Detection Of MTB Research Articles

Advancing proficiency testing for ultra in resource-limited settings using dried tube specimen: A study by SRL-Uganda.

Proficiency testing (PT) has been hard to set up due to cost limitations and technical capacity. Conventional Xpert MTB/RIF PT programs use liquid and culture spots which require stringent storage and transportation conditions with cross-contamination chances prevalent. These setbacks prompted the use of dried tube specimens (DTS) for Ultra assay PT. For continuity of PT provision, stability of DTS and compatibility with testing protocols when kept for a long period needs to be established. DTS were prepared from known isolates inactivated using a hot air oven at 85°C. 100μl of bacterial suspensions were aliquoted and dried inside a Biosafety cabinet. Panel validation was done to establish the baseline Deoxyribonucleic acid (DNA) concentration in terms of cycle threshold (Ct) value. DTS aliquots were shipped to participants to test and report within six weeks. The remaining DTS were kept at 2-8°C and room temperature for one year with testing at six months. Twenty (20) DTS samples per set remaining at one year were heated at 55°C for two weeks before testing. The means of the different samples were compared to validation data using paired t-tests. Boxplots were designed to visualize the differences in the medians of the DTS. Overall mean Ct value increased by 4.4 from the validation to testing after one year at the different storage conditions. Samples heated at 55°C showed a 6.4 Ct difference from validation data. Testing done at six months on 2-8°C stored items showed no statistical difference. At all the remaining testing times and conditions, P-values were less than 0.008 although the absolute mean Ct when compared showed slight increments and accommodated differences for the detection of MTB and rifampicin resistance. Median values for samples stored at 2-8°C were lower compared to those at room temperature. DTS stored at 2-8°C remain more stable for one year compared to higher temperatures and can be consistently used as PT materials in more than one PT round for biannual PT providers.

A Comparative Study on Visual Detection of Mycobacterium tuberculosis by Closed Tube Loop-Mediated Isothermal Amplification: Shedding Light on the Use of Eriochrome Black T.

Loop-mediated isothermal amplification is a promising candidate for the rapid detection of Mycobacterium tuberculosis. However, the high potential for carry-over contamination is the main obstacle to its routine use. Here, a closed tube LAMP was intended for the visual detection of Mtb to compare turbidimetric and two more favorable colorimetric methods using calcein and hydroxy naphthol blue (HNB). Additionally, a less studied dye (i.e., eriochrome black T (EBT)) was optimized in detail in the reaction for the first time. Mtb purified DNA and 30 clinical specimens were used to respectively determine the analytical and diagnostic sensitivities of each method. The turbidimetric method resulted in the best analytical sensitivity (100 fg DNA/reaction), diagnostic sensitivity and specificity (100%), and time-to-positivity of the test (15 min). However, this method is highly prone to subjective error in reading the results. Moreover, HNB-, calcein-, and EBT-LAMP could respectively detect 100 fg, 1 pg, and 1 pg DNA/reaction (the analytical sensitivities) in 30, 15, and 30 min, while the diagnostic sensitivity and specificity were respectively 93.3% and 100% for them all. Interestingly, EBT-LAMP showed the lowest potential for subjective error in reading the results. This report helps judiciously choose the most appropriate visual method, taking a step forward toward the field applicability of LAMP for the detection of Mtb, particularly in resource-limited settings.

Evaluation of the GeneXpert Mycobacterium tuberculosis/Rifampicin Assay for Early Detection of Extrapulmonary Tuberculosis and Rifampicin Resistance in Aligarh Region of Northern India

Purpose: Early diagnosis of extrapulmonary tuberculosis (TB) with simultaneous detection of rifampicin (RIF) resistance (RR) directly from the sample is a revolution brought by GeneXpert Mycobacterium TB (MTB/RIF) assay. Line probe assay (LPA) is not recommended for extrapulmonary/nonsputum specimens; GeneXpert MTB/RIF provides a diagnostic option for these. Hence, the following study aimed to evaluate the performance of GeneXpert MTB/RIF assay in nonsputum samples. Materials and Methods: A total number of 642 extrapulmonary and nonsputum respiratory samples were subjected to Ziehl–Neelsen (ZN) staining technique, fluorescent microscopy (FM), and GeneXpert MTB/RIF Assay. All samples were cultured on a solid Lowenstein–Jensen (LJ) medium and culture-positive samples were subjected to GenoType MTBDRplus LPA and drug susceptibility testing by 1% proportion method on a solid LJ medium. Results: GeneXpert MTB/RIF assay detected MTB in 25.9% (166/642) of the samples. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of ZN staining technique and FM were 21.52%, 99.35%, 91.89%, and 78.77% and 27.22%, 99.78%, 97.73%, and 80.07%, respectively. Sensitivity, specificity, PPV, and NPV of GeneXpert MTB/RIF in the detection of MTB were 93.75%, 96.53%, 90.36%, and 97.80%, and RIF RR was 92.0%, 97.74%, 88.46%, and 98.48%, respectively. LPA performed on positive culture growth showed 100% sensitivity and specificity in the detection of RIF and isoniazid resistance. Conclusion: GeneXpert MTB/RIF showed high sensitivity and specificity in detecting MTB and simultaneous RIF RR among the nonsputum samples which had a good concordance to LPA. It provides a judicious option for early detection of multidrug-resistant TB in extrapulmonary and nonsputum respiratory samples.

Trends of Xpert MTB/RIF in the diagnosis of Mycobacterium tuberculosis and rifampicin resistance in Southwest Nigeria: A 4-year retrospective study

Objectives: In recent years, there has been an increased uptake of Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) for the diagnosis of tuberculosis (TB), with added benefits for detecting rifampicin-resistant TB (RR-TB). We set out to determine the trends in notification of MTB and RR-TB over 4 years in a tertiary hospital in Southwest Nigeria. Materials and Methods: This is a retrospective analysis of single early morning sputum/gastric washing sample for presumed TB in a tertiary health center between January 2016 and December 2019. Xpert MTB/RIF was used to detect (MTB) and RR-TB. Results: The mean age of the 4625 presumptive TB patients was 43.4 (18.8) years, with the majority aged 31–45 (30.6%). Males totaled 2247 (49.6%). Human immunodeficiency virus (HIV) coinfection was found in 756 (16.7%) of them. Using the Xpert MTB/RIF assay, the overall MTB notification was 12.9% (584/4526) and was associated with HIV status, P < 0.001. MTB notification trends increased from 5.6% in 2016 to 23.8% in 2019, P < 0.001. The overall yield of RR-TB was 7.5%. The proportion of RR-TB from TB cases declined from 28% in 2016 to 4.6% in 2019. Forty-two of the 44 cases of the RR-TB (42/572; 7.3%, [95% CI: 5.31, 9.75]) were new cases, with TB treatment failure and relapse cases accounting for 10.1% (2/11; 10.1%, [95% CI: 0.35, 42.5]) of all RR-TB cases (P < 0.01). RR-TB was associated with being older than 45 years (adjusted odds ratio = 2.046, [95% CI: 1.046, 4.004]). HIV infection status and gender had no effect on RR-TB status. Conclusion: This study found an increase in MTB detection with Xpert MTB/RIF utilization. Ages >45 years have 2–4-fold increased risk of developing RR-TB and should be targeted for drug resistance prevention.

Culture filtrate supplementation can be used to improve Mycobacterium tuberculosis culture positivity for spinal tuberculosis diagnosis.

Culture remains the gold standard to diagnose spinal tuberculosis (STB) despite the paucibacillary nature of the disease. Current methods can take up to 42 days to yield a result, delaying the ability to rapidly detect drug resistance. Studies have demonstrated the use of supplementation with culture filtrate (CF) from an axenic culture of Mycobacterium tuberculosis (Mtb) as a source of growth factors to improve culture rates. Our objective was to test a modified culture assay, utilizing CF supplemented media (CFSM), to improve culture positivity rates for suspected STB. Twelve patients with suspected STB were assessed by conventional culture (BACTEC™ MGIT 960), GeneXpert™ and standard histopathological examination. Spinal biopsies were taken from areas of diseased vertebral tissue or abscess, predetermined from MRI. Additional biopsies were obtained to assess CFSM for improved detection and faster culture of Mtb. All cases were diagnosed as STB and treated empirically for tuberculosis based on either bacteriological evidence (GeneXpert™, MGIT and/or CFSM positive), or based on clinical presentation. 5 specimens (45.45%) were positive for Mtb DNA as detected by GeneXpert™ and 1 specimen (8.33%) was cultured using MGIT (time to detection; 18 days). CFSM was able to culture 7 specimens (58.3%), with all CFSM positive specimens yielding a culture within 14 days. Two samples were positive only using the CFSM assay pointing to additional yield for diagnostic workup. Modification of standard culture can improve detection of Mtb and reduce time to positivity in individuals with STB where culture material is a requirement.

Role of GeneXpert MTB/RIF assay for the diagnosis of cervical lymph node tuberculosis and rifampicin resistance.

Tuberculosis (TB) of lymph node (TB lymphadenitis) is one of the most common forms of extrapulmonary TB (EPTB) whose diagnosis is critically challenging. Although new diagnostic methods have been developed, especially in patients without a history of TB, the cervical tuberculous lymphadenitis diagnosis is still elusive. This study assessed the applicability of GeneXpert in early diagnosis of EPTB, especially cervical lymphadenopathy. The study was conducted in a tertiary care hospital from January 2018 to December 2020 at the department of microbiology. All the samples of cervical lymph node tissue and lymph node aspirate were followed as per the routine protocol for mycobacterial identification. The sample was divided into two parts: one was used for the new molecular-based GeneXpert MTB/RIF assay and the second one was tested by direct and concentrated acid-fast bacilli microscopy by Z-N staining and culture for the detection of MTB. Among the 145 samples tested, the GeneXpert detected the DNA of MTB in 89 samples (61.37%), whereas the culture test was positive in 42 (28.93%) specimens. GeneXpert also detected 7 rifampicin resistance cases. GeneXpert sensitivity and specificity results were assessed according to culture results. The sensitivity and specificity of the GeneXpert assay were 85.71% and 48.54%, respectively. GeneXpert MTB/RIF should be used in conjunction with clinical presentation and other molecular investigation in nonrespiratory specimens.

Review on Drug Resistant Tuberculosis

Abstract Introduction: An estimated 9.9 million people fell ill with tuberculosis globally in 2020 with India and China contributing a major percentage to the burden of TB. India is grouped under high TB, high HIV associated TB and MDR TB burden countries and 1.24 lakh fell ill with drug resistant TB out of which 56000 were started on second line treatment in 2020. Annually India accounts for 27% of missing TB cases. Diagnosis: The major forms of drug resistant TB that are of clinical importance are INH monoresistant TB, multidrug resistant TB, pre- XDR TB and XDR TB.WHO approved newer molecular tests for MTB detection and drug susceptibility tests. Treatment: Few newer drugs and few previously used drugs are showing promise when used in combination which have come up in recent years. Bedaquiline based regimens are showing improved cure rates. Conclusion: Guidelines based regimens should be strictly adhered to by both public and private TB case treating physicians.

Effect of Laser Bio stimulation and Bone Graft in Treatment of a Large Periapical Lesion – A CBCT Analysis

India accounts for 23% of global TB burden. Of these 38% are of Extra Pulmonary Tuberculosis. Lymph node TB is the most common EPTB, pleural effusion is second most common type of EPTB in India. The bacteriological confirmation to diagnose EPTB is more difficult due to its paucibacillary nature. This study aims assessing the comparison of ADA with Gene- Xpert MTB RIF in pleural fluid in the diagnosis of tubercular pleural effusion. Study was a cross-sectional study carried out at the Department of Respiratory Medicine and Department of Internal Medicine, Muzaffarnagar Medical College, and School of Medical Science and Research, Greater Noida, Gautam Budh Nagar, UP from January 2018 to March 2019. Paired and unpaired Student's t-test, Chi square test, P < 0.05 was considered statistically significant; SPSS 18.0 software was used for analysis. The detection of MTB and rifampicin resistance using the Gene-Xpert MTB/RIF with pleural fluid ADA assay was assessed in 66 specimens. With ADA cut off as 40IU/L, 60 cases had an ADA > 40IU/L and 6 cases had ADA < 40IU/L. Pleural fluid Gene-Xpert detected MTb in 22 samples while in 44 samples MTB not detected. All pleural fluid MTb detected by Gene-Xpert were rifampicin sensitive. Gene-Xpert detected 2 tubercular pleural effusion in which ADA level was less than 40. Study shows that there was no statistically significance in diagnostic value between Gene- Xpert and ADA pleural fluid however Gene-Xpert MTB/RIF is useful method for rapid detection of MTB and Rifampicin resistance simultaneously. Estimation of ADA in pleural fluid is rapid inexpensive laboratory investigation where the diagnosis is uncertain. We found no significant connection between CBNAAT positivity and ADA level. In developing country lymphocyte predominant effusions with high ADA levels (> 40 U/L) is better option but definitely combined use of ADA and Gene-Xpert is better than using individually for the diagnosis of Tubercular pleural effusion

Xpert MTB/RIF Ultra outperformed the Xpert assay in tuberculosis lymphadenitis diagnosis: a prospective head-to-head cohort study

ObjectivesXpert Mycobacterium tuberculosis/rifampin (MTB/RIF) Ultra (Xpert-Ultra) has shown better sensitivity in comparison with Xpert MTB/RIF (Xpert) in extrapulmonary tuberculosis (TB), whereas the head-to-head comparison of these methods in TB lymphadenitis had barely been performed. MethodsPatients with undiagnosed lymphadenopathy were recruited prospectively and consecutively, and fine-needle aspiration (FNA) biopsy or lymph node tissue was collected. The specimen was subjected to smear, culture, Xpert, and Xpert-Ultra assays. Culture and/or smear for acid-fast bacilli (AFB) or AFB observed on histopathology were performed as a reference. ResultsA total of 106 participants were recruited, including 41 confirmed TB, 33 probable TB, and 32 non-TB lymphadenopathies. The head-to-head comparison for MTB detection showed that Xpert-Ultra produced the highest sensitivity when compared with smear, culture, and Xpert (75.7% vs 5.4 %, 13.5%, and 48.7%). When Xpert-Ultra outcomes were integrated for diagnosis, the percentage of confirmed TB lymphadenitis cases increased from 55.4% (41/74) to 85.1% (63/74). The sensitivities of Xpert-Ultra and Xpert on tissue were 73.6% (95% CI: 59.4-84.3) and 39.6% (95% CI: 26.8-54.0), respectively. The sensitivity of Xpert-Ultra on FNA samples (81.0%, 95% CI: 57.4-93.7) was higher than that of Xpert (71.4%, 95% CI: 47.7-87.8). ConclusionXpert-Ultra detected significantly more TB lymphadenitis cases than Xpert or culture. This superiority was particularly distinct using lymph node tissue than FNA detection.

Next-Generation Metagenome Sequencing Shows Superior Diagnostic Performance in Acid-Fast Staining Sputum Smear-Negative Pulmonary Tuberculosis and Non-tuberculous Mycobacterial Pulmonary Disease.

In this study, we explored the clinical value of next-generation metagenome sequencing (mNGS) using bronchoalveolar lavage fluid (BALF) samples from patients with acid-fast staining (AFS) sputum smear-negative pulmonary tuberculosis (PTB) and non-tuberculous mycobacterial pulmonary disease (NTM-PD). Data corresponding to hospitalized patients with pulmonary infection admitted to the hospital between July 2018 and July 2021, who were finally diagnosed with AFS sputum smear-negative PTB and NTM-PD, were retrospectively analyzed. Bronchoscopy data as well as mNGS, Xpert, AFS (BALF analysis), and T-SPOT (blood) data, were extracted from medical records. Thereafter, the diagnostic performances of these methods with respect to PTB and NTM-PD were compared. Seventy-one patients with PTB and 23 with NTM-PD were included in the study. The sensitivities of mNGS, Xpert, T-SPOT, and AFS for the diagnosis of PTB were 94.4% (67/71), 85.9% (61/71), 64.8% (46/71), and 28.2% (20/71), respectively, and the diagnostic sensitivity of mNGS combined with Xpert was the highest (97.2%, 67/71). The specificity of Xpert was 100%, while those of AFS and T-SPOT were 73.9% (17/23) and 91.3% (21/23), respectively. Further, the 23 patients with NTM-PD could be identified using mNGS, and in the population with immunosuppression, the sensitivities of mNGS, Xpert, T-SPOT, and AFS were 93.5% (29/31), 80.6% (25/31), 48.4% (15/31), and 32.3% (10/31), respectively, and the diagnostic sensitivity of mNGS combined with Xpert was the highest (100%, 31/31). The specificities of Xpert and T-SPOT in this regard were both 100%, while that of AFS was 40% (2/5). Furthermore, using mNGS, all the NTM samples could be identified. Thus, the analysis of BALF samples using mNGS has a high accuracy in the differential diagnosis of MTB and NTM. Further, mNGS combined with Xpert can improve the detection of MTB, especially in AFS sputum smear-negative samples from patients with compromised immune states or poor responses to empirical antibiotics.

Evaluation of Aptamer Fluorescence Microscopy in the Diagnosis of Pulmonary Tuberculosis.

ABSTRACTSputum smear microscopy for tuberculosis diagnosis has stood the test of time. However, due to its low sensitivity, the positive detection rate for tuberculosis in clinical specimens is not high. To improve the sensitivity of microscopic observation in Mycobacterium tuberculosis (MTB) detection, we developed the MTB-specific aptamer MA1. To further improve the binding reactivity of MA1 with MTB, we constructed a new derivative aptamer with a pocket-stem-loop-structure, MA1-39, and identified it to have high binding reactivity with the MTB reference strain. We developed an aptamer fluorescence microscopy test for MTB based on MA1-39 and evaluated its feasibility for diagnosing pulmonary tuberculosis. Among 56 tested strains, MA1-39 was proven to effectively discriminate MTB from the control strains, including 12 non-tuberculosis mycobacterial (NTM) reference strains, 6 NTM isolates, and 7 other bacteria. Next, this approach was applied to 169 clinical sputum samples from suspected tuberculosis patients and non-tuberculosis controls. Molecular tests together with both clinical and bacteriological identification were used as a protocol to evaluate the efficacy of aptamer detection. Compared with the traditional acid-fast staining light microscope, the aptamer fluorescence microscope showed a higher detection rate for MTB in clinical specimens (48.8% versus 32.6%), and the specificities of the two techniques had almost no significant difference (90.4% versus 94%). In addition, aptamer fluorescence microscopy showed the same positive predictive value (PPV) as staining (84% versus 84.9%), but a higher negative predictive value (NPV; 63% versus 57.3%). In conclusion, the newly established aptamer fluorescence microscopy approach is likely to be a feasible method for microbiological diagnosis of tuberculosis.IMPORTANCE We established an aptamer fluorescence microscopy approach for rapid detection of MTB in clinical sputum samples. The use of aptamer probes was proven to significantly increase the sensitivity of sputum smear microscopy. In resource-limited countries, microscopy is currently a fast, simple, and very common test method in many laboratories, and it will remain the primary means of microbiological diagnosis of tuberculosis in the foreseeable future. Improving detection techniques can further enhance the clinical application value of this ancient diagnostic method. Since aptamer fluorescence microscopy can provide rapid and sensitive results, it may be a feasible and useful method in resource-limited settings.

Mycobacterium tuberculosis antigen-specific T-cell responses in smear-negative pulmonary tuberculosis patients.

Despite recent improvements in microbial detection, smear-negative TB remains a diagnostic challenge. In this study, we investigated the potential discriminatory role of polychromatic flow cytometry of M. tuberculosis antigen-specific T cells to discriminate smear-negative TB from health controls with or without latent TB infection, and non-TB respiratory illnesses in an endemic setting. A cross-sectional study was conducted on HIV negative, newly diagnosed smear-positive PTB (n = 34), smear-negative/GeneXpert negative PTB (n = 29) patients, non-TB patients with respiratory illness (n = 33) and apparently healthy latent TB infected (n = 30) or non-infected (n = 23) individuals. The expression of activation (HLA-DR, CD-38), proliferation (Ki-67), and functional (IFN-γ, TNF-α) T-cell markers using polychromatic flow cytometry was defined after stimulation with PPD antigens. Sputum samples were collected and processed from all patients for Mtb detection using a concentrated microscopy, LJ/MGIT culture, and RD9 typing by PCR. Our study showed CD4 T cells specific for PPD co-expressed activation/proliferation markers together with induced cytokines IFN-γ or TNF-α were present at substantially higher levels among patients with smear-positive and smear-negative pulmonary TB than among healthy controls and to a lesser extent among patients with non-TB illness. Our study conclude that smear-negative TB can be distinguished from non-TB respiratory illness and healthy controls with a flow cytometric assay for PPD-specific T cells co-expressing activation/proliferation markers and cytokines.

Turner syndrome and tuberculosis in adolescent: a case report

Background: Turner syndrome is characterized by abnormality of one X chromosome. Those with Turner syndrome are at risk of developing tuberculosis infection due to decreased immune function. Case: A 17-year-old girl came with complaints of short stature, amenorrhea, and no signs of secondary sexual growth. She also felt weak. On physical examinations, she weighed 24 kg, and her height was 119 cm. Her conjunctiva looked pale. She had a low set hairline, small mandible, short 4th metacarpal, and wide-spaced nipple. Her breast and genitalia did not grow (1st stage of Tanner classification). There was a lesion between her breasts. Laboratory results showed Hemoglobin 7 g/dL, MCV 62,6 fL, MCH 18,2 pg, MCHC 29 g/L, FSH >170 IU/L, LH 95,3 IU/L, Estradiol 11 pg/mL, Iron Ferrozine 7 mg/dL, TIBC 186 μg/dL, and Ferritin 50 ng/mL. Her Mantoux test showed induration with a diameter of 20-23 mm, and the Xpert MTB-RIF test showed low detection of MTB. Her bone age was appropriate for a 13-year-old girl. Histopathology test of her lesion showed chronic granulomatous inflammation. Her chromosome analysis showed Mos 45,X(56)/46,XX(44). She was diagnosed with Turner syndrome and tuberculosis. She received Progynova® 2 mg every 24 hours for 21 days, stopped for 10 days, and continued again for 21 days. She also got anti-tuberculosis drugs (pediatric regime). Conclusion: The clinical spectrums of Turner syndrome vary widely. Decreased immune function in Turner syndrome can lead to infections such as tuberculosis. Early diagnosis and treatment can prevent further morbidity.

Integration of cytopathology with molecular tests to improve the lab diagnosis for TBLN suspected patients.

BackgroundTuberculosis lymphadenitis (TBLN) diagnosis is often challenging in most resource poor settings. Often cytopathologic diagnosis of TBLN suspected patients is inconclusive impeding timely clinical management of TBLN suspected patients, further exposing suspected patients either for unnecessary use of antibiotics or empirical treatment. This may lead to inappropriate treatment outcome or more suffering of suspected patients from the disease. In this study, an integrated diagnostic approach has been evaluated to elucidate its utility in the identification of TBLN suspected patients.MethodsA cross-sectional study was conducted on 96 clinically diagnosed TBLN suspected patients, where fine needle aspirate (FNA) samples were collected at the time of diagnosis. FNA cytology, Ziehl-Neelsen (ZN), Auramine O (AO) staining, GeneXpert MTB/RIF and Real time PCR (RT-PCR) were performed on concentrated FNA samples. Considering culture as a gold standard, the sensitivity, specificity, positive and negative predictive values were calculated. Cohen’s Kappa value was used to measure interrater variability and level of agreement and a P-value of <0.05 was considered as statistically significant.ResultOut of the 96 FNA sample, 12 (12.5%) were identified to have Mycobacterium tuberculosis (Mtb) using ZN staining, 27 (28.1%) using AO staining, 51 (53.2%) using FNAC, 43 (44.7%) using GeneXpert MTB/RIF, 51 (53.1%) using Real time PCR (RT-PCR) and 36 (37.5%) using Lowenstein-Jensen (LJ) culture. Compared to LJ culture, the sensitivities of GeneXpert MTB/RIF, RT-PCR, and FNAC were 91.7%, 97.2%, and 97.2%, respectively and the specificities were 83.3%, 73.3%, and 68.3%, respectively. GeneXpert MTB/RIF and RT-PCR when combined with FNAC detected 61 (63.5%) cases as having Mtb, and the sensitivity and specificity was 100% and 58.3%, respectively.ConclusionFNA cytology and RT-PCR detected more TBLN cases compared to other Mtb detection tools and the detection sensitivity even improved when FNA cytology was combined with GeneXpert MTB/RIF, performed on concentrated FNA sample, suggesting the combined tests as an alternative approach for improved diagnosis of TBLN.

Spinal cord involvement in tuberculous meningitis: a case report and brief review of literature

Introduction: Tuberculosis (TB) continues to pose a significant public health problem worldwide. Tuberculous meningitis (TBM) is the most devastating form of extrapulmonary TB however other forms of central nervous system (CNS) disease include tuberculoma and spinal arachnoiditis. TBM carries high mortality even for a patient who is already receiving treatment. The difficulty in diagnosis often leads to a delay in treatment and subsequent mortality. The emergence of Xpert ultra has improved the rapid detection of MTB and rifampicin resistance in CSF and is the preferred diagnostic tool in TBM. Case: In this case report we present a 33years patient of concern who presented with progressive lower limb weakness associated with pain and paresthesia for 4 months, admitted via the Orthopedic unit with a diagnosis of spinal mass (meningioma, neurofibroma, or nerve sheath tumor) for which biopsy was done and revealed a chronic inflammatory process, necrotic bone lesions with no granulomas and no malignancy, he was later diagnosed with tuberculous meningitis and promptly started anti-tuberculous therapy with a dramatic recovery and improvement in neurological function. Conclusion: Tuberculous meningitis conditions have high morbidity and mortality yet diagnosis and start of treatment continue to experience an important delay. Clinicians should keep in mind the limitations of clinical presentation due to pleiotropy and current diagnostics and should employ a combination of diagnostic modalities in addition to a high index of suspicion to prevent morbidity in patients with TBM.

Determination of the predictive factors for diagnostic positivity of nucleic acid amplification tests for diagnosing pulmonary tuberculosis

BackgroundTuberculosis (TB) remains a major threat to human health, and TB diagnostic methods remain unsatisfactory. Nucleic acid amplification tests (NAATs) show higher sensitivity compared with culture for the diagnosis of pulmonary TB (PTB). However, NAATs are expensive and cannot be easily implemented outside major medical centers. To improve the sensitivity of NAATs for PTB diagnosis, we investigated the predictive factors that might optimize NAAT utilization. MethodsA total of 1263 patients with suspected PTB were enrolled for evaluation. The sensitivity, specificity, and accuracy of methods including smear-microbiology, culture of Mtb and NAAT for Mycobacterium tuberculosis (Mtb) detection in sputum and bronchoalveolar lavage fluid samples were compared. Odds ratios and 95% confidence intervals were used to assess variables that might be associated with positive NAAT results for sputum and bronchoalveolar lavage fluid from patients with suspected PTB. ResultsNAAT showed higher sensitivity for Mtb detection (61.1%) when compared with smear (9.0%) and Mtb culture (47.8%). We found that an elevated erythrocyte sedimentation rate, the presence of cavities, and positive interferon-γ release assay (IGRA) results were indicative of positive Mtb detection by NAAT. Moreover, individuals who had all three of these characteristics showed an 86% diagnostic positivity for PTB from Mtb detection by NAAT. ConclusionsOur study suggests that an elevated erythrocyte sedimentation rate, a positive IGRA result, and the presence of pulmonary cavities are helpful factors for predicting positive Mtb detection by NAAT. Patients with the three positive clinical markers should undergo NAAT for Mtb detection because they are the most likely individuals to be bacteriologically confirmed as having TB.

Interaction of Host Pattern Recognition Receptors (PRRs) with Mycobacterium Tuberculosis and Ayurvedic Management of Tuberculosis: A Systemic Approach.

Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis (Mtb), infects the lungs' alveolar surfaces through aerosol droplets. At this stage, the disease progression may have many consequences, determined primarily by the reactions of the human immune system. However, one approach will be to more actively integrate the immune system, especially the pattern recognition receptor (PRR) systems of the host, which notices pathogen-associated molecular patterns (PAMPs) of Mtb. Several types of PRRs are involved in the detection of Mtb, including Toll-like receptors (TLRs), C-type lectin receptors (CLRs), Dendritic cell (DC) -specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), Mannose receptor (MR), and NOD-like receptors (NLRs) related to inflammasome activation. In this study, we focus on reviewing the Mtb pathophysiology and interaction of host PPRs with Mtb as well as adverse drug effects of anti-tuberculosis drugs (ATDs) and systematic TB treatment via Ayurvedic medicine.

AutoCellANLS: An Automated Analysis System for Mycobacteria-Infected Cells Based on Unstained Micrograph.

The detection of Mycobacterium tuberculosis (Mtb) infection plays an important role in the control of tuberculosis (TB), one of the leading infectious diseases in the world. Recent advances in artificial intelligence-aided cellular image processing and analytical techniques have shown great promises in automated Mtb detection. However, current cell imaging protocols often involve costly and time-consuming fluorescence staining, which has become a major bottleneck for procedural automation. To solve this problem, we have developed a novel automated system (AutoCellANLS) for cell detection and the recognition of morphological features in the phase-contrast micrographs by using unsupervised machine learning (UML) approaches and deep convolutional neural networks (CNNs). The detection algorithm can adaptively and automatically detect single cells in the cell population by the improved level set segmentation model with the circular Hough transform (CHT). Besides, we have designed a Cell-net by using the transfer learning strategies (TLS) to classify the virulence-specific cellular morphological changes that would otherwise be indistinguishable to the naked eye. The novel system can simultaneously classify and segment microscopic images of the cell populations and achieve an average accuracy of 95.13% for cell detection, 95.94% for morphological classification, 94.87% for sensitivity, and 96.61% for specificity. AutoCellANLS is able to detect significant morphological differences between the infected and uninfected mammalian cells throughout the infection period (2 hpi/12 hpi/24 hpi). Besides, it has overcome the drawback of manual intervention and increased the accuracy by more than 11% compared to our previous work, which used AI-aided imaging analysis to detect mycobacterial infection in macrophages. AutoCellANLS is also efficient and versatile when tailored to different cell lines datasets (RAW264.7 and THP-1 cell). This proof-of concept study provides a novel venue to investigate bacterial pathogenesis at a macroscopic level and offers great promise in the diagnosis of bacterial infections.

Immunological role of cluster of differentiation 56 and cluster of differentiation 19 in patients infected with mycobacterium tuberculosis in Iraq

Tuberculosis (TB) is one of the most dangerous infection mainly caused by gram positive bacteria Mycobacterium tuberculosis (Mtb) and cause many deaths every year worldwide especially in developing countries and activate the humoral and cellular immune response. The aim of this study was to investigate the role of CD56 and CD19 as diagnostic and immune markers in patients infected with Mtb. A total number of 26 patients infected Mtb and 27 patients infected with Multi-Drug resistance Mtb (MDR-Mtb) and 30 healthy individuals were included in this study. Mtb has been diagnosed by acid fast stain and MDR-Mtb were diagnosed by GeneXpert® heminested real time PCR for simultaneous detection of Mtb and rifampicin (RIF) resistance as MDR-Mtb. Serum concentration of CD56 and CD19 has been measurement by Enzyme-Linked Immune-sorbent Assay (ELISA) method. Results of this study showed a significant increase in the serum concentration of CD56 (P value = 0.0001) in Mtb-patients (1.95 ± 0.08 ng/ml) as compared with healthy individuals (1.17 ± 0.09 ng/ml). The same significant increases in the serum concentration of CD56 (2.24 ± 0.12 ng/ml) was reported in MDR-Mtb patients as compared to the healthy individuals. On the other hand, there was significant increase (P value 0.003) in serum concentration of CD19 in Mtb-patients as compared with MDR-Mtb-patients. Results of the study can be concluded that Mtb and MDR-Mtb infection stimulates the cellular and humoral immune response which resulted in the increased production of CD56 and CD19.

Diagnostic utility of GenoType MTBDRsl assay for the detection of moxifloxacin-resistant mycobacterium tuberculosis, as compared to phenotypic method and whole-genome sequencing.

Recently, moxifloxacin (MFX)-resistant results of Mycobacterium tuberculosis (Mtb) obtained by GenoType MTBDRsl (second-line line probe assay [SL-LPA]) have been stratified to determine their resistance level; however, its accuracy has not been well studied. Therefore, the study aimed to evaluate the diagnostic accuracy of SL-LPA, with phenotypic drug susceptibility testing (pDST) and whole-genome sequencing (WGS) for the detection of MFX-resistant Mtb and their resistance level. A total of 111 sputum samples were subjected to SL-LPA according to the diagnostic algorithm of the National Tuberculosis Elimination Program. Results were compared with pDST of MFX (at critical concentration [CC, 0.25 μg/ml] and clinical breakpoint [CB, 1.0 μg/ml] using BACTEC mycobacterial growth indicator tube-960), and WGS. At CC, SL-LPA and pDST yielded concordant results of MFX for 104 of 111 (94%). However, at CB, 23 of 30 (77%) isolates carrying gyrA mutation known to confer low-level resistance to MFX were scored as susceptible by pDST. Among 46 Mtb isolates carrying gyrA mutations known to confer high-level resistance to MFX, 36 (78%) isolates yielded concordant results, while 10 (22%) isolates were scored as susceptible at CB by pDST. WGS identified gyrA mutations in all isolates suggested by SL-LPA. It is concluded that the stratification of MFX-resistant results by SL-LPA/genotypic method is not very well correlated with pDST (at CB), and hence, pDST may not be completely replaced by SL-LPA. gyrA D94G and gyrAA90V are the most prevalent mutations in MFX-resistant Mtb.