Lowenstein-Jensen Research Articles

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 of Ziehl - Neelsen (ZN) Staining and Fluorescent (FL) Staining in Suspected Cases of Tuberculosis

A total of 200 clinically suspected cases of tuberculosis were included in the study. All the cytological specimens procured were smeared and stained for both ZN and FL staining. A part of the sample was used for Lowenstein Jensen (LJ) culture. Patients on ATT were excluded from the study.

Comparative Studies of the Methods of Detecting Rifampicin Resistant Mycobacterium tuberculosis among Tuberculosis Patients Attending Aminu Kano Teaching Hospital, Kano

This study was conducted to compare the specificity and sensitivity of GeneXpert MTB/Rif and Lowenstein-Jensen (LJ) Proportion methods of detecting rifampicin-resistant Mycobacterium tuberculosis among Acid Fast Bacilli (AFB) positive patients attending the Directly Observed Treatment (DOT) Centre of Aminu Kano Teaching Hospital Kano. A total of 150 AFB positive samples were collected and processed according to the guideline given by National TB and Leprosy Control Program (2015) and WHO (2012), The result revealed that rifampicin-resistant Mycobacterium tuberculosis (RR-TB) from the samples was very high; 66.7% and 60.8% for GeneXpert MTB/Rif and Lowenstein-Jensen (LJ) Proportion methods respectively. Cohen’s Kappa (interrater reliability) statistical analysis indicated a substantial agreement between GeneXpert and LJ Proportion specificity and sensitivity (Kappa value = 0.73).

Performance of GenoType MTBDRsl assay for detection of second-line drugs and ethambutol resistance directly from sputum specimens of MDR-TB patients in Bangladesh.

BackgroundRapid and early detection of drug susceptibility among multidrug-resistant tuberculosis (MDR-TB) patients could guide the timely initiation of effective treatment and reduce transmission of drug-resistant TB. In the current study, we evaluated the diagnostic performance of GenoType MTBDRsl (MTBDRsl) ver1.0 assay for detection of resistance to ofloxacin (OFL), kanamycin (KAN) and ethambutol (EMB), and additionally the XDR-TB among MDR-TB patients in Bangladesh.MethodsThe MTBDRsl assay was performed directly on 218 smear-positive sputum specimens collected from MDR-TB patients and the results were compared with the phenotypic drug susceptibility testing (DST) performed on solid Lowenstein-Jensen (L-J) media. We also analyzed the mutation patterns of gyrA, rrs, and embB genes for detection of resistance to OFL, KAN and EMB, respectively.ResultsThe sensitivity and specificity of the MTBDRsl compared to phenotypic L-J DST were 81.8% (95% CI, 69.1–90.9) and 98.8% (95% CI, 95.6–99.8), respectively for OFL (PPV: 95.7% & NPV: 94.1%); 65.1% (95% CI, 57.5–72.2) and 86.7% (95% CI, 73.2–94.9), respectively for EMB (PPV: 94.9% & NPV: 39.4%); and 100% for KAN. The diagnostic accuracy of KAN, OFL and EMB were 100, 94.5 and 69.6%, respectively. Moreover, the sensitivity, specificity and diagnostic accuracy of MtBDRsl for detection of XDR-TB was 100%. The most frequently observed mutations were at codon D94G (46.8%) of gyrA gene, A1401G (83.3%) of rrs gene, and M306V (41.5%) of the embB gene.ConclusionConsidering the excellent performance in this study we suggest that MTBDRsl assay can be used as an initial rapid test for detection of KAN and OFL susceptibility, as well as XDR-TB directly from smear-positive sputum specimens of MDR-TB patients in Bangladesh.

Efficacy of GeneXpert in Diagnosing Smear Negative Pulmonary Tuberculosis and Comparison to Culture

Objectives: Aim of present study is to observe the efficiency of GeneXpert MTB/RIF Assay in comparison to MTB culture on Lowenstein Jensen media in diagnosis of smear negative pulmonary tuberculosis cases.
 Methods: This descriptive study was carried out in The University of Lahore in collaboration with King Edward Medical University/Mayo Hospital Lahore 11th September, 2020 to 10th April 2021. Smear negative for acid fast bacilli patients enrolled for anti TB treatment were the target population. After taking informed consent, patients were asked to submit first morning sputum sample for culture on Lowenstein Jensen Medium and GeneXpert.
 Results: A total of 345 smear negative TB patients were diagnosed clinically and/or on the basis of radiological findings with mean age of 38.28±17.93,consisting of 47.5% male and 52.5% females recruited in this study. History of TB contact was present among 41.4% patients whereas history of smoking and diabetes remained to be 27.2% and 17.4% respectively. Culture showed significantly higher rate (35.1%) (p-value <0.05) of detection of MTB as compared to GeneXpert (21.5%). A sensitivity of 56.01% was calculated for GeneXpert whereas for culture on LJ medium it was 60.63%.
 Conclusion: Sensitivity of GeneXpert MTB/RIF Assay is a bit low in diagnosing the SNPT patients as compared to the culture but still registers itself as a handsome tool in terms of promptness and definite detection of MTB complex. Further provision of rifampicin susceptibility is bonus in same time.

Mycobacterium Tuberculosis and Avium Complex Investigation among Malaysian Free-Ranging Wild Boar and Wild Macaques at Wildlife-Livestock-Human Interface.

Simple SummaryThis study targeted a small epidemiological area of a selected wildlife-livestock-human interface in Selangor to detect important veterinary and public health mycobacteria in free-ranging wild boar (Sus scrofa) and wild macaques (Macaca fascicularis) using a combination of diagnostic methods, tuberculosis-like lesion (TBLL) detection and nucleic acids detection by conventional and molecular analyses. Conventional PCR on wild boar tissues showed that 75% (9/12) of the lymph node samples were positive for Mycobacterium bovis (95% CI: 46.8–91.1). For macaques, 33.3% (10/30) were positive for Mycobacterium avium (95% CI: 19.2–51.2).Wild animals are considered reservoirs, contributing to the transmission of emerging zoonotic diseases such as tuberculosis (TB). A cross-sectional study was conducted by opportunistic sampling from fresh carcasses of free-ranging wild boar (n = 30), and free-ranging wild macaques (n = 42). Stained smears from these tissues were tested for acid-fast bacilli (AFB) with Ziehl–Neelsen staining. Mycobacterial culture was conducted using Lowenstein–Jensen media and Middlebrook 7H11 agar media. Polymerase chain reaction (PCR) was performed through the detection of the 16S rRNA gene, with multiple sets of primers for the detection of Mycobacterium tuberculosis complex (MTBC) and Mycobacterium avium complex (MAC). In wild boars, 30% (9/30; 95% Confidence Interval: 16.7–47.9%) of examined samples showed gross tuberculosis-like lesions (TBLLs). Multiple nodular lesions that were necrotic/miliary with cavitation were found in the submandibular lymph nodes, tonsils, lungs, kidney and liver, while single nodular lesions were found in the mediastinal lymph nodes, spleen and mesenteric lymph nodes. Conventional PCR on the submandibular lymphoid tissues of wild boar (nine samples with TBLLs and three non-TBLL samples) showed that 75% (9/12) were positive for Mycobacterium bovis (95% CI: 46.8–91.1), and 91% (CI: 64.6–98.5) were positive for Mycobacterium avium. For macaques, 33.3% (10/30) were positive for M. avium (95% CI: 19.2–51.2) but negative for MTBC.

Prevalence and molecular identification of Mycobacteria isolated from animals slaughtered at Sokoto modern abattoir, Sokoto State, Nigeria


 
 
 
 This study investigated the molecular epidemiology of Mycobacteria isolated from animals slaughtered at Sokoto modern abattoir. During meat inspection, 104 suspected tuberculosis lesions were sampled from a total of 102,681 animals slaughtered between November 2016 and January 2018. These samples were subjected to Ziehl Neelsen staining, followed by culture on Lowenstein-Jensen media. Subsequently, polymerase chain reaction (PCR) and sequencing of the 65KDa heat shock protein (hsp65) gene were performed to identify and phylogenetically characterize the cultured organisms. Because sequencing of the hsp65 gene was unable to distinguish between Mycobacterium bovis (M. bovis) and M. tuberculosis, PCR was performed to amplify a genomic region-specific to M. bovis in order to differentiate them from M. tuberculosis. Results showed that, 14 samples yielded growth after culture. Furthermore, hsp65 was detected in 9 out of the 14 isolates screened, 5 of the amplicons were successfully sequenced. Similarity search using NCBI BLAST tool showed the five sequences to share highest identities with Mycobacterium novocastrense (95.99%), M. canettii (94.54%), and M. tuberculosis/M. bovis (100%). Two out of the 5 isolates were confirmed to be M. bovis after PCR amplification using M. bovis specific primers. Phylogenetic tree further confirmed the identity of these isolates by placing them close to species of their kind. Further studies should be conducted to establish the transmission dynamics of the zoonotic Mycobacteria between animals and their owners, to facilitate control and eradication of tuberculosis.
 
 
 

Immunohistochemical evaluation of infiltrating immune cells in endometrial biopsy of female genital tuberculosis

BackgroundFemale Genital Tuberculosis (FGTB) causes infertility in a large number of females in developing countries. Presence of granuloma on histopathological examination of endometrial samples is diagnostic of FGTB. But immunohistochemical evaluation of endometrial aspirates has not been explored before. AimTo evaluate the immunohistochemical delineation of immune cells in FGTB. Methods1515 infertile women from 20 to 35 years were enrolled and underwent endometrial aspiration (EA), which was subjected to microbiological and histopathological examination along with PCR. Patients positive for conventional tests like granulomas, acid fast bacilli, mycobacterial culture on LJ medium or liquid (MGIT) culture were started on antitubercular therapy. Conventional test negative but PCR positive patients were posted for laparoscopy. Immunohistochemistry (IHC) for LCA, CD68, CD3, CD4, CD8, CD 20, CD138, IFN gamma and IL10 were evaluated. Result38/1515 (2.5%) subjects tested positive for conventional methods. PCR-TB was positive in 615/1515 samples (40.59%). On IHC, the number of CD45 (LCA) positive immune cells (p = 0.03) and IFN gamma (p = 0.002) and IL10 expression (p = 0.012) at 1 + level were higher in the PCR positive samples. Laparoscopy done in 418/463 patients and 89/418 (21.3%) showed definitive findings of tuberculosis. CD3, CD4, CD8, CD20, CD68 and CD138 showed no correlation with PCR and laparoscopy. ConclusionIncreased IFN gamma and IL 10 expressing immune cells in PCR positive EA suggests subclinical early changes, and can be useful as a research tool but have no role in diagnosing FGTB.

Fast, simple and cheap: method modified from conventional cultivation for tuberculosis diagnosis allows seeding on Löwenstein-Jensen of any swab-embedded pulmonary samples decontaminated with sodium hydroxide.

BackgroundFew tuberculosis (TB) control programmes in low-income countries have access to culture facilities in their primary care diagnostic centres and this scenario may have worsened with the coronavirus disease 2019 pandemic. Thus the aim was to develop and evaluate a simpler TB test that allows seeding on Löwenstein–Jensen (LJ) medium of several swab-embedded samples decontaminated with sodium hydroxide (NaOH).MethodsA cotton swab containing each sample was decontaminated in NaOH before being dipped into a slightly acidic solution to neutralize the pH in order to allow the culture to develop on LJ medium. Samples (n=543) from suspected or confirmed pulmonary TB were analysed in two phases: standardization (n=167) and evaluation of the study method (n=376).ResultsThe study method showed sensitivity >95% and specificity >93% using Ogawa–Kudoh (OK) and modified Petroff (MP) as standards and was comparable to MP-LJ (p>0.05) and slightly superior to OK (p=0.03) for sputum culture and more comprehensive than the latter for other pulmonary specimens.ConclusionsThis article reports a more comprehensive, simpler and less costly method for diagnosing TB in the laboratory with fewer economic resources and biosafety equipment. Thus a patent application was filed (BR1020190103841).

Primary Isolation of Mycobacterium ulcerans.

Primary isolation of Mycobacterium ulcerans is the separation and growth of the bacterium from a mixed population either in clinical specimen or environmental specimen in pure cultures. It is a crucial activity as it can be used to monitor antimicrobial treatment, surveillance for antimicrobial resistance, and molecular epidemiology studies toward understanding pathogen ecology and transmission as well as pathogen biology. The process involves removal of unwanted fast-growing bacteria using 5% oxalic acid, inoculation on Lowenstein-Jensen medium supplemented with glycerol, and incubation at temperatures between 30°C and 33°C.

Scrofuloderma: A Rare Case Report of Sequelae of Intestinal Tuberculosis

Abstract Introduction: Scrofuloderma is caused by Mycobacterium tuberculosis and is the most common form of cutaneous tuberculosis. It remains a significant medical challenge because its clinical manifestations mimic those of various other conditions, making diagnosis difficult. Here, we report a 28-year-old man who had been diagnosed with intestinal tuberculosis but discontinued treatment. Case presentation: He subsequently presented with a 5-month history of multiple non-healing ulcerative lesions on the abdomen with no other significant clinical findings. Ziehl-Neelsen staining of an impression smear taken from the lesions was performed as the preliminary investigation and confirmed the presence of acid-fast bacilli. Histopathologic examination of a skin biopsy specimen, PCR, and the Mantoux test also confirmed the presence of Mycobacterium tuberculosis. The patient began treatment with anti-tubercular drugs and his lesions gradually regressed. Discussion: The most common finding of scrofuloderma is a chronic, painless ulcerative lesion with purulent discharge. Cervical lymph nodes are frequently involved. These clinical features mimic several other bacterial and fungal conditions. However, Ziehl-Neelsen staining of the discharging ulcers can be used as a preliminary test to demonstrate acid fast bacilli in most of the cases. Histopathologic examination of skin biopsy specimen, culture on Lowenstein-Jensen media and PCR can confirm the diagnosis. Conclusion: Although Scrofuloderma is a rare cutaneous manifestation, it is curable with early diagnosis and prompt intervention of antitubercular drugs.

Genotypic and phenotypic diversity of Mycobacterium tuberculosis complex genotypes prevalent in West Africa.

Findings from previous comparative genomics studies of the Mycobacterium tuberculosis complex (MTBC) suggest genomic variation among the genotypes may have phenotypic implications. We investigated the diversity in the phenotypic profiles of the main prevalent MTBC genotypes in West Africa. Thirty-six whole genome sequenced drug susceptible MTBC isolates belonging to lineages 4, 5 and 6 were included in this study. The isolates were phenotypically characterized for urease activity, tween hydrolysis, Thiophen-2-Carboxylic Acid Hydrazide (TCH) susceptibility, nitric oxide production, and growth rate in both liquid (7H9) and solid media (7H11 and Löwenstein–Jensen (L-J)). Lineage 4 isolates showed the highest growth rate in both liquid (p = 0.0003) and on solid (L-J) media supplemented with glycerol (p<0.001) or pyruvate (p = 0.005). L6 isolates optimally utilized pyruvate compared to glycerol (p<0.001), whereas L5 isolates grew similarly on both media (p = 0.05). Lineage 4 isolates showed the lowest average time to positivity (TTP) (p = 0.01; Average TTP: L4 = 15days, L5 = 16.7days, L6 = 29.7days) and the highest logCFU/mL (p = 0.04; average logCFU/mL L4 = 5.9, L5 = 5.0, L6 = 4.4) on 7H11 supplemented with glycerol, but there was no significant difference in growth on 7H11 supplemented with pyruvate (p = 0.23). The highest release of nitrite was recorded for L5 isolates, followed by L4 and L6 isolates. However, the reverse was observed in the urease activity for the lineages. All isolates tested were resistant to TCH except for one L6 isolate. Comparative genomic analyses revealed several mutations that might explain the diverse phenotypic profiles of these isolates. Our findings showed significant phenotypic diversity among the MTBC lineages used for this study.

English

The control of tuberculosis (TB) has become a global health challenge due to the emergence of multidrug-resistant tuberculosis (MDR-TB) in Mycobacterium tuberculosis (MTB). This highlights the need for faster and more accurate detection of tuberculosis cases. The study aims to detect MDR-TB strains of pulmonary tuberculosis using resistance ratio method and to compare the diagnostic value of drug susceptibility testing (DST) with line probe assay (LPA) using Genotype MTBDRplus. All the sputum samples were tested for Acid Fast Bacilli (AFB) by Ziehl-Neelsen&rsquo;s staining method and were cultured on Lowenstein-Jensen (L-J) media. The identification and confirmation of M. tuberculosis were done using various biochemical tests. DST was carried against the first line of anti-TB drugs. MTB positive samples were subjected to LPA. A total of 57 samples were subjected to DST and LPA for the detection of drug resistance of MTB to RIF and INH after conventional detection methods were applied to all the samples. Among these 57 MTB samples, 11 (19.29%) were resistant to INH, 4 (7.14%) were resistant to RIF; eleven (19.29%) isolates were identified as MDR-TB. LPA revealed 54 MTB positive among 57 MTB culture-positive samples and 3 showed invalid results. In LPA, MDR-TB was found in10 samples (17.54%) in which one was RIF-resistant. The study concludes risk factors that resulted in the development of TB are biomedical, socio-cultural, and behavioral interactions. LPA can be used as a rapid diagnostic technique for the detection of MDR-TB. Key words: Tuberculosis (TB), multidrug resistance (MDR-TB), drug susceptibility testing (DST), line probe assay (LPA). &nbsp

Performance of QuantaMatrix Microfluidic Agarose Channel system integrated with mycobacteria growth indicator tube liquid culture.

The QuantaMatrix Microfluidic Agarose Channel (QMAC) system was used for rapid drug susceptibility testing (DST). Here, we performed DST using QMAC integrated with the mycobacteria growth indicator tube (MGIT) liquid culture employing a specially designed cross agarose channel for the tuberculosis chip. MGIT-, QMAC-, and Löwenstein-Jensen (LJ)-DSTs were performed using 13 drugs. The protocol for QMAC-DST was optimized using the inoculum obtained after the disaggregation of Mycobacterium tuberculosis clumps in MGIT culture. The completion times of QMAC-DST and MGIT-DST were analyzed, and the results of all three DSTs were compared. Discrepant results were analyzed using line probe assays and DNA sequencing. Nontuberculous mycobacteria were distinguished using the ρ-nitrobenzoic acid inhibition test. The overall agreement rate of QMAT-DST and LJ-DST was 97.0% and that of QMAT-DST and MGIT-DST was 86.3%. An average turnaround time for DST was 5.4 days, which was considerably less than the time required for MGIT-DST. The overall time required to obtain DST results using QMAC-DST integrated with MGIT culture was an average of 18.6 days: 13.2 days for culture and identification and 5.4 days for DST. Hence, QMAC-DST integrated with liquid culture can be used to perform DSTs with short turnaround times and effective detection. KEY POINTS: • QMAC system can simultaneously perform phenotypic DST with 13 anti-TB drugs and PNB. • An optimized DST protocol led to a marked decrease in clumping in MGIT culture. • QMAC system integrated with MGIT liquid culture system reduced the turnaround time.

A simple, efficient and cost-effective method for medium- to longterm maintenance and storage of Mycobacterium abscessus complex organisms

Traditional culture of non-tuberculous mycobacteria (NTMs) has involved egg-based formulations (Lowenstein-Jensen medium, Ogawa Egg medium) or defined media (Middlebrook formulations), which have disadvatages of composition complexity, availability and cost. Previously, the commercial agar formulation, Standard Plate Count (SPC) agar [Yeast extract 2.5 g/L, pancreatic digest of casein 5.0 g/L, glucose 1.0 g/L, agar 15.0 g/L, pH 7.0 ± 0.2 at 25 °C] has been shown to be an effective solid medium for the enumeration and laboratory manipulation of Mycobacterium abscessus complex organisms. Given its relative simplicity, commercial availability and inexpensive cost, we wished to evaluate its utility for the medium- to longterm maintenance/storage of these organisms. M. abscessus complex organisms (n = 33), were inoculated onto SPCA slopes and stored undistubed in the dark at ambient temperature for six months. Following this, slopes were broken out and culture of the NTM attempted. All slopes maintained NTM culture viability and were able to initiate growth six months later. We therefore advocate the cost-effective employment of SPCA slopes for the medium- to longterm maintenance of M. abscessus organisms, without the need for complex media, availability of sterile blood and requirements for continuous -80 °C freezing.

Diagnostic efficacy of Light-Emitting Diode (LED) Fluorescence based Microscope for the diagnosis of Tuberculous lymphadenitis.

BackgroundThe comparatively straightforward and cheaper light-emitting diode fluorescent microscope (LEDFM) was suggested by WHO to replace conventional microscope in tuberculosis (TB) laboratories. However, the comparable efficacy of each of those techniques differs from laboratory to laboratory. We investigated the efficacy of LEDFM for the diagnosis of tuberculous lymphadenitis (TBLN) patients.MethodsA cross-sectional study was conducted on 211 samples from clinically suspected tuberculous lymphadenitis patients. Three smears were prepared from FNA on microscope slides for cytomorphology study, Auramine O (AO), and for Ziehl-Neelsen (ZN) staining. The left-over samples were inoculated onto Lowenstein-Jensen (LJ) media. Statistical analysis was done using STATA version 11. The sensitivity, specificity, positive and negative predictive values were calculated by considering the culture results as the gold standard using a 95% confidence interval.ResultsAmong 211 samples 49.7% (105) were positive by cytomorphology, 32.7% (69) by LEDFM, 23.69% (50) by LJ culture, and 13.7% (29) by ZN. Compared to the gold standard sensitivity of ZN, LEDFM, and cytomorphology were 30% [95% CI: 17.9–44.6], 66% [95% CI: 51.2–78.8] 78% [95% CI: 64–88.5], respectively. The specificity of ZN, LEDFM, and cytomorphology was 91.3% [95% CI: 85.8–95.2], 77.6% [95% CI: 70.4–83.8], 58.8% [95% CI: 50.7–66.5], respectively.ConclusionLED fluorescence microscopy gives a legitimate option in contrast to conventional ZN techniques in terms of its higher sensitivity, a bit lower specificity, time-saving, and minimal effort.

Prevalence of Multidrug-Resistant Tuberculosis Using Phenotypic Drug Susceptibility Testing and GeneXpert MTB/RIF with Characterization of Non-tuberculous Mycobacteria Using MALDI-TOF

Background: Multidrug-resistant tuberculosis (MDR-TB) and infections by nontuberculous mycobacteria (NTM) are diseases of major public health concern. Objective: The aim of the present work is to study the prevalence and patterns of MDRTB as well as the characterization of isolated NTM species. Methodology: All samples (1069) were subjected to smear microscopy, culture on Lowenstein-Jensen (LJ) media, and phenotypic drug susceptibility testing (DST) of MTB to isoniazid (INH), rifampin (RF), streptomycin (S), and ethambutol (E). GeneXpert was used for direct detection of MTB and RF resistance. Matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS) was utilized for characterizing isolated NTM species. Results: M.tuberculosis (MTB) was isolated at a rate of 95.3% (1019/1069). MDR-TB was detected at rate of 7.16% with significant patterns for INH + RF + S + E (46.5%) and INR + RF (24.6%) (P-value &lt;0.001). RF resistance was detected at a rate of 27.2% by GeneXpert. Seven NTM species (0.6%) were isolated in culture of which M.porcinum and M.fortuitum had confident identification by MALDI-TOF (score ≥1.8). Conclusion: MDR-TB rate was found to be 7.16% with significant dominance for INH + RF + S + E and INR + RF resistance patterns, while NTM rate was 0.6%.

Antimicrobial activity of Centella asiatica and Gigantochloa apus.

Antibiotic treatments can create multi-drug resistance among several pathogens. There is a need for an antibiotic alternative to overcome this problem. In Indonesia, Centella asiatica (Asiatic pennywort) and Gigantochloa apus (string bamboo) are two common medicinal plants used to treat tuberculosis, diarrhea, and other symptoms. This study was done to compare the antimicrobial activity of C.asiatica and G.apus against five pathogenic bacteria, i.e.,Mycobacterium tuberculosis H37Rv strain, Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Salmonella typhi. The ethanol extracts of C.asiatica, and G.apus shoot were obtained by using speed extractor, pressure, and temperature extraction. The phytochemical contents of each extract were screened. The ethanol extract's antimycobacterial activity was determined using Lowenstein Jensen (LJ) medium and antibacterial activity was determined using Kirby-Bauer methods on Mueller Hinton agar (MHA). The phytochemical analysis showed that G.apus extract contains alkaloids and tannins, whereas C.asiatica extract contains flavonoids, alkaloids, saponins, and tannins. This study showed that G.apus inhibited the growth of M.tuberculosis H37Rv strain and S.typhi. C.asiatica showed antimicrobial activity against all pathogenic bacteria tested, except B.subtilis. Both medicinal plants extract can inhibit the growth of five pathogenic bacteria tested, thus, have the potential as an alternative treatment, or complementary, to treat the pathogenic bacterial infection.

Baseline Xpert MTB/RIF ct values predict sputum conversion during the intensive phase of anti-TB treatment in HIV infected patients in Kampala, Uganda: a retrospective study

BackgroundIn resource-limited settings, sputum smear conversion is used to document treatment response. Many People living with HIV (PLHIV) are smear-negative at baseline. The Xpert MTB/RIF test can indirectly measure bacterial load through cycle threshold (ct) values. This study aimed to determine if baseline Xpert MTB/RIF could predict time to culture negativity in PLHIV with newly diagnosed TB.MethodsA subset of 138 PLHIV from the ‘SOUTH’ study on outcomes related to TB and antiretroviral drug concentrations were included. Bacterial load was estimated by Mycobacterium Growth Indicator Tubes (MGIT) culture time-to-positivity (TTP) and Lowenstein Jensen (LJ) colony counts. Changes in TTP and colony counts were analyzed with Poisson Generalised Estimating Equations (GEE) and multilevel ordered logistic regression models, respectively, while time to culture negativity analysed with Cox proportional hazard models. ROC curves were used to explore the accuracy of the ct value in predicting culture negativity.ResultsA total of 81 patients (58.7%) were males, median age 34 (IQR 29 ̶ 40) years, median CD4 cell count of 180 (IQR 68 ̶ 345) cells/μL and 77.5% were ART naive. The median baseline ct value was 25.1 (IQR 21.0 ̶ 30.1). A unit Increase in the ct value was associated with a 5% (IRR = 1.05 95% CI 1.04 ̶ 1.06) and 3% (IRR = 1.03 95% CI 1.03 ̶ 1.04) increase in TTP at week 2 and 4 respectively. With LJ culture, a patient’s colony grade was reduced by 0.86 times (0R = 0.86 95% CI 0.74 ̶ 0.97) at week 2 and 0.84 times (OR = 0.84 95% CI 0.79 ̶ 0.95 P = 0.002) at week 4 for every unit increase in the baseline ct value. There was a 3% higher likelihood of earlier conversion to negativity for every unit increase in the ct value. A ct cut point ≥28 best predicted culture negativity at week 4 with a sensitivity of 91. 7% & specificity 53.7% while a cut point ≥23 best predicted culture negativity at week 8.ConclusionBaseline Xpert MTB/RIF ct values predict sputum conversion in PLHIV on anti-TB treatment. Surrogate biomarkers for sputum conversion in PLHIV are still a research priority.

Diagnostic utility of GeneXpert MTB/RIF assay versus conventional methods for diagnosis of pulmonary and extra-pulmonary tuberculosis

BackgroundTuberculosis (T.B) is one of the major infectious diseases in the developing countries. The diagnosis of extrapulmonary T.B (EPTB) remains problematic and emergence of resistant strains poses a significant threat. Improved diagnosis of tuberculosis is a global priority for proper control. The study aimed to assess the diagnostic accuracy of GeneXpert MTB/RIF assay for diagnosis of pulmonary TB (PTB) and EPTB and to evaluate the performance of GeneXpert system for demonstrating rifampicin resistance among the studied patients.MethodsA total of 582 clinical samples (449 pulmonary; 430 sputum and 19 bronchoalveolar lavage (BAL) and 133 extra-pulmonary origins; 26 pleural fluid, 62 CSF, 19 ascetic fluid, 12 pus and 14 urine) were collected from patients under clinical and radiological assessment of either PTB or EPTB who were admitted to Menoufia Chest Hospital over a period of three years. Clinical samples were processed and investigated for detection of Mycobacterium tuberculosis (MTB) by both Xpert assay and the conventional methods including Ziehl-Neelsen (ZN)/acid-fast bacillus (AFB) smear microscopy and Lowenstein-Jensen (LJ) culture. Patients′ demographic, clinical characteristics and risk factors for acquiring rifampicin resistance were analyzed.ResultsThe sensitivity, specificity, false- negative rate and total accuracy of AFB smear microscopy respectively were 72.1 %, 81.3 %, 27.9 and 78.8 % for PTB. However for EPTB, they were 63.2 %, 70.5 %, 36.8 and 68.4 % respectively in relation to LJ culture as the gold standard. GeneXpert MTB/RIF revealed better performance for PTB than EPTB. For PTB, it showed 90.2 % sensitivity, 86.9 % specificity, and 9.8 % false- negative rate. For EPTB, the assay showed a sensitivity of 81.6 %, specificity of 78.9 % and false- negative rate of 18.5 %. Multivariate regression analysis showed that presence of EPTB and contacts with known TB cases were independent risk factors for developing rifampicin resistance.ConclusionsGeneXpert MTB/RIF assay is a rapid and highly sensitive technique for diagnosis of PTB or EPTB. Its simplicity and accuracy make this new method a very impressive tool for diagnosis of MTB and rifampicin resistance.