Utilization of Truenat chips in defining XDR, pre-XDR and MDR in tuberculous meningitis.

SETTING AND OBJECTIVE: To develop and evaluate newer molecular tests that identify drug resistance according to contemporary definitions in Tuberculous meningitis (TBM), the most severe form of EPTB. DESIGN: 93 cerebrospinal fluid (CSF) specimens [41 culture-positive and 52 culture-negative], were subjected to Truenat MTB Plus assay along with chips for rifampicin, isoniazid, fluoroquinolones and bedaquiline resistance. The performance was compared against phenotypic drug susceptibility testing (pDST), Line probe assay (LPA) and gene sequencing. RESULTS: Against pDST, Truenat chips had a sensitivity and specificity of 100%; 94.47%, 100%; 94.47%, 100%; 97.14% and 100%; 100%, respectively for rifampicin, isoniazid, fluoroquinolones and bedaquiline. Against LPA, all Truenat chips detected resistant isolates with 100% sensitivity; but 2 cases each of false-rifampicin and false-isoniazid resistance and 1 case of false-fluoroquinolone resistance was reported. Truenat drug chips gave indeterminate results in ∼25% cases, which were excluded. All cases reported indeterminate were found to be susceptible by pDST/LPA. CONCLUSION: The strategic drug resistance chips of Truenat Plus assay can contribute greatly to TB elimination by providing rapid and reliable detection of drug resistance pattern in TBM. Cases reported indeterminate require confirmation by other phenotypic and genotypic methods.

Fe-based MOFs as promising adsorbents and photocatalysts for re-use water contained arsenic: Strategies and challenges.

Arsenic (As) contaminated water, especially groundwater reservoirs, is a major issue worldwide owing to its hazardous consequences on human health and the global environment issues. Also, irrigating agricultural fields with As-contaminated water not only produces an accumulation of As in the soil but also compromises food safety due to As entering into agricultural products. Hence, there is an urgent need to develop an efficient method for As removal in water. Fe-based MOFs have attained special attention due to their low toxicity, high water stability, better physical and chemical properties, and high abundance of iron. The arsenic species removal by Fe-MOF follows the adsorption and oxidation mechanism where As (III) converts into As (V). Moreover, the adsorption mechanism is facilitated by electrostatic interactions, H-bonding, acid-base interaction, hydrophobic interactions, van der Waals forces, π-π stacking interactions, and coordinative bindings responsible for Fe-O-As bond generation. This review thoroughly recapitulates and analyses recent advancements in the facile synthesis and potential application of Fe-based MOF adsorbents for the elimination of As ions. The most commonly employed hydro/solvothermal, ultrasonic, microwave-assisted, mechanochemical, and electrochemical synthesis for Fe-MOF has been discussed along with their adsorptive and oxidative mechanisms involved in arsenic removal. The effects of factors like pH and coexisting ions have also been discussed. Lastly, the article also proposed the prospects for developing the application of Fe-based MOF in treating As-contaminated water.

Role of MicroRNAs as post transcription regulators of matrix metalloproteinases and their association in tuberculous meningitis.

Matrix metalloproteinases (MMPs) have a role in driving neuroinflammation in infectious as well as non-infectious diseases; however, recent reports have potentiated the role of microRNAs in regulating MMPs at post-transcriptional levels, leading to dysregulation of crucial MMP functions like tissue remodelling, blood brain barrier integrity, etc. In present study, microRNAs regulating MMPs (MMP2 and MMP3) were selected from database search followed by literature support. Expression of these microRNAs i.e., hsa-miR-495-3p, hsa-miR-132-3p and hsa-miR-21-5p was assessed by RT-PCR and the protein levels of MMPs were assessed by ELISA in the cerebrospinal fluid (CSF) of tuberculous meningitis (TBM) patients, healthy controls (HC) and non-infectious neuroinflammatory disease (NID) patients. The expression of hsa-miR-495-3p and hsa-miR-132-3p showed downregulation in TBM while hsa-miR-21-5p was overexpressed as compared to healthy controls. Moreover, MMP levels were found to be deranged with a significant increase in MMP3 levels in the TBM and NID patients compared to HC group. These observations highlight dysregulated microRNAs (hsa-miR-495-3p, hsa-miR-21-5p and hsa-miR-132-3p) levels might impair the levels of MMPs (MMP2 and MMP3) leading to neuroinflammation in TBM and NID population. These findings can further be applied to target these microRNAs for developing newer treatment modalities for better complication management.

Diagnosing osteoarticular tuberculosis and detecting rifampicin resistance: A comparative analysis of Truenat MTB Plus vs GeneXpert Ultra.

SETTING: Diagnosing osteoarticular tuberculosis (OATB) and detecting drug resistance is a challenge in an endemic country like India. OBJECTIVE: Truenat MTB Plus assay (TruPlus), a chip-based portable machine, was compared with GeneXpert Ultra (GxUltra) for diagnosing drug-resistant OATB. DESIGN: 115 synovial fluid and pus specimens [22 culture-positive confirmed, 58 culture-negative clinically-suspected, 35 non-TB controls] processed between 2017 and 2023 were subjected to TruPlus, GxUltra and multiplex-PCR for diagnosing OATB. They were further screened for rifampicin resistance using TruRif chip. The performance was evaluated against composite reference standard, phenotypic drug susceptibility testing and rpoB gene sequencing. RESULTS: TruPlus, GxUltra and MPCR detected 77.5 %, 71.25 %, and 83.75 %, cases of OATB, respectively. TruPlus detected five additional cases missed by GxUltra. The performance of TruPlus was comparable to GxUltra (p = 0.074) and to MPCR (p = 0.074), while performance of GxUltra was significantly inferior to MPCR (p = 0.004). The overall agreement with reference standard was substantial for TruPlus and MPCR and moderate for GxUltra. Both TruRif and GxUltra reported 4 cases as rifampicin resistant. CONCLUSION: TruPlus along with TruRif offers better sensitivity than GxUltra. Its compact and portable platform allows wider application in peripheral settings, thus making it a pragmatic solution for diagnosing OATB and its drug resistance.

Allium sativum Essential Oil Supplementation Reverses the Hepatic Inflammation, Genotoxicity and Apoptotic Effects in Swiss Albino Mice Intoxicated with the Lead Nitrate.

Prolonged lead (Pb) exposure impairs human health due to its interference with physiological and biochemical processes. Therefore, it is necessary to investigate natural therapeutics to alleviate Pb-induced intoxication. In the current investigation, essential oil extracted from the fresh bulbs of Allium sativum was considered as a natural remedy. Initially, in vitro antioxidant and anti-inflammatory activity of A. sativum essential oil (ASEO) were explored. The results reported that ASEO exhibits potent antioxidant and anti-inflammatory potential. Additionally, an in vivo study was conducted to elucidate its preventive role against Lead-nitrate (LN)-induced hepatic damage in Swiss albino mice. The experimental mice were allocated into six groups: Control, LN-intoxicated group (50 mg/kg), LN + ASEO (50 mg/kg), LN + ASEO (80 mg/kg), LN + Silymarin (25 mg/kg), and LN + vehicle oil control group. The entire duration of the study was of 30 days. From the results, it was determined that LN exposure elevated the Pb content in hepatic tissues which subsequently increased the serum biomarkers, inflammatory cytokines (NF-kB, TNF-α, IL-6) as well as apoptotic factors (caspase-3, BAX), all of which contribute to DNA damage. Meanwhile, it reduced anti-inflammatory (IFN-γ and IL-10) and anti-apoptotic factors (Bcl-2). Furthermore, Pb accumulation in hepatic tissues changed the histological architecture, which was linked to necrosis, central vein dilation, inflammatory cell infiltration and Kupffer cell activation. In contrast to this, ASEO administration decreased the Pb content, which in turn reduced the level of serum biomarkers, inflammatory and apoptotic factors. At the same time, it increased the anti-inflammatory and anti-apoptotic factors, thereby reduced DNA damage and restored the hepatic histology. In conclusion, exhaustive research is of the utmost demand to elucidate the precise defense mechanisms of ASEO against LN-induced hepatotoxicity.

Mycobacterium abscessus Complex-Associated Chronic Meningitis: Time to Think Beyond Tuberculosis.

Background: Mycobacterium abscessus complex (MabC) has emerged as an important cause of human infections, including meningitis. In the absence of correct microbiological identification, cases of MabC meningitis are treated with conventional anti-tubercular therapy, thereby worsening the outcome. Objective: The current study was conducted to determine the clinical features, antimicrobial susceptibility, and outcome of patients with MabC meningitis. Material and Methods: Cerebrospinal fluid specimens processed between 2011 and 2021 were subjected to smear, culture, MALDI TOF identification, hsp65 gene sequencing, and susceptibility testing using Sensititre™ RAPMYCOI plates along with a literature review. Results: 12 cases of MabC meningitis were identified between 2011 and 2021, 11 of which were M. abscessus subspecies abscessus on hsp65 gene sequencing. A pioneer case of meningitis with M. abscessus subspecies bolletii was also identified. The common predispositions were TB elsewhere, HIV positivity, and head injury. Two patients had dual infections, both MabC and TB. Ten patients succumbed to infection with a mean survival of 11 months. All isolates were susceptible to amikacin and tigecycline and subspecies bolletii had a higher minimum inhibitory concentration (MIC) than subspecies abscessus. A combined analysis with the available literature, reporting 19 more cases, revealed that the overall mortality of MabC meningitis was 61.3% (19/31) and that of shunt-associated/neurosurgical intervention-related MabC meningitis was 66.7% (12/20). To date, out of 20 MabC meningitis isolates in which subspecies identification was carried, 13 were M. abscessus, six were M. massiliense, and one was M. bolletii. Conclusion: MabC is an important differential diagnosis of chronic meningitis. Prompt identification and speciation are imperative for targeted therapy, thus improving the overall patient outcome.

Changing Clinical Profile and Predictors of Mortality in Patients of Acute Febrile Encephalopathy from North India.

Introduction: Acute encephalitis syndrome (AES) or acute febrile encephalopathy is a clinical condition characterized by altered mental status occurring after or along with a short febrile illness. In developing countries, infections are the predominant cause of AES. Prominent infections known to cause AES include viruses (such as herpes simplex virus [HSV], Japanese Encephalitis [JE] virus, dengue, enteroviruses [EVs]), bacteria, fungus, and parasites. In the present study, we aim to analyze the etiology, clinical features, and predictors of mortality in patients presenting with acute febrile encephalopathy or acute encephalitic syndrome. The present study was a prospective observational study conducted at Post Graduate Institute of Medical Education and Research a tertiary care center in Chandigarh, India. Methods: A total of 105 patients with ≥18 years of age with fever (body temperature >101° F for duration ≤14 days) and altered sensorium (Glasgow coma scale [GCS] score ≤10) lasting for more than 24 h, either accompanying the fever or following it were enrolled. Demographic and clinical details were recorded on pro forma. Cerebrospinal fluid (CSF) analysis was performed for all the enrolled patients at admission for cytology, CSF glucose to blood glucose ratio, protein levels, gram stain and culture sensitivity, adenosine deaminase levels, polymerase chain reaction for HSV/EV/mycobacterium tuberculosis (TB) and immunoglobulin M Enzyme-linked immune assay for JE. Computed tomography of the brain was done in all patients while magnetic resonance imaging (MRI) of the brain was carried out in 75 patients. Results: Among the 105 patients, tubercular meningitis was seen in 27 (25.7%) patients followed by acute pyogenic meningitis in 18 (17.1%) patients. Probable viral encephalitis was present in 12 (11.4%) cases. Septic encephalopathy (n = 10) and scrub typhus encephalitis (n = 8), HSV encephalitis (n = 6), dengue encephalitis (n = 4), leptospirosis (n = 3) were the other infections causing acute febrile encephalitis in our study. In addition to fever and altered sensorium common symptoms observed were headache (52.4%), vomiting (35.2%), and seizures (29.5%). The factors predicting increased mortality were female gender, fever of more than 38°C at admission, GCS <7, MRI showing disease-related findings like altered signal intensity bilateral medial temporal and insular area in herpes simplex encephalitis, etc., changes, and the group of patients where a definite diagnosis could not be established during the hospital stay. Conclusions: Tubercular meningitis/central nervous system TB is the predominant cause of acute febrile encephalopathy in developing countries. Scrub and dengue encephalitis are emerging as an important cause of acute febrile encephalopathy and occur predominantly in postmonsoon seasons. Acute febrile encephalopathy remains an important cause of mortality in patients presenting to Emergency Department (ER). The strongest predictors of mortality are low GCS and undiagnosed cases of AES.

Role of vascular endothelial growth factor in tuberculous meningitis: A prospective study from a tertiary care center in North India.

INTRODUCTION: The status of vascular endothelial-derived growth factor (VEGF) in the pathogenesis of tuberculous meningitis (TBM) remains far from clear. We prospectively evaluated the role of serum and cerebrospinal fluid (CSF) VEGF in TBM. PATIENTS AND METHODS: This prospective study was conducted at a tertiary care center in North India from January 2018 to June 2019. Consecutive drug-naive patients (n = 82) of TBM diagnosed on the basis of modified Ahuja's criteria were included in the study. The results were compared with 49 control subjects (n = 49). Serum and CSF VEGF were done in all the cases and controls. Follow-up serum VEGF levels were done in 34 patients after 3 months of completion of antitubercular therapy. The VEGF levels were estimated using the human VEGF enzyme-linked immunosorbent assay kit. RESULTS: The mean age was 29.9 ± 13.1 years. The study group consisted of 33 (40.2%) men and 49 (59.8%) women. BACTEC MGIT960 was positive in 15 (18%) patients while multiplex tuberculosis polymerase chain reaction was positive in 73 (89%) patients. Levels of VEGF in serum and CSF of TBM patients were not elevated when compared to controls. There was no association between final outcome in TBM and decrease in serum levels of VEGF at follow-up. CONCLUSION: VEGF may not be playing a significant role in the pathogenesis of TBM. Future studies with larger sample size may clarify the status of VEGF further in TBM.

Knock-out of vasotocin reduces reproductive success in female zebrafish, Danio rerio.

The vertebrate nonapeptide vasotocin/vasopressin is evolutionarily highly conserved and acts as neuromodulator and endocrine/paracrine signaling molecule. Circumstantial and mechanistic evidence from pharmacological manipulations of the vasotocin system in several teleost fishes suggest sex- and species-specific reproductive roles of vasotocin. While effects of vasotocin on teleost reproductive physiology involve both courtship behaviors and the regulation of the hypothalamic-pituitary-gonadal (HPG) axes, comprehensive studies investigating behavioral and physiological reproductive consequences of genetic ablation of vasotocin in a genetically tractable fish model, such as the zebrafish, are currently lacking. Here, we report the generation of homozygous CRISPR/Cas9-based vasotocin gene knock-out zebrafish. Breeding pairs of vasotocin knock-out fish produce significantly fewer fertilized eggs per clutch compared to wildtype fish, an effect coincident with reduced female quivering courtship behavior. Crossbreeding experiments reveal that this reproductive phenotype is entirely female-dependent, as vasotocin-deficient males reproduce normally when paired with female wild-type fish. Histological analyses of vasotocin knock-out ovaries revealed an overall reduction in oocytes and differential distribution of oocyte maturation stages, demonstrating that the reproductive phenotype is linked to oocyte maturation and release. Ovarian hormone quantification and gene expression analysis in mutant fish indicated reduced synthesis of Prostaglandin F2α, a hormone involved in ovarian maturation, egg release and regulation of female courtship behavior in some cyprinids. However, acute injection of vasotocin did not rescue the female mutant reproductive phenotype, suggesting a contribution of organizational effects of vasotocin. Together, this study provides further support for emerging roles of vasotocin in female teleost reproduction in an important teleost model species.

Determining the diagnostic potential of Truenat MTB Plus for Tubercular lymphadenitis and detection of drug resistance and a comparison with GeneXpert Ultra.

SETTING: Tubercular lymphadenitis (TBLA), the most common form of extrapulmonary tuberculosis, is a diagnostic challenge. OBJECTIVE: Truenat MTB Plus (TruPlus) along with Truenat Rif assay (TruRif) was evaluated for detection of TBLA and rifampicin resistance and compared with GeneXpert Ultra (Xpert Ultra). DESIGN: 100 fine-needle aspirated specimens [50 confirmed by culture/smear/cytology, 20 clinically suspected, and 30 controls], processed in the mycobacteriology division of department of microbiology were subjected to TruPlus and TruRif, Xpert Ultra and multiplex PCR. The results of TBLA detection were compared against composite reference standard (CRS) and those of rifampicin resistance were compared against phenotypic drug susceptibility testing and rpoB gene sequencing. RESULTS: In comparison to CRS, the diagnostic yield of TruPlus, Xpert Ultra and MPCR was 77.14%, 59.18% and 84.28%, respectively; with substantial agreement for TruPlus (k = 0.66) and MPCR (k = 0.76) and moderate for Xpert Ultra (k = 0.60). TruRif reported four cases as RifR and Xpert Ultra reported two. On comparing with phenotypic DST and gene sequencing, only two cases of RifR were confirmed, hence TruRif reported false-RifR in two cases. CONCLUSION: TruPlus could be used as a reliable tool for diagnosing TBLA. The reporting of RifR by TruRif should be confirmed by phenotypic DST or gene sequencing.

Association of Micronutrients with Tuberculosis Development in HIV Infected Patients.

Human immunodeficiency virus (HIV) infection associated with weakened immune system due to decreased CD4 T cell count favors development of tuberculosis. Effector immune responses are also associated with micronutrient status due to their prominent role in maintaining immune functions. Micronutrient deficiencies are quite common among HIV patients that further result into compromised immunity thus making the conditions even more favorable for mycobacteria to establish disease. So, current study was designed to assess association of different micronutrients with development of TB in HIV patients. Micronutrient levels were measured in asymptomatic HIV patients who were monitored for the development of TB during follow up period (incident TB) within one month to one year and also in symptomatic microbiologically confirmed HIV-TB patients. Among various micronutrients assessed, levels of ferritin were found to be significantly increased (p < 0.05) with significant decreased zinc (p < 0.05) and selenium (p < 0.05) levels in incident TB group as well as in HIV-TB subjects compared to asymptomatic HIV patients who did not develop TB in the follow up period. Importantly, increased levels of ferritin and decreased levels of selenium were significantly associated with development of tuberculosis in HIV patients.

Molecular diagnosis of Tuberculous meningitis: sdaA-based multi-targeted LAMP and GeneXpert Ultra.

SETTING: Nucleic acid amplification techniques like GeneXpert and GeneXpert Ultra (Xpert Ultra), the first-line tests for diagnosing Tuberculous meningitis (TBM), are expensive and depend on sophisticated equipment. OBJECTIVE: The diagnostic potential of multitargeted loop-mediated isothermal assay (MLAMP), a low-cost simple test using novel gene combination, was evaluated for TBM. DESIGN: 300 CSF specimen (200 TBM patients, 100 controls) processed between January 2017 and December 2021 were subjected to MLAMP (using sdaA, IS1081 and IS6110 gene targets), sdaA PCR and Xpert Ultra. The performance was evaluated against uniform case definition as per Marais criteria, and against culture. RESULTS: Uniform case definition classified 50 as definite TBM and 150 as probable or definite TBM. Against this uniform case definition, the sensitivity and specificity of MLAMP was 88% and 100%, respectively. The sensitivity was 96% against culture-positive cases and 85.3% against culture-negative cases. The sensitivity of sdaA-LAMP, IS1081-LAMP, IS6110-LAMP, Xpert Ultra and sdaA-PCR was 82.5%, 80.5%, 85.3%, 67% and 71%, respectively against uniform case definition. sdaA-LAMP detected additional two cases and IS1081-LAMP detected nine. 11 of 134 (8.2%) cases were reported rifampicin resistant by Xpert Ultra. CONCLUSION: MLAMP, incorporating sdaA and IS1081, is a cheap, easy and accurate first-line diagnostic test for TBM.

Comparison of Protein B Polymerase Chain Reaction (PCR) With IS6110 PCR for Diagnosis of Tuberculous Meningitis Patients.

Purpose Tuberculous meningitis (TBM) is a diagnostic challenge. With the conventional staining and culture techniques being too insensitive and time-consuming, and the commercial detection systems being costly, polymerase chain reaction (PCR) seems lucrative for routine laboratories. The purpose of this study was to evaluate the diagnostic potential of protein b antigen polymerase chain reaction (Pab PCR) for TBM, in comparison to IS6110. Another purpose was to compute a cut-off at which adenosine deaminase (ADA) could diagnose TBM. Material and methods This is a prospective case-control study to measure the diagnostic accuracy of PCR, BACTEC culture, Lowenstein-Jensen (LJ) culture, ADA, and acid-fast bacilli (AFB) smear tests in TBM. CSF from 50 TBM patients (10 confirmed, 40 clinically suspected) and 40 controls was subjected to Pab PCR and IS6110 PCR, and performance was compared against culture and composite reference standards. Results The overall sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of Pab PCR in diagnosing TBM were 82%, 100%, 100%, and 81.63%, respectively, and that of IS6110 PCR were 74%, 100%, 100%, and 75.47%, respectively. Both PCRs outperformed culture (p<0.001). Though performance of both PCRs was comparable (p=0.335) with excellent agreement (k=0.86), Pab PCR detected four additional cases, one culture-positive and three culture-negative clinically suspected. ADA of 6.5 IU/L was able to differentiate between TBM and non-TBM with 86% sensitivity and 63% specificity. Conclusions Molecular tools such as PCR have the potential to increase the clinician's ability to diagnose tuberculous meningitis. Pab PCR is a rapid and reliable method for diagnosing TBM in routine microbiology laboratories.

Evaluating Truenat Assay for the Diagnosis of Ocular Tuberculosis and Detection of Drug Resistance.

BACKGROUND: Truenat MTB Plus assay was evaluated for diagnosing ocular tuberculosis (OTB) and detecting multi-drug resistant (MDR) and extremely-drug resistant (XDR) OTB. METHODS: A total of 75 vitreous fluid specimens [five confirmed OTB, 40 clinically suspected OTB and 30 controls] were subjected to Truenat MTB Plus, multiplex PCR, and Xpert Ultra. Chips of Truenat were used for detecting rifampicin, isoniazid, fluoroquinolone and bedaquiline resistance. The performance was compared against culture, composite reference standard, and gene sequencing. RESULTS: The overall sensitivity of TruePlus, MPCR, and Ultra in diagnosing OTB was 66.6%, 73.3%, and 55.5%, respectively. Out of six cases with mutations in rpoB gene, RifR was detected in five by TrueRif and four by Ultra. Three MDR and one XDR-OTB were reported by Truenat. CONCLUSION: Truenat assay along with its strategic chips is a rapid and reliable tool for diagnosis of OTB and detection of drug resistance, including MDR and XDR-OTB.Abbreviations: OTB: Ocular tuberculosis; XDR: Extremely drug resistant; Ultra: Xpert MTB/RIF Ultra; Xpert: Xpert MTB/RIF; PCR: polymerase chain reaction; NAATs: Nucleic acid amplification tests; MDR: Multi Drug Resistant; NSP: National Strategic plan for elimination of tuberculosis; FqR: Fluoroquinolone resistant; BdqR: bedaquiline resistant; TrueRif: Truenat MTB Rif Dx; TruePlus: Truenat Plus; INH: Isoniazid; DST: Drug susceptibility testing; MGIT: Mycobacterial growth indicator tube; CRF: Composite reference standard; PPV: positive predictive value; NPV: negative predictive value; EPTB: extrapulmonary tuberculosis; VF: vitreous fluid; DNA: deoxyribonucleic acid; ATT: antitubercular therapy; RifR: Rifampicin resistance; RifS: Rifampicin susceptible; RifI: Rifampicin indeterminate.

MTBDRplus and MTBDRsl for simultaneous diagnosis of gastrointestinal tuberculosis and detection of first-line and second-line drug resistance.

BACKGROUND AND AIM: Emergence of drug resistance, especially to second-line drugs, hampers tuberculosis elimination efforts. The present study aimed to evaluate MTBDRplus and MTBDRsl assays for detecting first-line and second-line drug resistance, respectively, in gastrointestinal tuberculosis (GITB). METHODS: Thirty ileocecal biopsy specimens, processed in the Department of Microbiology between 2012 and 2022, that showed growth of Mycobacterium tuberculosis on culture were included in the study. DNA, extracted from culture, was subjected to MTBDRplus and MTBDRsl (Hain Lifescience GmbH, Nehren, Germany), following manufacturer's instructions. Their performance was compared against phenotypic drug susceptibility testing (pDST) and gene sequencing. RESULTS: Out of the 30 specimens, 4 (13.33%) were mono-isoniazid resistant, 4 (13.33%) were multidrug resistant (MDR), 2 (6.67%) were pre-extensively drug resistant (pre-XDR), and 2 (6.67%) were mono-fluoroquinolone resistant. The results were 100% concordant with pDST and gene sequencing. CONCLUSIONS: In the wake of growing drug resistance in all forms of extrapulmonary tuberculosis, including GITB, MTBDRplus and MTBDRsl are reliable tools for screening of resistance to both first-line and second-line drugs.

Laboratory Investigations in Infectious Uveitis.

Laboratory investigations can play a significant role in the diagnosis and decision-making of infectious uveitis. Though direct demonstration of the infective organism remains the gold standard of diagnosis, it is not always possible with ocular tissues. Recent advancements in molecular techniques have made it possible to overcome these limitations and to identify the genomic DNA of pathogens associated with infectious uveitis. Techniques such as next-generation sequencing can analyze all DNA-based lifeforms, regardless of whether they are bacteria, fungi, viruses, or parasites and have been used in the laboratory diagnosis of intraocular inflammation. On the other hand, serological tests, though they dominate the diagnostic landscape of various infectious etiologies in uveitis in routine clinical practice, have varied specificities and sensitivities in different infectious uveitis. In this review, we focus on various methods of laboratory diagnosis of infectious uveitis and discuss the recent advances in molecular diagnosis and their role in various infectious clinical entities.

Diagnosing osteo-articular tuberculosis and multidrug resistance using real-time polymerase chain reaction and high-resolution melt-curve analysis.

The study evaluated real-time quantitative polymerase chain reaction (qPCR) and high-resolution melt-curve analysis (HRM) for simultaneous diagnosis of osteo-articular tuberculosis (OATB) and drug resistance. Two hundred and fifty synovial fluid and pus specimens (20 confirmed OATB by culture, 130 suspected OATB, and 100 controls) processed in the Department of Medical Microbiology, PGIMER were subjected to qPCR using rpoB, MPB64, and IS6110 genes. All OATB positive specimens were subjected to HRM for detecting resistance to rifampicin and isoniazid. qPCR detected 129/150 OATB cases with a sensitivity of 86% (95% for confirmed and 84.6% for suspected OATB cases) and specificity of 100%. rpoB and MPB64 genes had higher sensitivity than IS6110 (86% vs. 74.6%). HRM reported eight multidrug resistant (MDR), two mono-rifampicin, and five mono-isoniazid resistant cases, all were concordant with gene sequencing. qPCR followed by HRM analysis offer a simple, accurate, and rapid platform for simultaneous detection of OATB and MDR.

Comparative Evaluation of GeneXpert MTB/RIF Ultra and GeneXpert MTB/RIF for Detecting Tuberculosis and Identifying Rifampicin Resistance in Pars Plana Vitrectomy Samples of Patients with Ocular Tuberculosis.

BACKGROUND: Xpert MTB/RIF Ultra (Ultra) was evaluated for the first time on Ocular tuberculosis (OTB) samples and compared with Xpert. METHODS: Seventy five vitreous fluid samples (3 confirmed OTB, 47 clinically suspected OTB, and 25 controls) were subjected to Ultra, Xpert and Multiplex-PCR and compared against culture, composite reference standard (CRS), and gene sequencing. RESULTS: The sensitivity of Ultra was 50% in diagnosing OTB (100% against culture and 46.8% against CRS). The overall sensitivity of Xpert and MPCR was 16% and 72%, respectively. Xpert missed three culture-positive cases and MPCR detected additional 11. Ultra and Xpert missed two and four cases of RifR, respectively. A total of 13(59%) cases were reported 'trace' by Ultra in which RifR could not be evaluated. CONCLUSION: Ultra outperformed Xpert in diagnosing OTB. The advantage of Ultra's simultaneous RifR detection is lost since the trace bacterial loads in the specimens cause indeterminate results of RifR testing.Abbreviations: OTB: Ocular tuberculosis; Ultra: Xpert MTB/RIF Ultra; Xpert: Xpert MTB/RIF, MPCR: multiplex polymerase chain reaction; NAATs: Nucleic acid amplification tests; MLAMP: multitargeted loop-mediated isothermal amplification; PPV: positive predictive value; NPV: negative predictive value; EPTB: extrapulmonary tuberculosis; VF: vitreous fluid; DNA: deoxyribonucleic acid; ATT: antitubercular therapy; RifR: Rifampicin resistance; RifS: Rifampicin susceptible; RifI: Rifampicin indeterminate.

Diffusion Tensor and Dynamic Contrast-Enhanced Magnetic Resonance Imaging Correlate with Molecular Markers of Inflammation in the Synovium.

Objectives: It is difficult to capture the severity of synovial inflammation on imaging. Herein we hypothesize that diffusion tensor imaging (DTI) derived metrics may delineate the aggregation of the inflammatory cells and expression of inflammatory cytokines and dynamic contrast-enhanced (DCE) imaging may provide information regarding vascularity in the inflamed synovium. Patients and methods: Patients with knee arthritis (>3-months duration) underwent conventional (T2-weighted fast spin echo and spin echo T1-weighted images) as well as DTI and DCE MRI and thereafter arthroscopic guided synovial biopsy. DCE and DTI metrics were extracted from the masks of the segments of the inflamed synovium which enhanced on post-contrast T1-weighted MRI. These metrics were correlated with immunohistochemistry (IHC) parameters of inflammation on synovium. Statistical analysis: Pearson's correlation was performed to study the relationship between DTI- and DCE-derived metrics, IHC parameters, and post-contrast signal intensity. Linear regression model was used to predict the values of IHC parameters using various DTI and DCE derived metrics as predictors. Results: There were 80 patients (52 male) with mean age 39.78 years and mean disease duration 19.82 months. Nineteen patients had tuberculosis and the rest had chronic undifferentiated monoarthritis (n = 31), undifferentiated spondyloarthropathy (n = 14), rheumatoid arthritis (n = 6), osteoarthritis (n = 4), reactive arthritis (n = 3), ankylosing spondylitis (n = 2), and juvenile idiopathic arthritis (n = 1). Fractional anisotropy (FA), a metric of DTI, had significant correlation with number of immune cells (r = 0.87, p < 0.01) infiltrating into the synovium and cytokines (IL-1ß, r = 0.55, p < 0.01; TNF-α, r = 0.42, p < 0.01) in all patients and also in each group of patients and adhesion molecule expressed on these cells in all patients (CD54, r = 0.51, p < 0.01). DCE parameters significantly correlated with CD34 (blood flow, r = 0.78, p < 0.01; blood volume, r = 0.76, p < 0.01) in each group of patients, a marker of neo-angiogenesis. FA was the best predictor of infiltrating inflammatory cells, adhesion molecule and proinflammatory cytokines. Amongst the DCE parameters, blood volume, was best predictor of CD34. Conclusion: DTI and DCE metrics capture cellular and molecular markers of synovial inflammation in patients with chronic inflammatory arthritis.