el diagnóstico es complejo. El objetivo del presente trabajo fue describir las formas de presentación clínica, frecuencia, métodos de diagnóstico empleados y respuesta al tratamiento en niños con TB atendidos en un hospital de tercer nivel. Métodos: Se diseñó un estudio retrospectivo, descriptivo, de una cohorte de casos consecutivos atendidos desde enero de 2010 hasta diciembre de 2013. Se revisaron 93 expedientes clínicos de niños con diagnóstico de TB de acuerdo con la definición de la NOM-006-SSA2-2013. Se utilizó estadística descriptiva para el análisis. Resultados: El 58% de 93 niños fueron pacientes de sexo masculino con una media de edad de 7 años. El 97% contaba con antecedente de vacunación BCG; el 6% tuvo contacto con algún caso de TB. Las formas clínicas más frecuentes fueron la TB pulmonar (30.1%), ganglionar (24.7%), miliar/diseminada (16.1%), meníngea (13%) y ósea (7.5%). Los síntomas más comunes fueron fiebre y pérdida de peso (50% y 40%, respectivamente). El BAAR y el cultivo fueron positivos en el 26% y el 7% de todos los casos, respectivamente. El estudio histopatológico fue concluyente en el 90%. El tratamiento fue exitoso en el 94.6%, sin mortalidad asociada. Conclusiones: La asociación del cuadro clínico con las alteraciones en la radiografía de tórax y PPD positivo son útiles para establecer el diagnóstico presuntivo e iniciar el manejo oportuno.

Abstract: Background: Tuberculosis (TB) remains a challenge because severe forms occur most frequently in children under 5 years of age and the diagnosis is complex. The objective of this paper was to describe the clinical presentation, frequency, diagnostic methods used and response to treatment in children with TB treated at a tertiary level hospital. Methods: The study was retrospective and descriptive of a cohort of consecutive cases treated from January 2010 to December 2013. Ninety-three medical records of children diagnosed with TB according to the definition of the NOM-006-SSA2-2013 were reviewed. Descriptive statistics were used for the analysis. Results: From 93 children, 58% were male (mean age of 7 years), 97% with a history of BCG vaccination, and 6% had contact with a TB case. The most frequent clinical forms were pulmonary (30.1%), lymph node (24.7%), miliary/disseminated (16.1%), meningeal (13%), and osteal TB (7.5%). The most common symptoms were fever and weight loss (50% and 40%, respectively). BAAR and culture were positive in 26% and 7% of all cases, respectively. The histopathological study was conclusive in 90% of the cases. The treatment was successful in 94.6%, with not associated mortality. Conclusions: The association of clinical symptoms with alterations in chest radiography and positive PPD are useful in establishing the presumptive diagnosis and an early and appropriate treatment.

Identification of novel Mycobacterium tuberculosis peptides as diagnostic reagents for tuberculosis among human immunodeficiency virus infected population / 中华传染病杂志

Objective To screen and validate the major histocompatibility complex class-Ⅰ(MHC-Ⅰ) restricted tuberculosis peptides as potential diagnostic reagents in tuberculosis interferon-gamma release assay (IGRA) used among human immunodeficiency (HIV)-infected population.Methods Candidate peptides were encoded by Mycobacterium tuberculosis (TB) RD (Region of difference).Computer software was used to predict and select CD8+ T cell epitopes restricted by MHC-Ⅰ molecules with high frequency and high affinity among HIV-infected people.Then peptides containing CD8+ T cell epitope were synthesized and screened in vitro.The sensitivity and specificity of IGRA using the above mixed peptides as stimulants were compared with those of IGRA using early secretory antigen target-6 (ESAT-6,molecular weight of 6 000) and culture filtrate protein-10 (CFP-10,molecular weight of 10 000) as stimulants among HIV-infected population.Results Eight overlapping peptides,including Rv0222176-191,Rv1980c122-138,Rv1985c105-120,Rv3425141-165,Rv3873133-151,Rv3873158-166,Rv387878-86,Rv3879c673-690,were obtained finally,which were able to stimulate the production of interferon-gamma from peripheral CD8+ T cells of tuberculosis patients,but not from peripheral blood mononuclear cells (PBMC) of healthy controls.Among the 25 patients with HIV/TB co-infection,the sensitivities of IGRA using the combination peptides (CP) and that using rESAT-6/CFP-10 (CE) were low (68％ vs 48％,x2 =2.052,P=0.152).However,the sensitivity increased to 92％ by using the combination of CP and CE,which was significantly higher than that using rESAT-6/CFP-10 alone (x2 =11.523,P ＜ 0.01),and the specificity was not affected.Conclusion These RD peptides with CD8+ T cell epitopes can increase the sensitivity of IGRA in detecting HIV/TB co-infection,which may improve the detection rate of tuberculosis in HIV infected population.

Cutaneous Atypical Mycobacterial Infection in a Body Scrubber (“Ddaemirri”) / 대한피부과학회지

No abstract available.

Diseño del kit de tinción Ziehl Neelsen del Instituto nacional de Higiene "Rafael Rangel" / Design Kit Ziehl Neelsen the Instituto Nacional de Higiene "Rafael Rangel"

La tinción de Ziehl Neelsen (ZN), es una técnica de coloración de microorganismos para la identificación de patógenos, como Mycobacterium tuberculosis causante de la tuberculosis, que requiere de tres (03) soluciones: Carbol Fucsina Fenicada (Fucsina Básica), Azul de Metileno al 1% y Solución Decolorante, que se elaboran en la Sección de Reactivos y Colorantes del Instituto Nacional de Higiene "Rafael Rangel" y se emplean en el diagnóstico de tuberculosis. Esta investigación surgió con el propósito de comprobar el tiempo de caducidad y condiciones de almacenamiento de dichos productos y presentarlos en un estuche tipo kit para su distribución, en apoyo a la Red Nacional de Laboratorios de Salud Pública y comercialización con otros entes. Se realizó el ensayo de tres (3) lotes del kit de Ziehl Neelsen; con su respectiva contramuestra que fue evaluada en el análisis final. Se registraron los parámetros físicos de temperatura y humedad relativa bajo condiciones normales de almacenamiento en el laboratorio, con las muestras protegidas de la luz. Se evaluó la funcionalidad por medio de la tinción ZN observada bajo microscopio, de tres (03) muestras con ATCC 700686: M. peregrinum y ATCC 29213: S. aureus por lote; tomando en cuenta el exceso de colorante, y la definición de las coloraciones. Estas evaluaciones se realizaron durante dos (02) años encontrándose como resultado que física y funcionalmente los productos contentivos en el kit se mantenían estables, fijándose un tiempo de caducidad de dos (02) años.

Ziehl Neelsen (ZN) is a staining technique of microorganisms for the identification of pathogens as Mycobacterium tuberculosis, causative of tuberculosis, which requires three (03) solutions: Carbol Fuchsin combined with Phenol (Basic Fuchsin), Methylene Blue 1% and Bleaching solution, which are prepared in Section of Reagents and Coloring of the Instituto Nacional de Higiene "Rafael Rangel" and are used in the diagnosis of tuberculosis. This investigation was made with the purpose of checking the shelf life and storage conditions of these products and present them in a kit type container for distribution in support of the National Network of Public Health Laboratories and marketing with other entities. The analysis was performed in three (3) batches of the Ziehl Neelsen kit; with their respective counter sample that was evaluated in the final analysis. The physical parameters of temperature and relative humidity were recorded in the laboratory under normal storage conditions with samples protected from light. The functionality was evaluated through ZN staining being observed under a microscope three (03) samples with ATCC 700686: M. peregrinum and ATCC 29213: S. aureus by Batch; taking into consideration the excess dye, and the definition of the colors. These evaluations were conducted for two (02) years found as main result that physically and functionally the products in the kit were stable, and can set an expiration time of two years.

Evaluation of mycobacterium tuberculosis IgG antibody protein chip in patients with active and inactive tuberculosis / 中国生化药物杂志

Objective To explore protein microarray chip diagnostic value for patients with active and inactive tuberculosis.Methods 178 cases of active patients tuberculosis and 79 cases of inactive tuberculosis patients and 92 cases of healthy control using protein microarray chip detection.Results Tuberculosis protein chip had a diagnostic value for tuberculosis and the positive rate is 58.4%; combined the diagnostic value of three kinds of proteins is higher than the diagnostic value of a single protein;16 kD protein of inactive tuberculosis positive rate was 16.4%, better than the positive rate of 3.4% for active tuberculosis (P<0.05).Conclusion Tuberculosis protein chip has a diagnostic value for active tuberculosis and inactive tuberculosis.16 kD protein positive rate more than 38 kD protein in patients with inactive tuberculosis (P<0.05).

Genotypic Detection of rpoB and katG Gene Mutations Associated with Rifampicin and Isoniazid Resistance in Mycobacterium Tuberculosis Isolates: A Local Scenario (Kelantan)

Background: Drug resistant tuberculosis (DR-TB) remains a public health issue that is of major concern on a global scale. The characterisation of clinical isolates may provide key information regarding the underlying mechanisms of drug resistance, and helps to augment therapeutic options. This study aims to evaluate the frequency of gene mutations associated with Rifampicin (RIF) and Isoniazid (INH) resistance among nine clinical isolates. Methods: A total of nine drug resistant Mycobacterium tuberculosis clinical isolates were screened for genetic mutations in rpoB and katG using polymerase chain reaction (PCR) amplification and DNA sequencing. Genotypic analysis was performed to detect the mutations in the sequence of the target genes. Results: Our findings reveal that 80% of the isolates possess mutations at codon 119 (His119Tyr) and 135 (Arg135Trp and Ser135Leu) within the rpoB gene; and 70% possess mutations in the katG gene at codon 238 with amino acid change (Leu238Arg). Conclusion: Findings from this study provide an overview of the current situation of RIF and INH resistance in a hospital Universiti Sains Malaysia (HUSM) located in Kelantan, Malaysia, which could facilitate molecular-based detection methods of drug-resistant strains. Further information regarding the molecular mechanisms involved in resistance in RR-/MDR-TB should be addressed in the near future.

PCR interna con ADN extraído directamente de portaobjetos positivos para confirmar o excluir el diagnóstico de tuberculosis: enfoque en la bioseguridad / In-house PCR with DNA extracted directly from positive slides to confirm or exclude the diagnosis of tuberculosis: focus on biosafety

The possibility to obtain DNA from smears is a valuable alternative to remedy the lack of samples when they are totally used for bacilloscopy; this technique solves the biosafety problem related to a possible accident with the transportation of flasks containing potentially transmissible clinical samples. Hence, the purpose of this study was to utilize the insertion sequence IS6110 for amplification of DNA from a smear-positive sample for tuberculosis (TB) diagnosis. Among the 52 positive bacilloscopies, sensitivity, specificity, positive predictive value and negative predictive value were 52.3%, 100%, 100% and 89.7%, respectively whereas accuracy was 90.7%. The IS6110-based PCR for TB diagnosis developed in DNA extracted from a positive smear is a fast, simple, specific, and safe method

La posibilidad de obtener ADN a partir de frotis es una valiosa alternativa para remediar la falta de muestras cuando estas son totalmente utilizadas para la baciloscopia; esta opción soluciona, además, el problema de bioseguridad asociado a la posibilidad de accidente al transportar frascos que contienen muestras clínicas potencialmente infectivas. Por lo tanto, el propósito de este estudio fue utilizar para el diagnóstico de la tuberculosis la secuencia de inserción IS6110 para amplificación del ADN a partir de frotis que resultaron positivos por baciloscopia. Del análisis de 52 baciloscopias positivas surge que la sensibilidad, la especificidad, el valor predictivo positivo y el valor predictivo negativo de esta técnica fueron, respectivamente, del 52,3%, del 100%, del 100% y del 89,7%; y la precisión fue del 90,7%. La PCR IS6110 para el diagnóstico de tuberculosis, desarrollada con ADN extraído de frotis positivos, es un método rápido, simple, específico y seguro

Comparative Study of Real-Time PCR with Culture in the Diagnosis of Tubercular Lymphadenopathy.

One-third of the world’s population is estimated to be infected with M.tuberculosis. Timely and accurate diagnosis is pivotal in the management of the disease. Conventional tests, although accurate, are time consuming as compared to the more promising latest molecular techniques like real-time PCR which are rapid, reproducible and reliable. Objective : The aim of the study is to compare the real-time PCR with culture method in the diagnosis of tubercular lymphadenopathy. Material and Methods: Present study included 40 patients belonging to the age group 0-40 years and presenting as cervical lymphadenopathy, attending the indoor and out-patient of Department of TB & Chest Diseases, S.N. Medical College, Agra (U.P.) from Jan’2009 to June’2010. All patients were subjected to routine investigations, e.g. hemogram, montoux test, Chest skiagram, AFB staining, and sputum examination. Lymph node aspirate were subjected to decontamination, DNA isolation and Real time PCR( q PCR). Also specimen were simultaneously put to culture on L.J. Media. Results of both modalities compared. Results: Conventional culture method was able to detect M.tuberculosis in 75% of the cases as compared to real-time PCR which was positive in 77.5% with comparable sensitivity (100% vs 96.7%) and specificity (100% vs 87.5%). Conclusion: Conventional culture method is gold standard in the diagnosis of tuberculosis since long but recent molecular assays like Real-time PCR is one of the latest addenda to the armamentarium for the rapid and accurate diagnosis of M.tuberculosis. Needless to say, early diagnosis is advantageous to the management of tuberculosis.

Management of severe gastrointestinal tuberculosis with injectable antituberculous drugs

Abdominaltuberculosis (TB) is generally responsive to medical treatment, and earlydiagnosis and management can prevent unnecessary surgical intervention. However, there is a needfor intravenous therapy for severe forms of tuberculosis with extensivegastrointestinal involvement. The authors report an immunocompetent patientwith gastrointestinal TB who was successfully managed with a combination ofsurgical intervention and anti-TB medications, and discuss the importance ofinjectable anti-TB medications in the management of severe gastrointestinal TB.The present case report illustrates a model for assessment and intervention in severeforms of gastrointestinal TB.

Management of Severe Gastrointestinal Tuberculosis with Injectable Antituberculous Drugs

Abdominal tuberculosis (TB) is generally responsive to medical treatment, and early diagnosis and management can prevent unnecessary surgical intervention. However, intravenous therapy is needed for severe forms of tuberculosis with extensive gastrointestinal involvement. The authors report an immunocompetent patient with gastrointestinal TB who was successfully managed with a combination of surgical intervention and anti-TB medications, and discuss the importance of injectable anti-TB medications in the management of severe gastrointestinal TB. The present case report provides a model for assessment and intervention in severe forms of gastrointestinal TB.

Drug Susceptibility Testing of M. tuberculosis to Isoniazid and Rifampicin- A Comparison between MTT Tube Method and LJ Proportion Method.

Aim: To evaluate the methodology of MTT tube assay and compare it with standard proportion method for detection of drug susceptibility of M. tuberculosis to rifampicin (RIF) and isoniazid (INH). Study Design: Prospective. Place and Duration of Study: Sher-i-Kashmir Institute of Medical Sciences, Kashmir, India. One year study. Methodology: MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay was performed on 60 clinical isolates of M. tuberculosis. An inoculum of 107CFU/ml prepared in Middlebrook 7H9 with OADC (Oleic acid, albumin, dextrose and catalase) was chosen as standard. For each drug three tubes were used, one drug containing (INH 0.2 μg/ml or RIF 1 μg/ml), second inoculum control and third blank control. The method was performed after incubating the tubes at 37°C for 4 days for RIF and 7 days for INH. Results were read visually and by spectrophotometer at 570 nm. Relative optical density units of 0.2, was taken as cutoff. Results of drug susceptibility were compared with those obtained by Lowenstein Jensen proportion method. Results: For RIF, sensitivity was 88.9% and 94.4%; specificity was 100% and 97.6% for visual MTT and MTT by RODU respectively. For INH similar sensitivity of 95.1% was seen while specificity was 97.0% and 95.0% by visual MTT and MTT by RODU respectively. There was almost perfect agreement between proportion and MTT method for both drugs. Turn-around time for MTT assay was 7 days. Conclusion: The MTT tube assay can be used for rapid drug susceptibility testing of M. tuberculosis to RIF and INH.

Clonal Diversity and Drug Resistance in Mycobacterium tuberculosis Isolated from Extra-pulmonary Samples in Central India - a Pilot Study.

In India, extrapulmonary tuberculosis (EPTB) accounts for 10 - 15% of all types of tuberculosis. To identify and compare predominant spoligotypes and drug‑resistance patterns in strains of Mycobacterium tuberculosis isolated from extrapulmonary and pulmonary specimens in central India, drug susceptibility testing and spoligotyping were carried out. Spoligotyping data was analyzed using SITVIT2 database. ST11/EAI3_Ind with 33% isolates among extrapulmonary specimens and ST26/ CAS1_DEL with 28% isolates among pulmonary specimens were the most predominant lineages. Multidrug resistance was found in 5.5% of the strains isolated from extrapulmonary specimens in contrast to 17% isolated from pulmonary specimens.

Rapid screening of rpoB and katG mutations in Mycobacterium tuberculosis isolates by high-resolution melting curve analysis.

Background: Early detection of multidrug‑resistant tuberculosis (MDR‑TB) is essential to prevent its transmission in the community and initiate effective anti‑TB treatment regimen. Materials and Methods: High‑resolution melting curve (HRM) analysis was evaluated for rapid detection of resistance conferring mutations in rpoB and katG genes. We screened 95 Mycobacterium tuberculosis 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. Nineteen M. tuberculosis isolates with known drug susceptibility genotypes were used as references for the assay validation. The nucleotide sequences of the target regions rpoB and katG genes were determined to investigate the frequency and type of mutations and to confirm HRM results. Results: HRM analysis of a 129‑bp fragment of rpoB allowed correct identification of 19 of the 20 phenotypically RIF‑R and all RIF‑S isolates. All INH‑S isolates generated wild‑type HRM curves and 18 out of 21 INH‑R isolates harboured any mutation in 109‑bp fragment of katG exhibited mutant type HRM curves. However, 1 RIF‑R and 3 INH‑R isolates were falsely identified as susceptible which were confirmed for 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. Conclusion: HRM was found to be a reliable, rapid and low cost method to characterise drug susceptibility of clinical TB isolates in resource‑limited settings.

Comparison of Rifampicin Resistance in Mycobacterium tuberculosis Isolates by Multiplex Allele Specific PCR (MAS-PCR) with Enzyme Linked Immunosorbent Assay (PCR-ELISA).

Aims: Detection of drug resistance M. tuberculosis isolates is one of the most important strategies to control the disease. Nowadays, with advances in molecular technology, various methods are available to detect drug resistant M. tuberculosis strains such as those based on capture specific probes. In this study, we aimed to investigate the frequency of mutation in the M. tuberculosis -rpoB gene by Polymerase Chain Reaction based on Enzyme Linked Immuno Sorbent Assay (PCR-ELISA) detection. Methodology: Thirty-three culture positive isolates were randomly selected for this study. All the isolates were subjected to a drug Susceptibility Test (DST) using the proportion method. Then the ability and the efficiency of Multiplex Allele Specific PCR (MAS-PCR) and PCR-ELISA to detect Rif resistant (Rifr) M. tuberculosis isolates was compared and evaluated. Results: Mutation of rpoB gene was detected in 19/33 isolates (57.6%) by PCR-ELISA. Hybridization with the specific mutant probes 516 and 526 codon occurred in 1/33 isolates each (3% respectively). Hybridization with the specific mutant probe 531 occurred in 13/33 isolates (39.4%). Three isolates (9.2%) showed simultaneous mutation in codons 516 and 531. The sensitivity and specificity of MAS-PCR in comparison to the Proportional Method was 100%. On the other hand, PCR-ELISA showed 75% sensitivity and 69.2% specificity. The positive predictive value for the PCR-ELISA method was 78.9% and the negative predictive value was 64.3%.The general efficacy of test was 72.7%. Conclusion: The study showed that the sensitivity and specificity of PCR-ELISA to detect mutations in the rpoB gene in Drug Resistant strains was low. Furthermore, this proved to be a complex, time consuming and expensive test. Therefore, this test is not recommended for determining Rifampicin resistance in M. tuberculosis strains.

Primary tuberculous myositis: a rare clinical entity.

Summary: Primary tuberculous myositis without underlying pathology has been sparingly reported in medical literature. We report a case of primary tuberculous myositis of left upper arm in a seven-year-old boy. He presented with gradually increasing swelling on the medial aspect of the left arm. Ziehl Neelsen staining of pus collected revealed acid fast bacilli morphologically resembling Mycobacterium tuberculosis and the same was grown on the culture. Histopathological findings were consistent with tuberculosis. The results were confirmed by Genotype MTBDRpluse line probe assay. He was treated with standard four-drug regimen to which he responded well with complete resolution of the lesion.