ZN Smear Research Articles

DETECTION OF PULMONARY AND EXTRA-PULMONARY TUBERCULOSIS FROM CLINICAL SAMPLES WITH RIFAMPICIN (RIF) RESISTANCE BY GENE-XPERT MTB/RIF ASSAY AT A TERTIARY CARE TEACHING HOSPITAL, UDAIPUR, RAJASTHAN

Tuberculosis (TB) is one of the major concerns of health policy and rapid detection of M. tuberculosis and detection of rifampicin (RIF) resistance in infected patients are essential for disease management. Multi-drug resistance (MDR) TB is dened as tuberculosis (TB) disease caused by a strain of Mycobacterium tuberculosis (MTB) that was resistant to at least isoniazid and rifampicin (RIF). The aim of this study was to evaluate the importance of GeneXpert MTB/RIF for detection of Mycobacterium tuberculosis (MTB) with detection of rifampicin (RIF) resistance. In this study 967suspected cases of pulmonary and extra-pulmonary TB were included. Their sputum and other (Pus, CSF, tissue, etc.,) samples were collected and subjected to smear microscopy test followed by GeneXpert MTB/RIF assay. Of total 967 samples, 470 (48.60%) were positive for Mycobacteria by GeneXpert MTB/RIF and 118 (12.20%) samples were positive by ZN smear. Among 967 positive cases, 818 (84.59%) were pulmonary, 149 (15.41%) were extra-pulmonary (EP). Two ZN smear positive cases were negative by GeneXpertMTB/RIF. Of the 470 GeneXpert positive samples for MTB, 29(6.17%) cases showed RIF resistance and diagnosed as MDR-TB. GeneXpert MTB/RIF assay is efcient and reliable technique for the rapid diagnostic of TB. The GeneXpert MTB/RIF assay is a simple, high sensitivity and specicity for RIF resistance detection implies for diagnosis of MTB and RIF resistance in MDR cases as well as suspected smear negative and extra-pulmonary cases.

DIAGNOSING NTM IN IMMUNOCOMPETENT CHRONIC LYMPHADENITIS: AN EMERGING ENITITY

Introduction: Cervical lymph node infection due to nontuberculous mycobacteria in a immunocompetent patients are emerging problem globally. It is presented as chronic enlargement of gland with pain and pus discharge sometimes. Traditionally diagnosis is done through clinical examination, lymph node aspirated samples pathological finding or ZN microscopy, which altogether indicate mycobacterial infection only, unable to differentiate MTB from NTM infection.Increasing incidence of LNTB cases not responding to antituberculous treatment prompted us to pinpoint the etiological agent by doing liquid culture of FNA samples in microbiology laboratory. Materials and Methods: In this study 154 cases were recruited having chronic cervical lymph node swelling of suspicion of mycobacterial infection during January - December, 2014. Therein detailed history and thorough clinical examination of patients were reviewed. All patients were subjected for fine needle aspiration (FNA) using 10 ml syringe fitted with 21 G needle. The aspirated samples were subjected to smear microscopy byZiehlNeelsen (ZN) stain examination, May Grunwald - Giemsa stain and MGIT culture. Positive cultures were primarily identified by cord formation in ZN smear examination, detection of MPT-64 antigen by immuno-chromatographic test (Capilia test). Results: A total 154 lymph node samples were received and processed for culture during the study period. Out of total suspects 7/154 (4.5%) were identified as non-tuberculous mycobacterial lymphadenitis. Clinical examination showed that all the seven cases were presented with lymph node swelling and fever and previous ATT history. Other features seen were pain (2/7 28%), pus discharge (3/7 42%) and montoux positive (3/7 42%). The most common species identified was M. chelonae (3/7, 42%) followed by M. fortuitum (2/7, 28%), M. abscessus (1/7, 14%) and M.terricomplex (1/7, 14%). Four patients were managed surgically, one with surgical resection and medical treatment and 2 patients were lost tofollowup. Conclusion: Definitive and detailed microbiological examination is vital for diagnosing the nontuberculous lymphadenitis. M.chelonae is an important etiological agent.NTMs are important definitive etiological agents in cervical lymph node swelling. Clinical and pathological findings help in suspecting the infection.

Screening of Human Immunodeficiency Virus (HIV) among Newly Diagnosed Tuberculosis Patients in Eastern Sudan.

Tuberculosis (TB) is a leading cause of death in patients infected with Human Immunodeficiency Virus (HIV), and HIV infection is the most potent risk factor for the development of active TB disease from a latent TB infection. This study aims to determine the seroprevalence of HIV among newly diagnosed TB patients in Kassala state eastern Sudan. This was a descriptive, hospital-based, cross-sectional study of 251 active and newly diagnosed TB patients, selected by simple random sampling. Blood samples and demographic data were collected from each patient. TB was diagnosed by direct ZN smear and molecular detection by Xpert MTB/RIF. The serum samples were tested for HIV using 4th generation enzyme-linked immunosorbent assay (ELISA). The prevalence of HIV was 13.9% (35/251), the infection rate among pulmonary TB was 17%, whereas that in extrapulmonary TB was 4.8%, the prevalence was (18.2%) in the males, and (7.2%) in the females. In conclusion: TB/HIV co-infection in the Eastern part of Sudan was high compared with the global prevalence, all TB patients should therefore be assessed for HIV risk factors and advised to undergo HIV testing.

USE OF CARTRIDGE BASED NUCLEIC ACID AMPLIFICATION TEST FOR RAPID DETECTION OF MYCOBACTERIUM TUBERCULOSIS IN PULMONARY AND EXTRAPULMONARY TUBERCULOSIS

Objective: Tuberculosis is an airborne infection caused by Mycobacterium tuberculosis. Timely diagnosis and treatment are important to prevent the spread of infection. Cartridge-based nucleic acid amplification test (CBNAAT) provides a valuable tool in the early detection of TB. This study is undertaken to evaluate the utility of CBNAAT for the detection of MTB. Comparison of cartridge-based nucleic acid amplification testing with ZN staining.
 Methods: This prospective observational study was carried out in the Department of Microbiology, BLDEDU’s Shri B. M. Patil Medical College, Hospital and RC and Dr. Karigoudar Diagnostic Laboratory, Vijayapur. A total of 129 samples from patients with the presumptive diagnosis of TB based on history, clinical presentation, and radiological findings were included in the study. All samples were subjected to ZN staining, and Cartridge-based nucleic acid amplification test and data were analyzed.
 Results: The present study showed ZN smear positivity of 7.75% and CBNAAT positivity of 19.38%. CBNAAT sensitivity and specificity were 90% and 86.55, respectively, compared with ZN staining with a significant P value of <0.001.
 Conclusion: CBNAAT helps diagnose TB and detect rifampicin resistance within 2-3 h with high sensitivity and specificity. Rifampicin resistance detection is of great concern, which otherwise leads to treatment failure and on time spread of multidrug resistance TB, leading to increased morbidity and mortality.

Extra-pulmonary tuberculosis (EPTB): Study of clinico-demographic profile, and comparison of microbiological diagnostic modalities with special emphasis on role of CBNAAT in detecting rifampicin resistance in fluid specimens

The purpose of our study was to evaluate the place of CBNAAT in the diagnosis of extra-pulmonary TB in fluid specimens, and also detecting drug resistance in comparison to the Gold Standard - MGIT Bactec Culture. Material and Methods: In prospective observational study of 2 yrs. among 165 EPTB cases, we studied the clinical presentation, compared the results of microbiological diagnostic modalities (ZN smear, CBNAAT, MGIT Culture) and evaluated the drug resistance. Statistics: Used Chi square test via software STATA 15 and calculated p value.Results: The positivity rate of CBNAAT (31.30%) was higher than ZN smear (23.92%), which was in turn higher than MGIT culture (9.9%) and CBNAAT was able to detect Rifampicin resistance in 16.66% EPTB cases. Conclusion: We concluded that, the role of CBNAAT in fluid specimens should not be undermined and should be preferred over the “Gold standard” MGIT culture.

Sensitivity of sputum exam with respect to gene expert in detection of MTB at tertiary care hospital

Background: According to the WHO Tuberculosis Report 2014, there were 9 million incident cases of TB worldwide, with the South–East Asia and Western Pacific areas accounting for 58 percent of the global burden and India accounting for 24 percent. Aims and Objective: To study Sensitivity of sputum exam with respect to gene expert in detection of MTB at tertiary care hospital Methodology: The present study is an observational cross-sectional study of the patients with pulmonary tuberculosis attending Department of Pulmonary Medicine at a tertiary care centre during period of 1 January 2019 to 30 June 2020 by the permission of Institutional Ethical Committee . Genexpert test / CBNAAT [ Xpert MTB/RIF Assay] Statistical analysis done buy of (version 20) for Windows package (SPSS Science, Chicago, IL, USA). Result: Ziehl and Neelsen staining was done for 200 samples of cases of pulmonary tuberculosis. Out of these 55(27.50%) sputa samples were ZN smear positive and 145(72.50%) were negative. Then all the samples were tested on Gene Xpert MTB/RIF assay. Out of these 200 patients, 186(93%) were MTB detected and 14(7.00%) were MTB not detected. The MTB/RIF assay detect the agent in 54 out of 55 ZN smear positive cases and 132 out of 145 AFB smear negative cases as shown in table No.10. So, MTB detection rate by Gene Xpert 186cases (93.00%) is more as compared to ZN staining 55cases (27.50%). Out of 55 sputum positive cases, 54 were positive for MTB by Gene Xpert but in 01 sputum positive case MTB was not detected by Gene Xpert assay. There was statistically significant better detection with Genexpert when compared to microscopy AFB test in pulmonary tuberculosis patients.The Sensitivity of Sputum was 29.03% and Specificity was 92.86%, Positive Likelihood Ratio4.06, Negative Likelihood Ratio 0.76, Positive Predictive Value (*) was 98.18% and Negative Predictive Value (*) was 8.97%. Conclusion: It can be concluded from our study that the Genexpert is highly sensitive test for the detection of the Mycobacteria and Xpert MTB/RIF assay can quickly identify possible multidrug-resistant TB. From our study we conclude that Gene Xpert MTB/RIF is simple and reliable technique for diagnosing extra pulmonary tuberculosis with high sensitivity and specificity not only in smear positive cases but also in smear negative cases. It is a game changer not only in pulmonary tuberculosis control but probably also in extra pulmonary tuberculosis.

GeneXpert® assay; and its performance for the diagnosis of HIV-associated tuberculosis: Is scale-up worth it?

Background: Synergistic Tuberculosis (TB) and human immunodeficiency virus (HIV) co-infections are responsible for epidemics in several nations and have established themselves as a tremendous health challenge to these nations in terms of patient point of care and economic burden on health care organizations. TB is the fundamental driver of HIV-related mortality around the world. Co-infection with these two pathogens results in an accelerated course for the two illnesses and raises complex challenges in diagnosis and therapy. TB and HIV both work in synergy to accelerate the deterioration of the immunological defense mechanisms often leading to death. This present study was designed to evaluate the performance of GeneXpert assay for the detection of Mycobacterium tuberculosis among HIV positive and negative TB patients. Methods and materials: The present prospective cross-sectional study was conducted in the Mycobacteriology laboratory, Allama Iqbal Medical College, Lahore, Pakistan from 2011–2017. A total of 3784 (HIV-ve n = 3568, HIV +ve n = 216) sputum samples were collected with complete history from patients strongly suspected for pulmonary TB (PTB) patients. HIV status was evaluated by PCR (Quantitative) and then referred for Flow-cytometry and GeneXpert assay for MTB Results: Out of a total of n = 3784 PTB, the prevalence of HIV was 5.7% (n = 216/3784). Of 216 HIV positive, 15.7% (n = 34) were also co-infected with TB. The sensitivity of the ZN smear and GeneXpert assay was considerably reduced among HIV and TB co-infected patients. The sensitivity and specificity of ZN smear was 63.7%, and 100% among HIV negative compared to 23.5% and 100% among HIV positive individuals respectively. Whereas sensitivity and specificity of the GeneXpert were found to be 94.5% and 100% for HIV negative individuals as compared to the 76.4%, and 100% respectively among the HIV positive patients. Furthermore, in the case of smear-negative samples, GeneXpert assay showed noticeable sensitivity and specificity 85.8%, 100% and 69.2%, 100%, respectively for HIV negative and positive patients. Conclusion: The GeneXpert assay showed an overall better sensitivity compared to the ZN smear for the diagnosis of TB, the sensitivity was much less in TB patients co-infected with HIV, compared to the HIV negative patients.

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.

Mutation in MPT64 gene influencing diagnostic accuracy of SD Bioline assay (capilia)

BackgroundSuccess of India’s TB control program relies on rapid case detection, monitoring, care and treatment of drug resistance. Patients on multidrug resistance (MDR) treatment are monitored by follow up cultures. Discordant results (culture and smear positive while capilia negative) are usually declared negative Mycobacterium tuberculosis complex (MTBC). This study was designed to understand the possible causes of discordant results.MethodsThe capilia kit was evaluated to test its utility among 4737 follow up MDR patients enrolled during a period of 1 year. A total of 889 were liquid culture positive, 3375 were negative and 473 were contaminated. Of the 889 cultures positive, 829 were found positive by ZN smear, capilia test and MTBDR plus assay. The cultures which gave a positive result on Mycobacterium Growth Indicator Tube 960 (MGIT 960) and ZN smear but were negative on capilia test with no growth on Brain Heart Infusion agar (BHI) were included in this study. The conflicting results of capilia were compared with other molecular techniques; MTBDR plus assay and DNA sequence analysis of MPT64 gene.ResultsOut of 889 culture positive, 60 (6.7%) were found positive on liquid culture and ZN smear but were negative on capilia. Of these 60 cultures, 10 (16.7%) were found positive by both MTBDR plus assay and PCR. The sequencing analysis revealed that all of the capilia negative isolates had mutations within the MPT64 gene.ConclusionRe-evaluation of culture positive but capilia negative isolates should be done before declaring them as Mycobacterium other than tuberculosis (MOTT) because such cases can act as chronic carriers of TB in the population which can lead to the rise of this lethal disease.

Diagnosis of paediatric tuberculosis by cartridge based nucleic acid amplification test and its effectiveness as compared to the other conventional diagnostic methods

Background: Childhood TB constitutes 10-20% of all TB cases in high burden countries like India and accounting for 8-20% of TB related deaths. Diagnosis of TB in children is difficult. One test, CBNAAT which was recently endorsed by WHO has the potential to lead a revolution in diagnosis of active TB disease.Methods: A cross sectional study in SCB MCH and SVPPGIP, Cuttack in all the suspected TB patients admitted during the period from January 2016 to October 2017.Results: A total of 100 suspicious patients admitted to the Department of Pediatrics in SCB MCH and SVPPGIP during the study period. Of these 45 were diagnosed TB and rest others were diagnosed otherwise than TB. Diagnosis of TB was established on basis of Microscopy, CBNAAT, culture, biochemistry, cytology, clinical findings, neuroimaging, FNAC/biopsy, USG abdomen. Out of 45 TB patients 30 were CBNAAT positive taking the body fluid samples other than blood, urine and stool with a sensitivity of 66.7% and specificity of 100%. Out of 45 TB patients 14 were having ZN Smear positive taking the same fluid sample with a sensitivity of 31.1% and specificity of 100%. Whereas out of these 45 TB patients 32 were MGIT culture positive taking the same sample with a sensitivity of 71.1% and specificity of 100%. When diagnostic performances of CBNAAT and MGIT culture were compared, it was found to be statistically insignificant with a P value 0.54.Conclusions: The CBNAAT is able to confirm a diagnosis of TB with 66.7% sensitivity and 100% specificity within 2 hours. We can use CBNAAT as a diagnostic method as it provides rapid result and simultaneous better sensitive result, it can be helpful in starting ATT in sick patients and also in outdoor patients.

Diagnosis of Pulmonary Tuberculosis in Resource Limited Setting of Rawalpindi

Introduction:Tuberculosis is an infectious disease with a high prevalence of about 9 million cases occurring annually. Ziehl Neelsen microscopy is the most widely used technique to detect Acid Fast Bacilli, but it is less sensitive. However, fluorescent microscopy is more helpful with simple diagnostic criteria. Gene Xpert®MTB/RIF assay is a rapid molecular assay that enables diagnosis of Tuberculosis with simultaneous detection of rifampicin resistance. Owing to this fact, we aimed at evaluating the diagnostic accuracy of Ziehl Neelsen microscopy, fluorescent microscopy and Xpert MTB/RIF keeping MTB culture (Mycobacterial Growth Indicator Tube) as a gold standard for the diagnosis of tuberculosis.Methodology:This study was carried out at a tertiary care hospital of Rawalpindi in the year 2016. Patients aged 18 to 70 years irrespective of gender with suspected TB based on history, clinical and radiological examination were included in the study. Respiratory clinical specimens including sputum, Broncho-Alveolar Lavage (BAL), and endobronchial washings were collected. Specimens were processed by MGIT (MTB culture), ZN microscopy, fluorescent microscopy and Gene Xpert MTB/RIF assay.Results:A total of 352 respiratory specimens were tested among which 160 (45%) samples were positive by culture. Out of culture positive samples, 158 samples (98.7%) were GeneXpert TB positive while 2 were negative. While only 49 (30.6%) were positive on ZN microscopy and 89 (55%) were positive on fluorescent microscopy. Out of the culture negative samples, 2 were positive with ZN microscopy, one was positive with fluorescent microscopy and 3 were positive on Gene Xpert. Sensitivity, specificity, Positive Predictive Value (PPV), Negative Predictive Value (NPV) and diagnostic accuracy of ZN Smear microscopy was 39%, 99.5%, 96%, 63% and 14.5% respectively. Sensitivity, specificity, PPV, NPV and diagnostic accuracy of fluorescent smear microscopy was 55% and 99.5%, 98%, 72% and 79% respectively. Sensitivity, specificity, PPV, NPV and diagnostic accuracy of Gene XPERT was 98% and 99%, 98%, 99% and 98% respectively.Conclusion:In countries like Pakistan where Tuberculosis is endemic, the diagnostic accuracy with highest sensitivity and specificity was Gene Xpert Polymerase Chain Reaction (PCR) MTB/RIF assay which can help in well-timed diagnosis of the disease.

The importance of diagnosing Mycobacterium tuberculosis by real time PCR Compared with the approved diagnostic methods in the Clinical Laboratory

Background: Tuberculosis remains a serious public health problem worldwide. Zheil-Neelsen stained smear and culture onLowenstein Jensen(LJ)media are conventional methods used for the diagnosis of Mycobacterium tuberculosis in most developing countries .PCR for the diagnosis of tuberculosis is not evaluated in developing countries. Aim: To evaluate the ability of PCR to diagnose MTB in pulmonary and extra-pulmonary samples, and to compare it with the results of ZN and LJ. Methods:Samples obtained from 96 patients of suspected TB (pulmonary and extra-pulmonary) were processed for detection of MTB by ZN smear examination , LJ medium culture and Real time PCR test. Results: The sensitivity of PCR was 100% compared to 17,4% for smear microscopy and 100% for LJ culture as a golden standard. PCR sensitivity in pulmonary and extra-pulmonary samples was 96.2% and 100% respectively. The mean detection time for MTB was 6 weeks by LJ medium culture, less than one day by PCR and 4 days by ZN for 96 samples. Conclusions:PCR is a rapid and sensitive method for the early diagnosis of pulmonary and extra- pulmonary tuberculosis.

Evaluation of loop mediated isothermal amplification (LAMP) assay in the diagnosis of tubercular lymphadenitis: A pilot study

Tubercular lymphadenitis (TBLA) contributes to 30–40% of extrapulmonary TB cases in the immunocompetent individuals and 40–50% in people with HIV. Current diagnostic methods for TBLA like Gene-Xpert or PCR are costly and conventional methods like fine needle aspiration cytology, histopathology lack sensitivity and specificity. Culture which is considered as gold standard require high turnaround time. Loop mediated isothermal amplification (LAMP) assay has been developed as a novel technique for nucleic acid amplification and has shown promising results in the diagnosis of pulmonary tuberculosis. Present study evaluated the Nu-LAMPTM TB Kit (RAS Life Sciences Pvt. Ltd, a bioMerieux group company) for diagnosis of TBLA comparing with conventional tests (cytology, ZN smear, culture). The sensitivity, specificity, PPV and NPV of LAMP assay was found to be 33.3%, 91.2%, 40% and 88.57% as compared to 100%, 76.5%, 42.9% and 100% of ZN staining and 100%, 73.5%, 40% and 100% of cytopathology. The low sensitivity of LAMP assay in the present study addresses the need for comparison and validation of the commercially available LAMP kits before used for patient diagnosis.

GeneXpert technology. A breakthrough for the diagnosis of tuberculous pericarditis and pleuritis in less than 2 hours.

Objectives:To evaluate the diagnostic validity of GeneXpert for the detection of Mycobacterium tuberculosis (MTB) in pericardial and pleural effusions samples.Methods:A cross sectional study was conducted at the Mycobacteriology Laboratory, Allama Iqbal Medical College, Lahore, Pakistan. A total of 286 (158 pleural and 128 pericardial fluids) samples were received from tuberculosis (TB) suspects between January 2014 and August 2016. Every sample was processed for Ziehl-Neelsen (Zn) smear, Lowenstein Jensen (LJ) culture, GeneXpert MTB/RIF assay according to standard protocols. Validity of GeneXpert assay for the detection of MTB was evaluated using LJ culture as gold standard.Results:Out of 286 effusions samples, MTB was isolated by LJ culture in 51 (17.8%) samples followed by GeneXpert in 43 (15%), and acid- fast bacilli (AFB) was detected by Zn smear microscopy in 11 (3.8%) samples. GeneXpert showed high sensitivity (84.3%), specificity (100%), with positive predictive value (100%), and negative predictive value (96.7%), while Zn smear showed sensitivity 18.3%, specificity 99.1%, positive predictive value 81.8%, and negative predictive value 85.4%. A strikingly high sensitivity of 72.2% was observed for pericardial fluid by GeneXpert.Conclusion:GeneXpert assay is an innovative tool, for prompt detection of MTB and drug resistance. It is definitely an attractive point of care test, with high sensitivity and specificity along with turn around time of 2 hours, which facilitates timely diagnosis and appropriate management of TB pleuritis and pericarditis.

TUBERCULOUS PERICARDITIS AND PLEURITIS;

Background: Diagnosis of Tubercles Pericarditis and Pleuritis remains thegreatest challenge for clinicians. WHO has recommended GeneXpert MTB/RIF assay as ascreening test for substitution of conventional methods for the initial diagnosis and prognosisof the extra pulmonary and pulmonary tuberculosis in developing countries. Objectives: Tofind out the diagnostic validity of GeneXpert assay for detection of Myco-bacterium tuberculosisin the pericardial and pleural effusions samples, keeping MTB culture as “Gold Standard”.Material and Methods: Total number of 286 samples of effusions (pericardial 128, pleural 158)were received, and processed for Zn smear microscopy, LJ culture, GeneXpert MTB/RIF assayaccording standard protocols. Efficacy for the detection of MTB was evaluated comparatively.Results: Out of 286effusions samples AFB was detected by Zn smear in 11 (3.8%) samples whileGeneXpert detected MTB in 43 (15.0%) and LJ culture 51 (17.8%). Zn smear showed sensitivity18.2%, specificity, 98.1%, Positive predictive value 81.8%, Negative predictive value 85.4 %, incomparison GeneXpert showed high sensitivity 84.3%, specificity 100%, with Positive predictivevalue 100%, and Negative predictive value 96.7%. Conclusion: GeneXpert assay is innovativetool in resource limited settings for prompt detection of MTB along with drug résistance. It isdefinitely an attractive point of care test, with High sensitivity and specificity along with turnouttime of two hours which facilitates timely diagnoses and appropriate management of tuberclePleuritis and Pericarditis.

English

Background: Diagnosis of Tubercles Pericarditis and Pleuritis remains thegreatest challenge for clinicians. WHO has recommended GeneXpert MTB/RIF assay as ascreening test for substitution of conventional methods for the initial diagnosis and prognosisof the extra pulmonary and pulmonary tuberculosis in developing countries. Objectives: Tofind out the diagnostic validity of GeneXpert assay for detection of Myco-bacterium tuberculosisin the pericardial and pleural effusions samples, keeping MTB culture as “Gold Standard”.Material and Methods: Total number of 286 samples of effusions (pericardial 128, pleural 158)were received, and processed for Zn smear microscopy, LJ culture, GeneXpert MTB/RIF assayaccording standard protocols. Efficacy for the detection of MTB was evaluated comparatively.Results: Out of 286effusions samples AFB was detected by Zn smear in 11 (3.8%) samples whileGeneXpert detected MTB in 43 (15.0%) and LJ culture 51 (17.8%). Zn smear showed sensitivity18.2%, specificity, 98.1%, Positive predictive value 81.8%, Negative predictive value 85.4 %, incomparison GeneXpert showed high sensitivity 84.3%, specificity 100%, with Positive predictivevalue 100%, and Negative predictive value 96.7%. Conclusion: GeneXpert assay is innovativetool in resource limited settings for prompt detection of MTB along with drug résistance. It isdefinitely an attractive point of care test, with High sensitivity and specificity along with turnouttime of two hours which facilitates timely diagnoses and appropriate management of tuberclePleuritis and Pericarditis.

Evaluation of genotype MTBDRplus VER 2.0 line probe assay for the detection of MDR-TB in smear positive and negative sputum samples

BackgroundMulti drug resistant tuberculosis (MDR-TB) poses formidable challenges to TB control due to its complex diagnostic and treatment challenges and often associated with a high rate of mortality. Accurate and rapid detection of MDR-TB is critical for timely initiation of treatment. Line Probe Assay (LPA) is a qualitative in vitro diagnostic test based on DNA-STRIP technology for the identification of the M. tuberculosis complex and its resistance to rifampicin (RMP) and/or isoniazid (INH). Hain Lifescience, GmbH, Germany has improved the sensitivity of Genotype MTBDRplus VER 2.0 LPA for the detection of MDR-TB; with the possibility of applying the tool in smear negative sputum samples.MethodA cross sectional study was conducted on 274 presumptive MDR-TB patients referred to the National TB Reference Laboratory (NTRL), Ethiopian Public Health Institute (EPHI) who submitted sputum samples for laboratory diagnosis of drug resistant-TB testing. Seventy-two smear and culture positive samples processed in smear positive direct LPA category and 197 smear negative sputum samples were processed for direct LPA. Among the smear negative samples 145 (73.6%) were culture negative and 26 (13.2%) were culture positive. All specimens were processed using NALC-NaOH method and ZN smear microscopy done from sediments. Genotype MTBDRplus VER 2.0 done from processed sputum sediments and the result was compared against the reference, BACTEC MGIT 960 culture and DST. Sensitivity, specificity, PPV and NPV of Genotype MTBDRplus VER 2.0 assay was determined and P-value <0.05 was considered as statistically significant.ResultsThe sensitivity, specificity, PPV and NPV of Genotype MTBDRplus VER 2.0 LPA were 96.4, 100, 100 and 96.9%, respectively for the detection of MDR-TB from direct smear positive sputum samples. The sensitivity, specificity, PPV and NPV of Genotype MTBDR plus VER 2.0 LPA were 77.8, 97.2, 82.4 and 97.2%, respectively, for the detection of M. tuberculosis from direct smear negative sputum samples. Fourteen (53.8%) samples had valid results with LPA among the 26 smear negative culture positive samples. The remaining 8 (30.8%) and 4 (15.4%) were invalid and negative with LPA, respectively. The sensitivity and specificity of Genotype MTBDRplus VER 2.0 LPA were 100% for the detection of MDR-TB among 14 direct smear negative and culture positive sputum samples.The most common mutations associated with RMP and INH resistance were S531L and S315TL, respectively. A single rare mutation (C15T/A16G) was detected for INH resistance.ConclusionThe diagnostic performance of Genotype MTBDRplus VER 2.0 LPA in direct smear positive sputum sample was highly sensitive and specific for early detection of MDR-TB. However, the diagnostic performance of this molecular assay in direct smear negative sputum sample was low and showed a high level of invalid results for detection of M. tuberculosis and its resistance to RMP and/or INH so it is unlikely to implement Genotype MTBDRplus VER 2.0 for the detection of MDR-TB in direct smear negative sample in our routine settings. The sensitivity of the assay should be improved for detection of MDR-TB in direct smear negative sputum specimens.

MYCOBACTERIUM TUBERCULOSIS RESISTANCE TO ISONIAZID AND RIFAMPICIN IN A HIV-1 ENDEMIC POPULATION IN WESTERN KENYA IN 2014

BackgroundThe spread of mono-resistant and multi-drug resistant tuberculosis (MDR-TB) has been enhanced by delays in the identification of resistant strains. However, resistance gene patterns and the extent and distribution of...

Concentration of fine needle aspirates similar to molecular method improves sensitivity of the diagnosis of tuberculous lymphadenitis in Addis Ababa, Ethiopia

BackgroundTuberculous lymphadenitis (TBLN) diagnosis has been a true challenge solely by clinical evidence in developing countries, due to limited the diagnostic facility on hand. However, the availability and affordability of available diagnostic tools in resource-limited settings like Ethiopia necessitates the quest for other techniques with added value over direct Z-N microscopy. Therefore, we aimed at to assess whether the concentration of lymph node aspirate similarly improves the detection rate of tuberculous lymphadenitis or not.Materials and methodsA cross-sectional study design was conducted on 132 individual subjects presumptive for tuberculous lymphadenitis from February to October 2013 in Addis Ababa, Ethiopia. Fine needle aspirate (FNA) samples were collected from the cases and cultured on Löwenstein-Jensen (LJ) slants. Identification of species and strains of mycobacteria was made by region of difference (RD) based polymerase chain reaction (PCR). Data entry and statistical analyses were performed by SPSS version 20. The confidence level of 95% was used for statistical significance.ResultA total of 132 study subjects were included in our study. Of these 56.1% (74/132) were positive for M. tuberculosis on culture. The detection rate of direct smear microscopy and the concentration method were 29.5 and 65.2% respectively. The sensitivity of direct smear microscopy was 43.2%, for concentrated smear microscopy 94.5%, for PCR 93.2% and for cytomorphology 95.4%. The level of agreement of concentrated ZN smear microscopy was 0.62 which was very similar with kappa of 0.58 of molecular (PCR) technique. AFB positivity by the concentration method and molecular method was increased in caseous aspirates as compared to purulent and hemorrhagic aspirates though it was not statistically significant (p-value = 0.18) and (p = 0.62) respectively.ConclusionThe concentration of FNA (Fine Needle Aspirate) aspirates for acid-fast smear microscopy similarly improves the sensitivity of acid fast bacilli in diagnosing of TBLN.