Extrapulmonary Samples Research Articles

Evaluation of xpert Mycobacterium tuberculosis rifampicin for tuberculosis diagnosis in a reference laboratory.

The Xpert Mycobacterium tuberculosis Rifampicin (MTB-RIF) is a technological innovation that presents precision and speed in the diagnosis of tuberculosis (TB). The study aimed to evaluate the performance of Xpert MTB/RIF in the TB diagnosis and compare its results with those of culture, species identification, Antimicrobial Susceptibility Testing (AST), and rpoB gene sequencing of discordant results in AST, used for the diagnosis of TB in a reference laboratory. Cross-sectional descriptive study of pulmonary and extrapulmonary samples requesting Xpert MTB/RIF and culture for TB diagnosis from 2015 to 2019 at Adolfo Lutz Institute-São Paulo/Brazil. The analysis was performed with Epi-Info 7.2.1, presenting the distribution of frequencies and, for comparative analyses, Pearson's Chi-square test and Fisher's exact test were used, considering P ≤ 0.05 as statistically significant. For variables agreement, the Kappa method was used. A total of 1575 pulmonary and extrapulmonary samples were analyzed using Xpert MTB/RIF and culture, of which 293 were positive for the MTB Complex in both methodologies with a sensitivity of 94.55%; specificity of 95.97%; accuracy 95.69%; positive predictive value 85.53%; negative predictive value 98.59%, substantial agreement by Kappa 0.87, and detection sensitivity even at lower levels of bacillary load (P < 0.05). The Xpert MTB/RIF and AST showed concordant results (P < 0.05). The study brings forward that the Xpert MTB/RIF shows substantial agreement with the results of culture and AST, contributing to the diagnosis of TB and the rapid detection of resistance. The sequencing of resistant cultures in Xpert MTB/RIF and sensitivity in AST identified the H526N mutation, characterized by a low level of resistance to RIF.

Use of Whole-Genome Sequencing to Explore Mycobacterium tuberculosis Complex Circulating in a Hotspot Department in France.

The Seine-Saint-Denis is the French metropolitan department with the highest incidence of tuberculosis (TB). Our aim was to explore epidemiological and phylogenetic characteristics of TB strains in this hotspot department. We performed WGS on 227 strains of Mycobacterium tuberculosis complex isolated from patients at the Avicenne Hospital from 2016 to 2021 and randomly selected to represent the clinical diversity of French TB localization. Clinical and demographic data were recorded for each TB patient. The mean age of patients was 36 years old. They came from Africa (44%), Asia (27%), Europe (26%) and America (3%). Strains isolated from extrapulmonary samples were associated with Asian patients, whereas strains isolated from pulmonary samples were associated with European patients. We observed a high level of lineage diversity in line with the known worldwide diversity. Interestingly, lineage 3 was associated with lymph node TB. Additionally, the sensitivity of WGS for predicting resistance was 100% for rifampicin, isoniazid and ethambutol and 66.7% for pyrazinamide. The global concordance with drug-susceptibility testing using the phenotypic approach was 97%. In microbiology laboratories, WGS turns out to be an essential tool for better understanding local TB epidemiology, with direct access to circulating lineage identification and to drug susceptibilities to first- and second-line anti-TB drugs.

Magnitude of Mycobacterium tuberculosis, drug resistance and associated factors among presumptive tuberculosis patients at St. Paul's Hospital Millennium Medical College, Addis Ababa, Ethiopia.

Mycobacterium tuberculosis (M. tuberculosis) remains one of the most significant causes of death and a major public health problem in the community. As a result, the aim of this study was to determine magnitude of Mycobacterium tuberculosis, its drug resistance, and associated factors among presumptive tuberculosis (TB) patients at St. Paul's Hospital Millennium Medical College, Addis Ababa, Ethiopia. Cross-sectional study was conducted at St. Paul's Hospital Millennium Medical College (SPHMMC), Addis Ababa, Ethiopia from January to July 2019. Demographic and clinical data were collected by structured questionnaire through face to face interview. Using microscopic examination and GeneXpert MTB/RIF assay and culturing in the Lowenstein-Jensen (LJ) culture media, we collected and analyzed both pulmonary and extra-pulmonary clinical samples. Data were analyzed by SPSS version 23. Binary logistic regression was done to identify the associated risk factors and p-value less than 0.05 was taken as significant association. Of the total 436 respondents, 223(51%) were male. The mean ±SD age of the participants was 38±17years. Overall, 27/436(6.2%) of the participants had confirmed Mycobacterium tuberculosis using the GeneXpert MTB/RIF assay and LJ culture media, and two isolates were resistant to RIF and one to INH medication, with two (0.5%) being MDR-TB. MTB infection was associated with previous TB contact history, patient weight loss, and CD4+ T-cell counts of 200-350/mm3 of blood. The magnitude of M. tuberculosis and MDR-TB in this study underscores the need for improved early case detection and management of MDR-TB in order to reduce transmission and patient suffering.

Accuracy of Xpert MTB/RIF Ultra for the diagnosis of tuberculosis in adult patients: a retrospective cohort study

Objectives: The value of the “trace” result in Xpert Ultra for diagnosing active tuberculosis (TB) remains unclear. Our study evaluated the diagnostic accuracy of Xpert MTB/RIF Ultra (Xpert Ultra) (Cepheid, Sunnyvale, USA) over Xpert MTB/RIF (Xpert) (Cepheid, Sunnyvale, USA) and mycobacterial culture when compared with a composite reference standard (CRS).Methods: A retrospective single-center observational study was conducted in a tertiary care hospital in South India. Over three months, patients (aged ≥15 years) data on Xpert Ultra tests and mycobacterial culture of pulmonary and extrapulmonary samples were extracted from their electronic medical records. Patients were defined as TB cases based on the CRS criteria. Sensitivity, specificity, positive and negative predictive values of diagnostic tests were calculated by comparing them to the CRS.Results: Xpert Ultra was more sensitive (87.8%) than Xpert (72.1%) and culture (44.1%). The specificity of Xpert Ultra was lower (98.1%) than those of Xpert (100%) and culture (100%). The sensitivity (92%) and specificity (100%) of Xpert Ultra were highest when performed on pus samples.Conclusions: Xpert Ultra with the trace category is superior to the conventional Xpert, and mycobacterial culture in identifying TB.

Agreement between CBNAAT, liquid culture and line probe assay for detection of Mycobacterium tuberculosis and anti-tubercular drug resistance in extrapulmonary samples

PurposeCartridge based nucleic acid amplification test (CBNAAT) has been endorsed by the WHO as the screening test for diagnosing extrapulmonary tuberculosis (EPTB). In the present study we report the agreement between CBNAAT (Xpert MTB/RIF), liquid culture (LC) and line probe assay (LPA) for diagnosis of Mycobacterium tuberculosis and detection of drug resistance among EPTB cases. MethodsThe EP samples were subjected to CBNAAT (Xpert MTB/RIF, Cepheid, USA) and wherever possible, to LC (MGIT 960, Becton Dickinson, USA) followed sequentially by first line and second line-LPA (FL-LPA, SL-LPA, Hain Lifescience, Germany) on the isolates. ResultsTotal 566/4080 (13.9%) EP samples were detected positive for M. tuberculosis on CBNAAT. Aspirates from lymph nodes were most often positive (11/30; 36.6%), followed by pus (240/873; 27.5%) and CSF samples (166/104; 15.8%). The detection of M. tuberculosis was more in adults than children except in tissue biopsy samples. Rifampicin resistance was also higher among adults except CSF in which resistance was more in children. Total 185 of 566 (32.7%) CBNAAT positive and 770 of 3510 (21.9%) CBNAAT negative samples could be cultured of which 110/185 (59.4%) and 33/770 (4.3%) respectively turned positive. FL-LPA and SL-LPA of 143 culture isolates showed that 27 isolates had drug resistance, of which 3 (2.1%) were XDR, 11 (7.7%) were Pre-XDR (FQ) and 13 (9.1%) were MDR. Of these 27 resistant isolates, 12 were negative by CBNAAT and two were mislabeled as Rifampicin sensitive or indeterminate based on the unique RpoB gene mutation patterns on LPA. The positive and negative agreements between LC and CBNAAT for detection of M. tuberculosis were 67.1% and 92.7% respectively and between LPA and CBNAAT for rifampicin resistance detection were 98.9% and 92.9% respectively. ConclusionsFor EPTB, CBNAAT should be accompanied with LC wherever possible irrespective of the CBNAAT result.

Comparative Study of PD-L1 Expression in Different Sites of Non-small Cell Lung Cancer

背景与目的程序性死亡配体1（programmed cell death ligand 1, PD-L1）作为非小细胞肺癌（non-small cell lung cancer, NSCLC）免疫治疗患者分层标志物已进入临床病理常规检测。然而PD-L1表达在肺内和肺外不同转移部位的空间异质性是困扰临床检测的难题。本研究旨在探讨NSCLC不同部位组织样本的PD-L1表达评分的差异，从而有助于晚期肺癌患者的PD-L1检测策略的制定。方法回顾性收集PD-L1（22c3抗体，Dako）临床病理连续检测的131例肺外转移性NSCLC以及同期非配对肺内肿瘤972例进行对照分析，对比肺外与肺内肿瘤组织样本检测的PD-L1肿瘤阳性比例评分（tumor proportion score, TPS）差异。结果肺外转移性NSCLC的PD-L1阳性表达率（TPS≥1%）为61.83%，TPS评分显著高于同期肺内肿瘤（P=0.03）。不同部位组织样本的PD-L1表达评分具有显著差异（P=0.007）。肝脏和肾上腺转移瘤的PD-L1阳性率高，分别为85.71%和77.78%，其TPS评分均显著高于肺内肿瘤（P < 0.05）。淋巴结、骨、脑、软组织和胸膜转移瘤的PD-L1表达率为40.00%-66.67%，TPS评分与肺内肿瘤无显著差异。组织学和样本类型分析显示，腺癌类型和手术切除的肺外样本PD-L1表达评分显著高于同类型肺内肿瘤。临床病理参数分析显示，PD-L1阳性表达和高表达均与患者男性、吸烟史以及表皮生长因子受体（epidermal growth factor receptor, EGFR）野生型显著相关。结论肺外转移性NSCLC样本的PD-L1表达评分高于肺内肿瘤，且不同部位组织样本的PD-L1表达阳性率存在差异。肺外转移性肿瘤与肺内肿瘤的PD-L1检测差异可能与不同转移部位、组织学和样本类型相关。

Diagnosis of Mycobacterium tuberculosis using GeneXpert MTB / RIF and TB-LAMP techniques from pulmonary and extra-pulmonary TB patients in Iraq

Tuberculosis is considered one globally public health problem, particularly in unindustrialized countries. The disease has an increasing threat to public health, causing high morbidity and mortality rates. So, rapid diagnosis is critical to diminish the death and interfere with transmission. In this study, GeneXpert MTB/RIF and Loop-Mediated Isothermal Amplification (TB-LAMP) assays were utilized for the rapid molecular diagnosis of Mycobacterium tuberculosis bacteria and compared sensitivity and specificity of Mycobacterium tuberculosis bacteria; these tests with acid-fast bacilli staining in pulmonary and extra-pulmonary clinical specimens from Iraqi patients in Baghdad capital. 141 pulmonary and extra-pulmonary samples were collected and tested for tuberculosis detection. Acid-fast bacilli, TB-LAMP and GeneXpert MTB/RIF methods were used to diagnose tuberculosis. Of 141 tuberculosis specimens, 58 were acid-fast bacilli smear-positive, while 90 samples were positive for Mycobacterium tuberculosis by GeneXpert MTB/RIF assay and 78 by TB-LAMP assay. The results reported a high sensitivity rate of GeneXpert MTB/RIF assay compared to the TB-LAMP and bacilli staining method. The sensitivity of GeneXpert was 63.83%, specificity 50%; positive and negative predictive values were 58.36% to 68.96% and 42.73% to 57.27%, respectively. TB-LAMP sensitivity was 55.32%, specificity 50%; positive and negative predictive values were 49.61% to 60.89% and 43.70% to 56.30%, respectively, while the sensitivity of bacilli staining was 41.13%, specificity 50%; positive and negative predictive values were 35.26% to 47.27% and 44.88% to 55.12%, respectively, these values are dependent on disease prevalence. We concluded that the GeneXpert MTB/RIF test is more sensitive and specific than the TB-LAMP and both techniques are more sensitive and specific than the conventional acid-fast staining method for both pulmonary and extra-pulmonary specimens. Despite culture remaining the gold standard for laboratory confirmation of TB, GeneXpert MTB/RIF and TB-LAMP should become standard practice for patients with suspected TB, and all clinicians and public health programs for TB control should have access to molecular testing for TB to shorten diagnosis time. Keywords. Mycobacterium tuberculosis; Pulmonary TB; Extrapulmonary TB; GeneXpert MTB/RIF; TB-LAMP; A.F.B

Diagnostic Performance of Xpert MTB/RIF Ultra Compared with Predecessor Test, Xpert MTB/RIF, in a Low TB Incidence Setting: a Retrospective Service Evaluation.

ABSTRACTThe aim of this study was to evaluate the performance of Xpert MTB/RIF Ultra (Ultra) compared with its predecessor, Xpert MTB/RIF (Xpert), in the diagnosis of tuberculosis (TB) in a low TB incidence country. Retrospective analysis was performed on 689 clinical samples received between 2015 and 2018, on which Xpert was performed, and on 715 samples, received between 2018 and 2020, on which Ultra was performed. Samples were pulmonary (n = 830) and extrapulmonary (n = 574) in nature, and a total of 264 were culture positive for Mycobacterium tuberculosis complex (MTBC). The diagnostic performance of both assays was analyzed using culture as the reference standard. The sensitivity of Ultra for culture positive (smear positive and smear negative) MTBC samples, was 93.2% (110/118) compared with 82.2% (120/146) for Xpert (P = 0.0078). In smear negative-culture positive samples, Ultra had a sensitivity of 74.2% (23/31) versus 36.11% (13/36) for Xpert (P = 0.0018). Specificity of both assays was comparable at 94.8% (566/597) for Ultra and 95.8% (520/543) for Xpert (P = 0.4475). The sensitivity of Ultra and Xpert assays among exclusively pulmonary samples was 95.3% (82/86) and 90.3% (84/93), respectively (P = 0.1955), and 87.5% (28/32) and 67.9% (36/53), respectively, among extrapulmonary samples (P = 0.0426). Ultra showed improved performance compared with Xpert in a low TB incidence setting, particularly in smear negative and extrapulmonary MTBC disease. The specificity of Ultra was lower than Xpert, however, this was not statistically significant.IMPORTANCE The study demonstrates the improved sensitivity of the Ultra compared with the Xpert, particularly in smear negative TB disease, for both pulmonary and extrapulmonary samples in a low TB incidence setting. Cycle threshold (Ct) value for both assays was found to positively correlate with time to TB culture positivity, suggesting that Ct and semiquantitative results could be used as indicators of sample MTBC bacillary burden, and thus, perhaps, of transmission potential. This may have implications for the designation of patient isolation precautions.

Isoniazid resistance in patients with Extrapulmonary Tuberculosis using line probe assay in a private multispecialty hospital in Bangalore, India

We aimed to study the rate of isoniazid (INH) resistance in Extrapulmonary Tuberculosis samples from a private care setting.A Line probe assay was performed on 74 culture isolates of Mycobacterium tuberculosis or directly on extrapulmonary samples received in our laboratory from 2018 to 2021.The INH mono-resistance among these extrapulmonary samples was 6.7%. (5 among 74) (95% CI: 1.04%–12.48%) Resistance to rifampicin was not detected.Increasing the availability and leveraging public private partnerships in hospitals for universal testing for INH resistance may increase detection of INH monoresistance in EP-TB and improve the strategy for TB elimination.

Combination of Xpert® MTB/RIF and DetermineTM TB-LAM Ag improves the diagnosis of extrapulmonary tuberculosis at Jimma University Medical Center, Oromia, Ethiopia.

Ethiopia is one of the high burden countries for extrapulmonary tuberculosis (EPTB); however, the prompt diagnosis of EPTB remains challenging. This study is aimed to evaluate the diagnostic performance of Xpert MTB/RIF and DetermineTM TB-LAM Ag (TB-LAM) for the prompt diagnosis of EPTB in Ethiopia. A total of 147 presumptive EPTB patients, including 23 HIV- positive participants were enrolled. Extra-pulmonary samples were collected from all presumptive EPTB cases and tested for Mycobacterium tuberculosis complex (MTBC) using fluorescent microscopy, Xpert MTB/RIF, and culture. Additionally, urine samples were also collected from 126 participants and were tested by DetermineTM TB-LAM Ag (Alere Inc, Waltham, USA). The Sensitivity and specificity of Xpert and TB- LAM tests were calculated by comparing with a composite reference standard (CRS), which comprises smear microscopy, culture and response to empirical anti-TB treatment. Of 147 patients, 23 (15.6%) were confirmed EPTB cases (culture-positive), 14 (9.5%) were probable EPTB (clinically, radiologically or cytologically positive and received anti-TB treatment with good response), and 110 (74.8%) were classified as "non- TB" cases. Compared to the composite reference standard (CRS), the overall sensitivity and specificity of Xpert MTB/RIF were 43.2% and 100%, respectively with the highest sensitivity for Lymph node aspirate (85.7%) and lower sensitivity for pleural fluid (14.3%) and 100% specificity for all specimen types. The sensitivity and specificity of TB-LAM were 33.3% and 94.4% respectively with the highest sensitivity for HIV co-infected participants (83.3%). The sensitivity of the combination of Xpert MTB/RIF and TB-LAM tests regardless of HIV status was 61.1% whereas the sensitivity was improved to 83.3% for HIV-positive cases. TB-LAM alone has low sensitivity for EPTB diagnosis; however, the combination of TB-LAM and Xpert MTB/RIF improves the diagnosis of EPTB particularly for countries with high EPTB and HIV cases.

Molecular studies on extra-pulmonary tuberculosis among patients attending Aminu Kano Teaching Hospital, Kano, Kano State, Nigeria

In 2010 World Health Organization reported that, one third of the World population had tuberculosis, and it is among the infectious disease with significant morbidity and mortality. Mycobacterium tuberculosis is a successful pathogen that has evolved several mechanisms to manipulate the host immune response. The purpose of this study was to determine the incidence of extra-pulmonary TB in patient that visited DOTs clinic, Aminu Kano Teaching Hospital, Kano Nigeria for treatment. A prospective cross-sectional laboratory based study that involved the use of questionnaire and consent/assent form prior to sample collection. Extra pulmonary tuberculosis samples were tested by MTB/RIF assay using GeneXpert. Descriptive statistics was used to analyzed the data obtained and results were given in charts and tables. Out of 71 samples analyzed along with positive control (H37RV) and molecular grade water as a negative control, 3 (4.2 %) samples had extra-pulmonary TB, among which 2 (2.8 %) were from urine samples and 1 (1.4 %) from acetic fluid. This study shows the existence of extra-pulmonary tuberculosis in the study area; and the tuberculosis was sensitive Mycobacterium tuberculosis. It is therefore important to make screening of patients with sign and symptoms of extra pulmonary tuberculosis using Xpert MTB/RIF assay as regular method for extra pulmonary tuberculosis diagnosis. This will be achieved through enlightenment of the clinicians in our health facilities, our States and local governments should strengthen the laboratory capacity for diagnosis and make the services available and reachable to the patients who need them.

RNA SARS-CoV-2 Persistence in the Lung of Severe COVID-19 Patients: A Case Series of Autopsies.

The exact role of viral replication in patients with severe COVID-19 has not been extensively studied, and it has only been possible to demonstrate the presence of replicative virus for more than 3 months in a few cases using different techniques. Our objective was to study the presence of RNA SARS-CoV-2 in autopsy samples of patients who died from COVID-19 long after the onset of symptoms. Secondary superimposed pulmonary infections present in these patients were also studied. We present an autopsy series of 27 COVID-19 patients with long disease duration, where pulmonary and extrapulmonary samples were obtained. In addition to histopathological analysis, viral genomic RNA (gRNA) and viral subgenomic RNA (sgRNA) were detected using RT-PCR and in situ hybridization, and viral protein was detected using immunohistochemistry. This series includes 26 adults with a median duration of 39 days from onset of symptoms to death (ranging 9–108 days), 92% of them subjected to immunomodulatory therapy, and an infant patient. We detected gRNA in the lung of all but one patient, including those with longer disease duration. SgRNA was detected in 11 out of 17 patients (64.7%) with illness duration up to 6 weeks and in 3 out of 9 patients (33.3%) with more than 6 weeks of disease progression. Viral protein was detected using immunohistochemistry and viral mRNA was detected using in situ hybridization in 3 out of 4 adult patients with illness duration of <2 weeks, but in none of the 23 adult patients with an illness duration of >2 weeks. A remarkable result was the detection of viral protein, gRNA and sgRNA in the lung cells of the pediatric patient after 95 days of illness. Additional pulmonary infections included: 9 acute bronchopneumonia, 2 aspergillosis, 2 cytomegalovirus, and 1 BK virus infection. These results suggest that in severe COVID-19, SARS-CoV-2 could persist for longer periods than expected, especially in immunocompromised populations, contributing to the persistence of chronic lung lesions. Additional infections contribute to the fatal course of the disease.

Detection of multidrug and extensively drug-resistance and mutation pattern in geriatric patients from North Indian referral institute

IntroductionGeriatric population are predisposed to reactivation to tuberculosis (TB) and multi-drug resistance (MDR) due to deteriorated immune system. Limited data is available in this population hence present study is undertaken to study drug resistance and associated mutations among geriatric presumptive DR-TB patients by genotypic methods MethodsFrom October 2011 to December 2018, demographic characteristics of enrolled patients was collected. Smear-positive processed sputum samples were subjected directly while cultures positive for Mycobacterium Tuberculosis (MTB) from smear-negative pulmonary and all extra-pulmonary samples were subjected to LPA. The LPA used were Genotype MTBDR plus (1st line LPA) for detection of susceptibility to rifampicin (RIF) and isoniazid (INH) and Genotype MTBDR sl (2nd line LPA), for susceptibility to fluoroquinolones (FQ) and aminoglycosides (AG). ResultsTotal of 2041 samples were received from presumptive MDR-TB patients above 60 years of age during study period, of which 1406; 68.9% were within 60–70 year followed by 495; 24.3% within 71–80 year and 140; 6.9% more than 80 years. Total of 1055 MTB were detected, of which those diagnosed as RIF resistant were 117/1055; 11.2% including 89/1055; 8.5% MDR-TB and resistance to INH was in 84/1055; 8%. Total 67, 2nd line LPA gave valid results, of which 19/67 (28.4%) isolates were resistant to only FQ, and one isolate was resistant to AG. ConclusionStudy finding highlights need for dedicated efforts for diagnosis, and treatment of geriatric tuberculosis. Suitable intervention at programmatic country level at country will help in strengthening tuberculosis control strategies in this population.

Machine learning analysis of SERS fingerprinting for the rapid determination of Mycobacterium tuberculosis infection and drug resistance

Over the past decades, conventional methods and molecular assays have been developed for the detection of tuberculosis (TB). However, these techniques suffer limitations in the identification of Mycobacterium tuberculosis (Mtb), such as long turnaround time and low detection sensitivity, etc., not even mentioning the difficulty in discriminating antibiotics-resistant Mtb strains that cause great challenges in TB treatment and prevention. Thus, techniques with easy implementation for rapid diagnosis of Mtb infection are in high demand for routine TB diagnosis. Due to the label-free, low-cost and non-invasive features, surface enhanced Raman spectroscopy (SERS) has been extensively investigated for its potential in bacterial pathogen identification. However, at current stage, few studies have recruited handheld Raman spectrometer to discriminate sputum samples with or without Mtb, separate pulmonary Mtb strains from extra-pulmonary Mtb strains, or profile Mtb strains with different antibiotic resistance characteristics. In this study, we recruited a set of supervised machine learning algorithms to dissect different SERS spectra generated via a handheld Raman spectrometer with a focus on deep learning algorithms, through which sputum samples with or without Mtb strains were successfully differentiated (5-fold cross-validation accuracy = 94.32%). Meanwhile, Mtb strains isolated from pulmonary and extra-pulmonary samples were effectively separated (5-fold cross-validation accuracy = 99.86%). Moreover, Mtb strains with different drug-resistant profiles were also competently distinguished (5-fold cross-validation accuracy = 99.59%). Taken together, we concluded that, with the assistance of deep learning algorithms, handheld Raman spectrometer has a high application potential for rapid point-of-care diagnosis of Mtb infections in future.

Evaluation the role of conventional and Xpert MTB/RIF assays as point-of-care tests of Mycobacterium tuberculosis infections, especially during the COVID-19 pandemic in Menoufia, Egypt

BackgroundTuberculosis (TB) is a destructive pulmonary disease, which was the most fatal infectious disease in the world for many years before the COVID-19 outbreak. During pandemic, COVID-19 was the main concern in every clinic and there were overlapping respiratory diseases resulting in delaying of the diagnosis and treatment of TB. Xpert MTB/RIF assay and Ziehl–Neelsen (ZN) stain are the most commonly used point-of-care test (POCT) assays for TB that were endorsed by WHO allowing a quick treatment turnaround time of a few minutes or hours, hence avoiding patient loss to follow-up. The aim of the study was to evaluate the role of Xpert MTB/RIF as a POCT for early, rapid, and accurate diagnosis of pulmonary and extrapulmonary TB during the COVID-19 pandemic and its role for exclusion of non-mycobacterial TB infections and to evaluate the proportion of patients with active pulmonary TB among COVID-19 patients and to study the difference of some inflammatory markers between patients with COVID-19, patients with pulmonary TB, and patients infected by both TB and COVID-19.Patients and methodsThis study was conducted from February 2018 to December 2021 (including the peak period of COVID-19 on 835 suspected TB patients (629 + 206 suspected COVID-19 patients six of them were proved pulmonary TB). Patients were from Shebin El-Kom Teaching Hospital and Chest hospital, Menoufiya). All 835 (pulmonary and extrapulmonary samples) patients were tested by gene Xpert MTB/RIF including 441 of them tested by ZN only. For detection of sensitivity, specificity positive predictive value (PPV), negative predictive value (NPV), and accuracy we selected 103 samples who were tested by the three methods (gene Xpert MTB/RIF, ZN staining, and culture on LJ media). For studying the difference of some inflammatory markers between patients with COVID-19, patients with pulmonary TB, and patients infected by both TB and COVID-19, 206 patients who were suspected of comorbid TB and COVID-19 during the pandemic were divided into three groups: group I positive for TB and COVID-19 (N = 6), group II positive COVID-19 only (N = 100), and group III were positive pulmonary TB only (N = 50) (NB: 50 patients were excluded due to incomplete data). Blood samples were taken for complete blood count, erythrocyte sedimentation rate, malondialdehyde, interleukin-6, C-reactive protein, D-dimer, ferritin, lactate dehydrogenase, calprotectin, and procalcitonin. Nasal swabs were needed for confirmation of COVID-19 by PCR.ResultsCompared with culture as a gold standard, sensitivity, specificity, PPV, and NPV for ZN smear were 77.1, 100, 100, and 53.8%, respectively. As regards the results of XPERT MTB/RIF assay, from the 103 samples examined, 89 (86%) were positive and 14 (14%) were negative. Eight false-positive results were recorded, compared with culture. The sensitivity was 98.8%, specificity was 61.9%, PPV was 91%, and NPV was 92.8%. There was a significant increase within groups in MDA, procalcitonin, ESR, and calprotectin with P value of 0.22, 0.015, 0.000, and 0.009, respectively.ConclusionXpert MTB/RIF as POCT for TB diagnosis is more sensitive and specific than traditional methods of diagnosis using ZN to overcome the challenges with weak testing infrastructure especially during the COVID-19 pandemic. Serum calprotectin was significantly increased in the COVID-19 group compared with the TB group C-reactive protein, which was significantly increased in the TB group compared with COVID-19 group.

COMPARISON BETWEEN FLUORESCENT MICROSCOPY AND DUPLEX PCR TO DETECT MYCOBACTERIUM BOVIS AND MYCOBACTERIUM TUBERCULOSIS IN TUBERCULOSIS SUSPECTED PATIENTS

Objective: The aim of this study was to compare the gold-standard fluorescent microscopy as a diagnostic tech­nique with the PCR test, an advance molecular technique. Methodology: A total of 200 suspected pulmonary and extra-pulmonary samples were taken and stored for analysis in Quetta city. Samples were tested for Mycobacterium tuberculosis and Mycobacterium bovis by both the gold standard fluorescent microscopy (auramine-O FM) and molecular technique duplex PCR. By duplex PCR, Mycobacterium tuberculosis’s 245bp sequence and Mycobacterium bovis’s 500bp sequence was detected by using specific primers. Results: Among 200 pulmonary and extra-pulmonary samples, fluorescent microscopy detected 31 positive cas­es, while PCR detected 60 and 2 positive cases for Mycobacterium tuberculosis and Mycobacterium bovis respec­tively. The PCR analysis showed 28% of male patients and 32% female patients as positive for M. tuberculosis. Moreover, about52/164 pulmonary samples and 8/36 Extra-pulmonary samples were detected to be positive by PCR analysis. Conclusion: The PCR results were more accurate, rapid, sensitive and specie specific for detection of tuberculosis showing 60 positive cases for Mycobacterium tuberculosis and 2 positives for Mycobacterium bovis with a sig­nificant p-value. On the other hand, FM detected Mycobacterium tuberculosis with comparatively lower sensitivity with only 31 positive cases and had failed to distinguish between species.

Susceptibility pattern of Mycobacterium tuberculosis over a period of five years at Indus Hospital and Health Network, Karachi, Pakistan.

Objective:To determine the susceptibility pattern and frequency of isolation of multidrug, pre-extensively drug and extensively drug resistant TB in a tertiary care hospital in Karachi, Pakistan.Method:A cross-sectional study was designed. Samples received in the lab were processed for growth and sensitivity testing of Mycobacterium tuberculosis. Isolation of MTB was done on Mycobacteria growth indicator tube (MGIT) followed by identification using MPT64. Samples were than evaluated for drug sensitivity against first and second-line antimycobacterial drugs. Statistical analysis was performed using SPSS version 24.0.Results:Of the 20014 samples received, 23.1% were identified as Mycobacterium tuberculosis. Drug sensitivity testing was performed on 95.9% isolates. Fifty-two percent samples were from males and 48% female patients. The study found statistically non-significant relationship between gender and likelihood of disease with drug-resistant (DR)-MTB organisms. The rate of isolation of MDR-TB was highest (43%) among ages 25-55 years and previously treated patients compared to newly diagnosed patients (62% vs 36%). Among MTB positive samples, 91.5% were pulmonary while 8.5% were extrapulmonary samples. Extrapulmonary samples were more likely to be sensitive to antimycobacterial drugs. The highest resistance was observed against Isoniazid (pulmonary=58%; extrapulmonary=12.7%), Rifampicin (pulmonary=58.7%; extrapulmonary=8.2%), and Levofloxacin (pulmonary=29.2%; extrapulmonary=20%).Conclusion:A considerable number of drug resistant tuberculosis cases were identified in the present study. It is essential to develop further strategies to reduce the spread of this disease.

Evaluation of the performance of the BD MAX MDR-TB test in the diagnosis of Mycobacterium tuberculosis complex in extrapulmonary and pulmonary samples

ABSTRACT Background The BD MAX MDR-TB is a recently marketed molecular test for detecting Mycobacterium tuberculosis complex (MTC), rifampin, and isoniazid drug resistance. Research Design and Methods This study aimed to evaluate the BD MAX MDR-TB test performance in 933 extrapulmonary and 774 pulmonary samples. Results Test MTC detecting sensitivity was 90.6%, 82.5%, and the specificity was 98.5%, 98.9%, in pulmonary and extrapulmonary samples, respectively. In smear-positive samples, sensitivity, and specificity were 100% for all samples. However, in smear-negative samples, the test’s sensitivity and specificity were 82.3%, 98.5% in pulmonary samples, and 76.7%, 98.9% in extrapulmonary samples. Test sensitivity in detecting isoniazid resistance was 71.4%, specificity 96.8%, and in detecting rifampin resistance was 100%, specificity 93.9%, respectively. Conclusions BD MAX MDR-TB is a reliable, rapid, user-friendly test for detecting MTC in extrapulmonary and pulmonary samples and its resistance toward isoniazid and rifampin. It can be used as an alternative to the Xpert system assays.

Performance of Real-Time Semi-Quantitative Polymerase Chain Reaction Assay for Optimum Diagnosis of Extrapulmonary Tuberculosis and Sensitivity to Rifampin in a Tertiary Care Center.

Tuberculosis represents a public health problem, with extrapulmonary disease occurring in 15% of incident cases annually. Early diagnosis is a challenge due to its paucibacillary nature. Recently, a molecular real-time semi-quantitative assay (GeneXpert Ultra) was developed to overcome limitations of the previous assay version (Xpert MTB/RF). The objective of the study was to assess the usefulness of the novel next-generation GeneXpert assay in extrapulmonary samples from different anatomic sites under routine diagnostic conditions at a university medical center. A total of 519 samples from patients with presumptive diagnosis of extrapulmonary TB were subjected to smear microscopy, culture, and molecular assay. Univariate analyses for demographic and microbiological characteristics were performed. The sensitivity, specificity, and Kappa index with a 95% confidence interval were determined. Molecular assay was positive in 53 samples (10.2%), of which 38 (71.6%) belonged to the "low" and "trace" semi-quantitative categories. The overall sensitivity and specificity were 86.4% (95% confidence intervals [CI]: 74.1-98.8) and 95.6% (95% CI: 93.7-97.6), respectively. Phenotypic drug susceptibility testing for rifampin was 100% concordant. Molecular assay showed significant results when compared to other standard tests, making it a useful tool that could lead in the improvement to a rapid diagnosis of extrapulmonary disease.

Tracing TB: Are there predictors for active TB disease in patients with Xpert Ultra trace results?

ObjectivesThe Xpert MTB/Rif Ultra (Xpert Ultra; Cepheid, USA) has increased sensitivity compared with its predecessor (Gene Xpert), due to the addition of multicopy amplification targets and a novel trace call. The World Health Organization suggests that tuberculosis (TB) treatment should be initiated in HIV, paediatric, and extra-pulmonary TB patients with trace results. However, other factors such as previous TB disease may complicate the interpretation of trace results in high-burden TB settings. This study aimed to clarify the positive predictors for active TB disease in patients with trace results and to investigate if previous TB disease influences TB culture positivity. MethodsA retrospective descriptive study was performed on 290 patients with trace results, to determine what the positive predictors for active TB are by comparing clinical factors to TB culture. ResultsThe key findings of this study were that extra-pulmonary TB samples (OR, 2.7; p=0.012), no previous TB disease (OR, 4.5; p=0.001) and symptoms suggestive of TB (OR, 6.4; p<0.001) are independent predictors for active TB disease. ConclusionThis study found readily available clinical predictors that can aid clinicians with TB management decisions in patients with trace results.