Automated Mycobacterium tuberculosis Detection in Multivariant Digitized Ziehl–Neelsen Staining Using Faster R‐CNN Method

Comparison of Fluorescence Microscopy with Ziehl-Neelsen Technique in the Examination of Sputum for Acid-Fast Bacilli Using Bleach Centrifugation Method

An atypical presentation of tuberculous gumma heralding a diagnosis of lymph node tuberculosis: Hindsight is 20/20

About one third of the world population is estimated to be infected with Mycobacterium tuberculosis (MTB), and this proportion is expected to be higher in countries with a high tuberculosis (TB) burden. The Philippines is both a high tuberculosis burden and a high multidrug resistant tuberculosis (MDR-TB) burden country. Though TB has been extensively described in the civilian population, there is limited data on TB in the military population. The objectives are: (1) To determine MTB/MDR-TB prevalence among military and civilian patients in the Philippines presenting with clinically suspected TB in a tertiary military hospital and (2) To determine performance of direct sputum smear microscopy (DSSM) using Ziehl-Neelsen (ZN) staining compared to Xpert MTB/RIF real-time reverse transcriptase polymerase chain reaction. Sputum samples were obtained from patients, clinically suspected with TB, and/or with TB associated signs/symptoms. Sputum specimens were tested using DSSM with ZN staining and Xpert MTB/RIF assay (Cepheid, Sunnyvale, California) and patient demographic and clinical data were collected. From March 2015 to December 2018, a total of 795 (173 military personnel [164 active duty and 9 retired]; 618 civilians; and 4 with no data on military/civilian status) patients with TB associated symptoms or clinically suspected with TB were tested. Overall, MTB prevalence was 81/795 (10%). MTB prevalence among active duty and retired military personnel were 27/164 (16%) and 4/9 (44%), respectively while MTB prevalence for civilian patients was 50/618 (8%) (p value=0.0003; OR=2.48 [95% C.I. 1.5-4]). Among active and retired military personnel who tested positive for MTB, rifampin resistance was 4/27 (15%) and 1/4 (25%), respectively, while rifampin resistance for civilian patients was 9/50 (18%) (p value=1; OR=0.88 [95% C.I. 0.26-2.90]). For active duty military personnel, average MTB prevalence (based on Xpert MTB/RIF) covering years 2015-2018 was 21% and ranged from 13% to 35%, while average rifampin resistance among MTB positive active duty military personnel was 15% and ranged from 0% to 25%. Overall sensitivity and specificity of DSSM compared to Xpert MTB/RIF were 70% and 96%, respectively. Positive and negative predictive values of DSSM to accurately categorize MTB in symptomatic cases (with Xpert MTB/RIF as "true positive" reference) were 74% and 95%, respectively. Performance of DSSM varied according to MTB load detected by Xpert MTB/RIF with increasing DSSM sensitivity observed as the MTB load detected by Xpert MTB/RIF increased (p=0.02). This report describes high MTB and MDR-TB prevalence rates among symptomatic military patients with military personnel having higher odds of MTB infection compared to the civilian patients in the study. Since DSSM (ZN) sensitivity greatly varied depending on MTB load, the Xpert MTB/RIF should be used as a first-line diagnostic tool to identify MTB and detect rifampin resistance among presumptive TB cases instead of DSSM (ZN) microscopy.

Background: Early secretory antigenic target protein 6-kDa (ESAT-6), culture filtrate 10-kDa (CFP- 10), and Mycobacterium tuberculosis (Mtb) 64 (MPT-64) antigens are secreted by actively growing Mtb in active tuberculosis (TB) patients. Increased levels of ESAT-6, CFP-10, and MPT-64 at the site of infection facilitate the entry of antigens into the systemic circulation. These low molecular weight proteins (< 67 kDa) can be excreted into the urine via the kidneys. Therefore, this study evaluated the urinary Mtb antigen cocktail (ESAT-6, CFP-10, and MPT-64) levels in active [pulmonary (P) and extrapulmonary (EP)] TB and latent tuberculosis infections (LTBI) individuals. Methods: This study was conducted in Dr. Hasan Sadikin General Hospital Bandung from June 2016 to June 2017, using 474 participants. The specimens for TB diagnosis were taken and investigated for acid-fast bacilli (AFB) using the Ziehl Neelsen (ZN) technique. The remaining sputum was cultured on Lowenstein- Jensen (LJ) medium, and TB Ag MPT-64 rapid tests were performed to identify Mtb. In addition, LTBI individuals were screened in a TB outpatient clinic who had close contact with patients with active TB and underwent an interferon g release assay (IGRA). Urine samples were collected for urinary Mtb antigen cocktail (ESAT-6, CFP-10, and MPT-64) testing using the quantitative immunochromatographic method. Results: The 99 patients were divided into three groups, PTB (n = 52), EPTB (n = 22) and LTBI (n = 25). There were significant differences in urinary Mtb antigen cocktail levels between PTB and EPTB (33.8 vs 13.6 ng/ml; p < 0.001), between PTB and LTBI (33.8 vs 0 ng/ml; p < 0.001), and between EPTB and LTBI (13.6 vs 0 ng/ml; p = 0.015). Conclusion: Urinary Mtb antigen cocktail levels were increased in patients with active TB and undetected in those with LTBI. This result was obtained using the urinary Mtb antigen cocktail as a potential marker for differentiating active TB and LTBI.

Background and Aims: Tuberculosis (TB) is severe infectious disease caused by Mycobacterium tuberculosis and imposes significant morbidity and mortality. Early diagnosis of active TB forms pulmonary and extrapulmonary is very important and the diagnostic methods such as culture test and Ziehl-Neelsen (ZN) stain displayed low diagnostic accuracy. Cartridge-based nucleic acid amplification test (CBNAAT) is widely used for the diagnosis of TB with good accuracy rate. Hence, the current study was commenced to assess the diagnostic usefulness of CBNAAT for the diagnosis of mycobacterium TB (MTB) and compared with ZN staining. Methods: This was a prospective study conducted on 250 sputum samples of pulmonary TB (new cases) and also patients who were receiving the treatment for TB. 5 mL of sputum sample including CSF, pericardial or ascitic fluid, pus, or lymph nodes aspirates were collected and subjected to ZN staining and CBNAAT. Results: Mean age observed was 48.12±8.76 years. 30 (12%) showed smear positive and 220 (88%) were smear negative for ZN staining. Further, samples have been subjected to CBNAAT showed 60 (24%) MTB positive, and 190 (74%) showed MTB negative. CBNAAT showed sensitivity and specificity of 91.5% and 89%. Out of 60 positives for MTB by CBNAAT, MTB has been identified in 28 patients. Conclusion: CBNAAT is a great tool in the early diagnosis of pulmonary and extrapulmonary TB.

Approximately one third of the global population is latently infected with tuberculosis (TB) and there are approximately 9.6 million new cases of TB disease per year resulting in 1.5 million deaths. Eleven percent of cases globally occur in children and 81% of the burden of TB disease is borne by the developing world and countries with emerging economies (BRICS). The African region accounts for 28% of new TB cases globally. TB remains a significant public health concern globally, particularly amongst children and immunocompromised individuals. Diagnosis of childhood TB is an on going challenge, as children usually do not present with the same signs and symptoms as adults, and are often misdiagnosed. Tuberculosis infection in children is seldom confirmed through sputum culture, as good sputum samples can rarely be collected. Only 15% of cases from children are sputum smear positive by acid-fast staining, and only 30%–40% are Mycobacterium tuberculosis (MTB) culture positive. Up to 40% of children present with extrapulmonary manifestations of TB disease. The most common manifestation is tuberculous lymphadenopathy. Good specimens for TB detection can be obtained from these cases through fine needle aspiration biopsy (FNAB), a cost-effective and practical out patient procedure for obtaining specimens from enlarged superficial lymph nodes. The conventional laboratory techniques that have been used globally for TB diagnosis are Ziehl-Neelsen (ZN) staining for acid fast bacilli (AFB) microscopy, culture and more commonly now LED microscopy, cytological determination with auto-fluorescence staining and molecular Xpert® MTB/RIF (Cepheid) detection. AFB-microscopy requires minimally 5000 AFB per millilitre of specimen to yield a consistently positive result and observation of between 100 and 300 microscopic fields in order to obtain accurate results. Culture for MTB is the gold standard diagnostic method but has a slow turnaround time and requires laboratory resources that are not available in most parts of the world. Recent systematic reviews of studies evaluating commercially available nucleic acid amplification test (NAAT) technologies confirm very high specificity, with sensitivity approaching, but not reaching, that of culture. The complexity and insufficient robustness of existing commercial NAAT protocols and their need for precision instruments, a high degree of technical support, and quality assurance make them unsuitable for most low resource TB endemic countries. In addition, none of these techniques have been fully validated for diagnosing TB in children and specifically not for extrapulmonary specimens. In light of these challenges, there is promise in two technologies that have been developed and under evaluation over the last few years; the Xpert® MTB/RIF kit (Cepheid) for rapid detection of MTB and rifampicin resistance endorsed for use by the WHO; and the Ustar EasyNATTM TB IAD kit (Ustar Biotechnologies, Hangzhou) for detection of MTB, selected by the WHO as a technology on assessment for use in TB endemic countries. In this study, their performance was assessed against the conventional laboratory diagnostic techniques of smear AFB microscopy, cytology and mycobacterial culture of fine needle aspirates from lymph nodes of children suspected of TB lymphadenopathy in Tanzania. Age defined clinical assessments were done for all 75 participants and TB treatment initiated based on these and/or laboratory diagnostic outputs. All laboratory diagnostic modalities were primarily assessed against TB culture as the conventional reference standard. As has been evidenced in earlier studies, the sensitivities for both smear microscopy and TB culture were very low in these extrapulmonary specimens. Lacking a true reference standard, composite reference standards (CRSs) were created to assess the performance of the test modalities under study. An alternative method for assessing diagnostic accuracy under these conditions is latent class analyses (LCA), which was utilized to further assess the performance of all diagnostic modalities in the study. The overall outcomes of the project demonstrated that cytomorphology was a feasible and effective technique for detection of TB in lymph node aspirates (sensitivity: LCA 100%, specificity: LCA 94.7%) that may complement TB culture (sensitivity: LCA 74.5%, specificity: LCA 90.3%). Further, it was shown that Xpert (sensitivity: CRS 58% LCA 70.7%, specificity: CRS 93% LCA 94.2%) was superior in performance to EasyNAT (sensitivity: CRS 19% LCA 29.2%, specificity: CRS 100% LCA 100%) and ZN (sensitivity: CRS 14% LCA 19.1%, specificity: CRS 100% LCA 100%) analyses, respectively. Combining two or more tests significantly improved the diagnostic efficacy, but including either EasyNAT testing or ZN microscopy to a diagnostic algorithm that already had Xpert testing added no value. These findings indicate that combining clinical assessment, cytology and Xpert MTB/RIF can provide for rapid and accurate diagnosis of childhood tuberculous lymphadenitis. Larger diagnostic evaluation studies on Xpert MTB/RIF would be required, to assess its use as a solitary initial test for tuberculous lymphadenitis in children.

Antimycobacterial Activities from Seagrass Enhalus sp. Associated Bacteria Against Multi Drug Resistance Tuberculosis (MDR TB) Bacteria

Tuberculosis (TB) is a public health problem caused by the Mycobacterium tuberculosis complex. An estimated 70% of TB patients living with HIV are from sub-Saharan Africa largely depends on direct smear microscopy light microscopy for TB diagnosis, which has low Sensitivity, especially among HIV patients. Hence, we conducted this study to evaluate the Performance of Auramine O LED Fluorescence microscopy and the Ziehl-Neelsen technique in Tuberculosis among HIV-positive patients. The study was conducted using a cross-sectional design among 107 consecutively selected HIV/AIDS patients with presumptive Tuberculosis attending ART clinic in Dalhatu Araf Specialist Hospital (DASH), Lafia, Nasarawa State. Three samples were collected from each patient in two visits. Each sample was examined using ZN, LED FM, and Gene-Xpert. Data on socio-demographic characteristics was collected from the subject participant. Univariate and bivariate analyses were done using MS Excel and Epi Info. The Sensitivity and Specificity were compared using McNemar’s chi-square. Seventy-three (68%) patients were female, 33 (30.8%) were in the age group 28-32 years, and 61 (56%) were employed. Ziehl-Neelsen and LED fluorescent microscopy respectively yielded 11 (10.3%) and 15 (14.0%) positive results, while Gene-Xpert gave 18 (16.3%). The Sensitivity for direct ZN and LED FM were 61.1% and 83.3 %, respectively (P-value 0.01) with 100% specificity for both methods. LED microscopy has a much higher sensitivity than ZN microscopy and would be a better alternative in the diagnosis of Tuberculosis in high HIV settings where the use and expertise with culture and Gene-Xpert are limited. Keywords: HIV, LED Fluorescence, Sensitivity, Specificity, Ziehl-Neelsen.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis. One way to diagnose this disease is by microscopic examination using the Ziehl Neelsen method. Bleach is a decontaminant solution that can help reduce microscopic dirt and sterilize microscopic fields of view. This study aims to determine the comparison of Ziehl Neelsen staining acid fast bacilli scores and Ziehl Neelsen Bleach 2% sputum specimens in patients with pulmonary tuberculosis. The design used is pre-experimental with 36 sputum samples taken. Data analysis is performed by bivariate analysis using the Wilcoxon test at value of 0.05. From this study, the results for Ziehl Neelsen staining are 30.6% negative, 11.1% scanty, 19.4% positive 1 (+1), 16.7% positive 2 (+2), 22.2% positive 3 (+3), while for Ziehl Neelsen staining Bleach 2% as much as 25% negative, 16.7% scanty, 19.4% positive 1 (+1), 16.7% positive 2 (+2), 22.2% positive 3 (+3). The results of bivariate analysis show a p value of 0.157 so it is concluded that there is no difference in the positivity rate of microscopic TB smear results between the addition of 2% bleach solution compared to ordinary Ziehl Neelsen staining. Keywords: Mycobacterium Tuberculosis, Staining of Acid-Fast Bacilli (AFB), Ziehl-Neelsen, Bleach. ABSTRAK Tuberkulosis (TB) adalah penyakit menular yang disebabkan oleh Mycobacterium tuberculosis. Salah satu cara untuk mendiagnosis penyakit ini yaitu dengan pemeriksaan mikroskopis metode Ziehl-Neelsen. Bleach merupakan larutan dekontaminan yang dapat membantu mengurangi kotoran mikroskopis dan mensterilkan lapang pandang mikroskopis. Penelitian ini bertujuan untuk mengetahui perbandingan skor basil tahan asam pewarnaan Ziehl Neelsen dan Ziehl Neelsen Bleach 2% spesimen sputum pada penderita Tuberkulosis paru.Desain yang digunakan yaitu pre-eksperimen dengan 36 sampel sputum yang diambil dari pasien TB di Rumah Sakit Santa Elisabeth Medan. Analisis data dilakukan dengan analisis bivariat menggunakan uji Wilcoxon pada nilai α 0,05. Dari penelitian ini didapatkan hasil pada pewarnaan Ziehl Neelsen sebanyak 30.6% negatif, 11.1% scanty, 19.4% positif 1 (+1), 16.7% positif 2 (+2), 22.2% positif 3 (+3), sedangkan pada pewarnaan Ziehl Neelsen Bleach 2% sebanyak 25% negatif, 16.7% scanty, 19.4% positif 1 (+1), 16.7% positif 2 (+2), 22.2% positif 3 (+3). Hasil analisis bivariat menunjukkan nilai p 0.157 sehingga disimpulkan bahwa tidak terdapat perbedaan tingkat kepositifan hasil BTA diagnostik mikroskopik TB antara penambahan larutan bleach 2% dibandingkan dengan pewarnaan BTA Ziehl Neelsen biasa. Kata kunci: Mycobacterium Tuberculosis, Pewarnaan Basil Tahan Asam (BTA), Ziehl- Neelsen, Bleach.

Institutional Control Measures for Tuberculosis in the Era of Multiple Drug Resistance: ACCP/ATS Consensus Conference

To assess the clinical value of polymerase chain reaction (PCR) in the diagnosis and differential diagnosis of joint tuberculosis (TB). PCR was used blindly to detect the DNA of Mycobacterium tuberculosis (M.TB) in five specimens of M.TB, 5 of BCG, and 10 of other bacteria. Then, M. TB in 98 samples from patients with joint TB and 100 samples from patients with non-tubercular joint disorders were detected by PCR, acid-fast staining and culture,. The sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of PCR were calculated. The χ2 test was used for statistical analysis of the frequency of various factors. At the same time, some problems with PCR were also systematically analyzed. (1) In the "standard samples", both M. TB and BCG showed positive while other bacteria were negative. (2) In 98 cases from patients with joint TB, 81 were positive by PCR, 6 by acid-fast staining, and 17 by culture. In 100 cases from patients with non-tuberculous joint disorders, 9 were positive by PCR, and none by either acid-fast staining or culture. Sensitivity, specificity, accuracy, positive and negative predictive value of PCR were 82.65% (81/98), 91.00% (91/100), 86.87% (172/198), 90.00% (81/90) and 84.26% (91/108), respectively. (3) The positive rates for PCR, acid-fast staining and culture in detection of M. TB were 82.65% (81/98), 6.12% (6/98), and 17.34% (17/98), respectively. There were statistically significant differences between the three methods (P < 0.001). (4) The process of PCR is automatic, and can be completed within 3 to 6 hours, whereas 4 to 8 weeks are required for the conventional culture of M. TB. PCR is a sensitive, specific, rapid, simple and minimally invasive method for detection of M. TB in samples from joint TB, and can play an important role in early and rapid diagnosis and differential diagnosis of joint TB. But it also has some limitations, such as false positivity and false negativity.

Background: Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (MTB). Most of healthcare facilities in Indonesia still use Ziehl Neelsen (ZN) staining to diagnose pulmonary tuberculosis (TB) rather than GeneXpert MTB/RIF. However, the agreement between both methods is not well established.Objective: This study aims to observe levels of agreement between ZN staining and GeneXpert MTB/RIF in diagnosing adult pulmonary TB at Dr. Mohammad Hoesin General Hospital (RSMH) Palembang.Methods: This study was a retrospective comparative analytical study using an agreement test. Its samples were patient specimens for a period of January 2016 - March 2017 which were examined by ZN staining and GeneXpert MTB/RIF to diagnose pulmonary TB at the Clinical Pathology and Microbiology Installation of RSMH Palembang. Its data were obtained from laboratory results of ZN staining and GeneXpert MTB/RIF. Then, Cohen’s Kappa was used to measure the levels of agreement between both the assays.Results: Of 150 specimens, 69.3% were from male patients and 30.7% were from female patients with mean age of 46.71±14.57. Both ZN staining and GeneXpert positivity were 57.3%. 0.05% of positive GeneXpert MTB/RIF were negative ZN smear, while 0.06% of negative GeneXpert were positive ZN smear. The Cohen’s Kappa coefficient was 0.893 indicating a very good agreement.Conclusion: This study found that there was a very good agreement between ZN staining and GeneXpert MTB/RIF in diagnosing adult pulmonary TB at the RSMH Palembang.

Current Concepts in the Management of Tuberculosis

Microbial Pathogenesis of Mycobacterium tuberculosis: Dawn of a Discipline

Background: Pakistan faces an immense burden of pulmonary tuberculosis (TB) due to large number of cases and limited resources. Despite the recent advancement in the diagnostic techniques for pulmonary TB, smear microscopy is still a useful technique for the diagnosis of this disease. This study was conducted in order to compare the diagnostic value of Auramine stain with the conventional Ziehl-Neelsen (ZN) stain on the sputum smear for the diagnosis of pulmonary TB, keeping GeneXpert MTB/RIF as the gold standard.&#x0D; Method: This cross-sectional study was conducted on 356 suspected patients of pulmonary TB referred to the Pathology laboratory from TB ward and OPD of District Head Quarter (DHQ) teaching hospital Rawalpindi, Pakistan. Sputum specimen were collected and two smears were prepared from each sputum sample on which Auramine phenol and Ziehl-Neelsen staining were carried out as per WHO recommendations. All these samples were further tested using Gene Xpert MTB/RIF technique. The sensitivity, specificity, Positive predictive value (PPV) and Negative predictive values (NPV) of ZN and Auramine stain were calculated and compared with GeneXpert MTB/RIF technique.&#x0D; Results: Out of the total 356 samples, 64(18%) were positive and 291(82%) were negative by GeneXpert which was taken as the gold standard. On comparison with GeneXpert, percentage of true positive was greater in case of Auramine than ZN stained samples (16.29% versus 12.92%), while the percentage of false positive was same for both staining techniques (0.28%). There were lesser false negative cases observed in samples stained by Auramine as compared to the ones stained by ZN (1.68% versus 5.05%). The sensitivity, specificity, positive predictive value and negative predictive values were 97.87%, 94.17%, 71.88 and 99.66%, respectively for the ZN staining and 98.31%, 97.98%, 90.63% and 99.66% respectively, for the Auramine phenol staining.&#x0D; Conclusion: Smear microscopy using Auramine phenol stain is a useful technique for the diagnosis of pulmonary TB. The Auramine phenol staining with fluorescent microscopy is found to be superior to ZN staining because of higher sensitivity and specificity.&#x0D; Keywords: Auramine phenol, Fluorescence microscopy, GeneXpert, Mycobacterium tuberculosis, Ziehl-Neelsen