The strain among caregivers of children with disabilities at the community-based rehabilitation centres in Kudat division of Sabah, Malaysia.

INTRODUCTION: The prevalence of children with disabilities (CWD) is increasing. CWD requires exceptional long-term attention, which often falls on the caregivers. Caring for a CWD affects caregivers in multiple ways, such as physical, social, personal, and financial aspects. Most of the CWD will be cared in Community-Based Rehabilitation (CBR) centres where the caregivers have to play a crucial role as they become partners in the respective service delivery. However, there is still insufficient information on the impact (strain) of a disabled child on the caregivers in the CBR centre in the Kudat division of Sabah, Malaysia specifically. Hence, this study aims to explore the strains among caregivers of CWD at the CBR centre in the Kudat. MATERIALS AND METHODS: A cross-sectional study through a purposive sampling method was employed, involving 142 caregivers of CWD at the selected CBR centre. Four CBR centres were selected in this study, which were located in the Kudat division of Sabah. The Malay version of the Modified Caregiver Strain Index Questionnaire (M-CSI-M) was used in this study to obtain information about the caregiver's strain. RESULTS: Findings from this study revealed that most caregivers experienced moderate strain (72.5%). Some of the leading causes of their strain were upsetting CWD's behaviour (10.6%), financial strain (9.2%), and overwhelmed (9.2%). In addition, there were significant differences between caregiver strain and their level of education, income, and the types of their child's disabilities (p ≤ 0.05). CONCLUSION: The well-being of the caregivers may significantly impact the effectiveness of rehabilitation for CWD, as caregivers are vital interdisciplinary team members.

Gender differences in COVID-19 knowledge, risk perception, and public stigma among the general community: Findings from a nationwide cross-sectional study in India.

Introduction: Individual and community characteristics predictive of knowledge, perception, and attitude on COVID-19, specifically on gender, have not been adequately explored. Objective: To examine the gender differences in COVID-19 knowledge, self-risk perception and public stigma among the general community and to understand other socio-demographic factors which were predictive of them. Method: A nationally representative cross-sectional multi-centric survey was conducted among adult individuals(≥18 yrs) from the community member (N = 1978) from six states and one union territory of India between August 2020 to February 2021. The participants were selected using systematic random sampling. The data were collected telephonically using pilot-tested structured questionnaires and were analyzed using STATA. Gender-segregated multivariable analysis was conducted to identify statistically significant predictors (p < 0.05) of COVID-19-related knowledge, risk perception, and public stigma in the community. Results: Study identified significant differences between males and females in their self-risk perception (22.0% & 18.2% respectively) and stigmatizing attitude (55.3% & 47.1% respectively). Highly educated males and females had higher odds of having COVID-19 knowledge (aOR: 16.83: p < 0.05) than illiterates. Highly educated women had higher odds of having self-risk perception (aOR: 2.6; p < 0.05) but lower public stigma [aOR: 0.57; p < 0.05]. Male rural residents had lower odds of having self-risk perception and knowledge [aOR: 0.55; p < 0.05 & aOR: 0.72; p < 0.05] and female rural residents had higher odds of having public stigma [aOR: 1.36; p < 0.05]. Conclusion: Our study findings suggest the importance of considering thegender differentials and their background, education status and residential status in designing effective interventions to improve knowledge and reduce risk perception and stigma in the community about COVID-19.

Pharmacokinetics of rifampicin, isoniazid & pyrazinamide during daily & intermittent dosing: A preliminary study.

Background & objectives: The National Tuberculosis (TB) Control Programme has transitioned from thrice-weekly to daily drug treatment regimens in India. This preliminary study was conceived to compare the pharmacokinetics of rifampicin (RMP), isoniazid (INH) and pyrazinamide (PZA) in TB patients being treated with daily and thrice weekly anti-TB treatment (ATT). Methods: This prospective observational study was undertaken in 49 newly diagnosed adult TB patients receiving either daily ATT (n=22) or thrice-weekly ATT (n=27). Plasma RMP, INH and PZA were estimated by high-performance liquid chromatography. Results: The peak concentration (Cmax) of RMP was significantly higher (RMP: 8.5 µg/ml vs. 5.5 µg/ml; P=0.003) and Cmax of INH was significantly lower (INH: 4.8 µg/ml vs. 10.9 µg/ml; P<0.001) in case of daily dosing compared to thrice-weekly ATT. Cmax of drugs and doses was significantly correlated. A higher proportion of patients had subtherapeutic RMP Cmax (8.0 µg/ml) during thrice-weekly compared to daily ATT (78% vs. 36%; P=0.004). Multiple linear regression analysis showed that Cmax of RMP was significantly influenced by the dosing rhythm, pulmonary TB and Cmax of INH and PZA by the mg/kg doses. Interpretation & conclusions: RMP concentrations were higher and INH concentrations were lower during daily ATT, suggesting that INH doses may need to be increased in case of a daily regimen. Larger studies are, however, required using higher INH doses when monitoring for adverse drug reactions and treatment outcomes.

Factors associated with COVID-19 stigma during the onset of the global pandemic in India: A cross-sectional study.

Objective: To assess factors associated with COVID-19 stigmatizing attitudes in the community and stigma experiences of COVID-19 recovered individuals during first wave of COVID-19 pandemic in India. Methods: A cross-sectional study was conducted in 18 districts located in 7 States in India during September 2020 to January 2021 among adults > 18 years of age selected through systematic random sampling. Data on socio demographic and COVID-19 knowledge were collected from 303 COVID-19 recovered and 1,976 non-COVID-19 infected individuals from community using a survey questionnaire. Stigma was assessed using COVID-19 Stigma Scale and Community COVID-19 Stigma Scale developed for the study. Informed consent was sought from the participants. Univariate and multivariate binary logistic regression analysis were conducted. Results: Half of the participants (51.3%) from the community reported prevalence of severe stigmatizing attitudes toward COVID-19 infected while 38.6% of COVID-19 recovered participants reported experiencing severe stigma. Participants from the community were more likely to report stigmatizing attitudes toward COVID-19 infected if they were residents of high prevalent COVID-19 zone (AOR: 1.5; CI: 1.2-1.9), staying in rural areas (AOR: 1.5; CI:1.1-1.9), belonged to the age group of 18-30 years (AOR: 1.6; CI 1.2-2.0), were male (AOR: 1.6; CI: 1.3-1.9), illiterate (AOR: 2.7; CI: 1.8-4.2), or living in Maharashtra (AOR: 7.4; CI: 4.8-11.3). COVID-19 recovered participants had higher odds of experiencing stigma if they had poor knowledge about COVID-19 transmission (AOR: 2.8; CI: 1.3-6.3), were staying for 6-15 years (AOR: 3.24; CI: 1.1-9.4) in the current place of residence or belonged to Delhi (AOR: 5.3; CI: 1.04-26.7). Conclusion: Findings indicated presence of stigmatizing attitudes in the community as well as experienced stigma among COVID-19 recovered across selected study sites in India during the first wave of COVID-19 pandemic. Study recommends timely dissemination of factual information to populations vulnerable to misinformation and psychosocial interventions for individuals affected by stigma.

Development & validation of scales to assess stigma related to COVID-19 in India.

Background & objectives: COVID-19 pandemic has triggered social stigma towards individuals affected and their families. This study describes the process undertaken for the development and validation of scales to assess stigmatizing attitudes and experiences among COVID-19 and non-COVID-19 participants from the community. Methods: COVID-19 Stigma Scale and Community COVID-19 Stigma Scale constituting 13 and six items, respectively, were developed based on review of literature and news reports, expert committee evaluation and participants' interviews through telephone for a multicentric study in India. For content validity, 61 (30 COVID-19-recovered and 31 non-COVID-19 participants from the community) were recruited. Test-retest reliability of the scales was assessed among 99 participants (41 COVID-19 recovered and 58 non-COVID-19). Participants were administered the scale at two-time points after a gap of 7-12 days. Cronbach's alpha, overall percentage agreement and kappa statistics were used to assess internal consistency and test-retest reliability. Results: Items in the scales were relevant and comprehensible. Both the scales had Cronbach's α above 0.6 indicating moderate-to-good internal consistency. Test-retest reliability assessed using kappa statistics indicated that for the COVID-19 Stigma Scale, seven items had a moderate agreement (0.4-0.6). For the Community COVID-19 Stigma Scale, four items had a moderate agreement. Interpretation & conclusions: Validity and reliability of the two stigma scales indicated that the scales were comprehensible and had moderate internal consistency. These scales could be used to assess COVID-19 stigma and help in the development of appropriate stigma reduction interventions for COVID-19 infected, and mitigation of stigmatizing attitudes in the community.

Comparison of performance indicators of MGIT primary culture to assess the impact of implementation of ISO 15189:2012 standards.

Performance indicators are key component and plays a major role for monitoring and continuous quality improvement of the test results. The NABL certificate of accreditation is issued in accordance with the standard ISO 15189:2012 requirements. As part of the accreditation process, the laboratory has acquired knowledge and implemented the quality system procedures. Present study analyzed the impact of the accreditation process on the "performance indicators" of MGIT primary culture and found that performance indicators have been improved significantly after implementation of NABL for almost all indicators which clearly indicate the importance of accreditation and implementation of quality procedures for reliability of valid test results.

Mycoplasma pneumoniae CARDS toxin exploits host cell endosomal acidic pH and vacuolar ATPase proton pump to execute its biological activities.

Mycoplasma pneumoniae is the leading cause of bacterial community-acquired pneumonia among hospitalized children in the United States. It is also responsible for a spectrum of other respiratory tract disorders and extrapulmonary manifestations in children and adults. The main virulence factor of M. pneumoniae is a 591 amino acid multifunctional protein called Community Acquired Respiratory Distress Syndrome (CARDS) toxin. The amino terminal region of CARDS toxin (N-CARDS) retains ADP-ribosylating activity and the carboxy region (C-CARDS) contains the receptor binding and vacuolating activities. After internalization, CARDS toxin is transported in a retrograde manner from endosome through the Golgi complex into the endoplasmic reticulum. However, the mechanisms and criteria by which internalized CARDS toxin is transported and activated to execute its cytotoxic effects remain unknown. In this study, we used full-length CARDS toxin and its mutant and truncated derivatives to analyze how pharmacological drugs that alter pH of intracellular vesicles and electrical potential across vesicular membranes affect translocation of CARDS toxin in mammalian cells. Our results indicate that an acidic environment is essential for CARDS toxin retrograde transport to endoplasmic reticulum. Moreover, retrograde transport facilitates toxin clipping and is required to induce vacuole formation. Additionally, toxin-mediated cell vacuolation is strictly dependent on the function of vacuolar type-ATPase.

Multicentric validation of indigenous molecular test Truenat™ MTB for detection of Mycobacterium tuberculosis in sputum samples from presumptive pulmonary tuberculosis patients in comparison with reference standards.

BACKGROUND & OBJECTIVES: : Early case detection is essential to interrupt transmission and to prevent further spread of tuberculosis (TB) in high endemic settings. Nucleic acid amplification tests (NAATs) with visual read-outs are ideal as point-of-care tests. Truenat™ MTB is an indigenous chip-based NAAT for detection of Mycobacterium tuberculosis, which involves extraction of DNA and real-time polymerase chain reaction (PCR) using portable, automated, battery-operated instruments. The current multicentric study was aimed to evaluate Truenat for detection of MTB in sputum samples obtained from patients with presumptive pulmonary TB with reference to culture as gold standard and Xpert as a comparator. METHODS: : The study was conducted at four sites, namely ICMR-National Institute for Research in Tuberculosis, Chennai; All India Institute of Medical Sciences, New Delhi; ICMR-National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra; and National Institute of TB and Respiratory Diseases, New Delhi. Patients suspected to have TB were screened for eligibility. Two sputum samples were collected from each patient. Tests included smear, Xpert and Truenat directly from the sputum sample and culture by Lowenstein-Jensen (L-J) medium and MGIT960 from decontaminated pellets. Sample used for Truenat assay was coded. Resolution of Truenat false positives was done using an in-house PCR with TRC4 primers. RESULTS: : The study enrolled 2419 presumptive TB patients after screening 2465 patients, and 3541 sputum samples were collected from the enrolled patients. Results of 2623 samples were available for analysis. Truenat showed a positivity rate of 48.5 per cent as compared to 37.0 per cent by Xpert. The sensitivities of Truenat and Xpert were was 88.3 and 79.7 per cent, respectively in comparison with culture. INTERPRETATION & CONCLUSIONS: : Truenat MTB identified more positives among culture-confirmed samples than Xpert and had higher sensitivity. In addition, other advantageous operational features of Truenat MTB were identified which would be useful in field settings.

TB diagnostic cascade among patients registered under the Revised National TB Control Programme in Chennai, South India.

OBJECTIVE: To discern and quantify the TB diagnostic cascade among patients registered under the Revised National TB Control Programme, Chennai city, Tamil Nadu, South India. METHODS: This cross-sectional study was conducted in metropolitan Chennai from February 2017 to March 2018. We interviewed TB patients retrospectively on their diagnostic attempt in different health facilities. RESULTS: Of 455 TB patients, only 4.4% received their diagnosis at their first health facility. Of 1250 visits to health facilities, the vast majority (79.4 vs. 20.6%) was in the public rather than the private sector. 56% of patients went to a public facility as the first point of care, of whom 1.6% shifted to private facilities subsequently. The remaining 54.4% shifted between up to five government health facilities. Male patients and those with a higher family income were more likely to shift from private to public. CONCLUSION: Most shifts between diagnostic facilities occurred in the public sector. This necessitates interventions at public health facilities for strengthening and extending services to TB patients at their first point of care.

OBJECTIF: Discerner et quantifier la cascade de diagnostic de la TB chez les patients enregistrés dans le Programme National Révisé de lutte contre la TB, dans la ville de Chennai, dans le Tamil Nadu, dans le sud de l'Inde. MÉTHODES: Cette étude transversale a été menée dans la région métropolitaine de Chennai de février 2017 à mars 2018. Nous avons interviewé rétrospectivement des patients TB sur leur tentative de diagnostic dans différents établissements de santé. RÉSULTATS: Sur 455 patients TB, seuls 4,4% ont reçu leur diagnostic dans le premier établissement de santé visité. Parmi 1250 visites dans les établissements de santé, la grande majorité (79,4 vs 20,6%) était dans le secteur public plutôt que le privé. Parmi les 56% des patients qui sont allés dans un établissement public comme premier point de soins, dont 1,6% sont ensuite passés dans des établissements privés. Les 54.4 restants se sont déplacés entre cinq établissements différents de santé publics. Les patients de sexe masculin et ceux dont le revenu familial était plus élevé étaient plus susceptibles de passer du privé au public. CONCLUSION: La plupart des changements entre les établissements de diagnostic se sont produits dans le secteur public. Cela nécessite des interventions dans les établissements de santé publique pour renforcer et étendre les services aux patients TB à leur premier point de soins.

Pharmacokinetics of isoniazid in children with tuberculosis-A comparative study at two doses.

AIM: To compare the pharmacokinetics of isoniazid (INH) at doses 5 and 10 mg/kg/day. METHODS: INH concentrations were estimated by high-performance liquid chromatography in 24 Indian children aged 1 to 15 years on antituberculosis therapy. Blood samples were collected at 0, 2, 4, 6, 8 hours after administration of INH. Patients were randomly given INH at 5 or 10 mg/kg/day and maximum concentrations (Cmax ) and area under the curve (AUC(0-8) ) were determined in each group. The 2-hour concentration of INH was used as Cmax for this study. RESULTS: Mean (standard deviation) Cmax was reached in 2 hours and was 2.68 ± 1.19 µg/mL in 5 mg/kg/day group and 8.86 ± 3.94 µg/mL in 10 mg/kg/day group (P < .05). The normal therapeutic range at 2-hour concentrations for INH in adults achieving good clinical response is between 3 and 5 µg/mL. Among 5 mg/kg/day, only 4 (33%) patients had INH concentrations within the 2-hour concentrations therapeutic range whereas in 10 mg/kg/day group, 11 (91%) patients achieved Cmax higher than the 2-hour concentrations therapeutic range and 1 (9%) patient had Cmax within the 2-hour concentrations therapeutic range. The mean AUC(0-8) in 5 mg/kg/day group was 10.04 ± 6.12 and 35.93 ± 25.37 µg·h/mL in 10 mg/kg/day group (P = .0001). CONCLUSION: Children on daily INH 10 mg/kg/day have higher AUC and Cmax than the required therapeutic range whereas over 65% of children with daily 5 mg/kg/day INH therapy failed to achieve the optimal therapeutic range.

Validation of an indigenous assay for rapid molecular detection of rifampicin resistance in presumptive multidrug-resistant pulmonary tuberculosis patients.

BACKGROUND & OBJECTIVES: There is a need for an affordable, easy, high-sensitivity test usable at the peripheral health facility for diagnosis of drug-resistant (DR) tuberculosis (TB) to interrupt disease transmission. Nucleic acid amplification tests (NAATs) for early detection of DR-TB are ideal to bring testing near to the patient. TruenatTM MTB (Mycobacterium tuberculosis) and TruenatTM MTB-RIF (rifampicin) is an indigenous chip-based real-time polymerase chain reaction (PCR) based test for detection of multidrug-resistant (MDR) TB. The test involves extraction of DNA using automated, battery operated Trueprep instrument and real-time PCR performed on the Truelab analyzer. We report here multicentric validation of Truenat MTB-RIF for detection of DR-TB in suspected DR-TB patients. METHODS: Consecutive patients aged 18-65 yr, with symptoms suggestive of TB and with a history of previous treatment, reporting to the National TB Elimination Programme (NTEP) clinics under four national institutes, namely AIIMS (All India Institute of Medical Sciences, New Delhi), NITRD (National Institute of Tuberculosis and Respiratory Diseases, New Delhi), NIRT (National Institute for Research in Tuberculosis, Chennai) and ICMR-National JALMA Institute for Leprosy and other Mycobacterial Diseases, Agra, were included in the study. Two sputum samples (one spot and one morning) were collected from each patient, after obtaining informed written consent. The samples were subjected to smear, GeneXpert and MGIT 960 culture (and drug susceptibility testing to RIF) (surrogate for MDR-TB) to serve as reference tests. The samples were coded to ensure blinding and subjected to Truenat MTB-RIF. Truenat MTB-RIF Version 1.5 was used for testing 1084 samples for RIF resistance, while Version 2.0 was used to test another 1201 samples. RESULTS: Truenat MTB-RIF Version 1.5 in comparison with comprehensive laboratory reference standards yielded sensitivity and specificity of 76.2 and 94.7 per cent, respectively for the detection of RIF resistance in 1084 samples, collected across four sites. Based on the analysis of discordant samples, Version 2.0 of Truenat was developed by the manufacturer and this was further tested on additional 1201 samples, yielding a sensitivity of 87.5 per cent and specificity of 99.5 per cent. INTERPRETATION & CONCLUSIONS: Multicentric trial of TruenatTM MTB-RIF demonstrated a great potential of this point of care NAAT for detection of MDR-TB. The test would be useful in limited resource settings and inaccessible areas without need for any additional infrastructure.

Association of single nucleotide polymorphisms of adiponectin and leptin genes with breast cancer.

Single nucleotide polymorphisms (SNPs) in adiponectin gene [rs1501299 (+276G/T) and rs266729 (-11377C/G)] and one SNP of leptin gene [rs7799039 (-2548G/A)] are known to influence plasma levels of adiponectin and leptin respectively. Literature is scarce on the association of adiponectin gene polymorphism rs266729 with breast cancer. The present study was taken up to study these polymorphisms and their association with breast cancer. Ninety-three patients diagnosed with malignant breast cancer were included as cases along with 186 age matched healthy controls. Adiponectin +276G/T, -11377C/G and leptin -2548G/A polymorphism were studied using polymerase chain reaction (PCR) based restriction fragment length polymorphism (RFLP). Adipokine levels in blood were measured using enzyme linked immunosorbent assay. Adiponectin +276G/T and leptin -2548G/A showed a significant increased risk for breast cancer even after adjusting for confounding variables like present age, age at menarche, age at first child birth and age at menopause. In the subset analysis, based on menopausal state, stronger association was observed between SNP in adiponectin gene +276G/T with the breast cancer in post-menopausal women after adjusting for all other variables. No association was found with adiponectin -11377C/G. No association of the gene polymorphisms with adipokine levels was observed. Also, no significant association was seen for the effect of gene-environment interaction i.e. presence of polymorphism with obesity and menopausal state for any of the SNPs studied. Adiponectin +276G/T is strongly associated with breast cancer in postmenopausal women while leptin -2548G/A polymorphisms is significantly associated with breast cancer irrespective of the menopausal state in south Indian subjects.

Pharmacokinetics of rifabutin during atazanavir/ritonavir co-administration in HIV-infected TB patients.

BACKGROUND & OBJECTIVE: Rifabutin (RBT) is the rifamycin that is recommended to treat tuberculosis (TB) in HIV-infected individuals during combination antiretroviral therapy (ART) containing HIV protease inhibitors (PIs). We studied the pharmacokinetics of rifabutin at doses of 300 mg thrice weekly and 150 mg daily during concomitant atazanavir/ritonavir (ATZ/r) administration in adult HIV-infected TB patients treated in the Revised National TB Control Programme (RNTCP) in India. METHODS: This was a multi-centric study conducted in 45 adult HIV-infected TB patients, who were being treated for TB with a RBT-containing regimen and an antiretroviral treatment regimen with ATZ/r, at doses of 300 mg thrice-weekly (n = 36) or 150 mg daily (n = 9). Serial blood draws at pre-dosing and at 1, 2, 4, 6, 8, 12 and 24 hours after drug administration were done. Plasma RBT was estimated by high pressure liquid chromatography (HPLC). RESULTS: The peak concentration (Cmax) of both doses were within the therapeutic range (0.45-0.90 µg/ml) of RBT. Proportion of patients having Cmax above or below the therapeutic range and trough concentration (Cmin) below the minimum inhibitory concentration of RBT did not significantly differ between the two doses. TB treatment outcomes were also similar at both doses. CONCLUSIONS: This is the first and only study from India reporting on the pharmacokinetics of RBT at 300 mg thrice weekly and 150 mg daily doses. Both doses yielded similar plasma RBT concentrations, outcomes and were well tolerated. RBT can be administered at either doses during ATZ/r co-administration in HIV-infected patients with TB.

Intrapatient variability in plasma rifampicin & isoniazid in tuberculosis patients.

Background & objectives: Large variability in anti-tuberculosis (TB) drug concentrations between patients is known to exist. However, limited information is available on intrapatient drug levels during the course of anti-TB treatment (ATT). This study was conducted to evaluate intrapatient variability in plasma rifampicin (RMP) and isoniazid (INH) concentrations during ATT at start of the treatment, at the end of intensive phase (IP) of ATT and at the end of ATT in adult TB patients being treated in the Revised National TB Control Programme (RNTCP). Methods: Adult TB patients (n=485), receiving thrice-weekly ATT in the RNTCP, were studied. Two-hour post-dosing concentrations of RMP and INH were determined at month 1, end of IP and end of ATT, after directly observed drug administration. Drug concentrations were estimated by high-performance liquid chromatography. Results: The median (inter-quartile range) RMP concentrations during the first month, at end of IP and end of ATT were 2.1 (0.4-5.0), 2.4 (0.6-5.5) and 2.2 (0.5-5.3) µg/ml, respectively. The corresponding INH concentrations were 7.1 (4.2-9.9), 7.2 (3.9-10.9) and 6.7 (3.9-9.5) µg/ml. None of the differences in drug concentrations obtained at different time points during ATT were significant. RMP and INH concentrations at different time points were significantly correlated. Age and body mass index caused significant variability in drug concentrations. Interpretation & conclusions: Plasma RMP and INH estimations in adult TB patients at two hours after drug administration remained unaltered during ATT. Clinicians can consider testing drug concentrations at any time point during ATT. These findings may assume significance in the context of therapeutic drug monitoring of anti-TB drug concentrations.

Mycoplasma pneumoniae Community-Acquired Respiratory Distress Syndrome Toxin Uses a Novel KELED Sequence for Retrograde Transport and Subsequent Cytotoxicity.

Mycoplasma pneumoniae is an atypical bacterium that causes respiratory illnesses in humans, including pharyngitis, tracheobronchitis, and community-acquired pneumonia (CAP). It has also been directly linked to reactive airway disease, asthma, and extrapulmonary pathologies. During its colonization, M. pneumoniae expresses a unique ADP-ribosylating and vacuolating cytotoxin designated community-acquired respiratory distress syndrome (CARDS) toxin. CARDS toxin persists and localizes in the airway in CAP patients, asthmatics, and trauma patients with ventilator-associated pneumonia. Although CARDS toxin binds to specific cellular receptors, is internalized, and induces hyperinflammation, histopathology, mucus hyperplasia, and other airway injury, the intracellular trafficking of CARDS toxin remains unclear. Here, we show that CARDS toxin translocates through early and late endosomes and the Golgi complex and concentrates at the perinuclear region to reach the endoplasmic reticulum (ER). Using ER-targeted SNAP-tag, we confirmed the association of CARDS toxin with the ER and determined that CARDS toxin follows the retrograde pathway. In addition, we identified a novel CARDS toxin amino acid fingerprint, KELED, that is required for toxin transport to the ER and subsequent toxin-mediated cytotoxicity.IMPORTANCEMycoplasma pneumoniae, a leading cause of bacterial community-acquired pneumonia (CAP) among children and adults in the United States, synthesizes a 591-amino-acid ADP-ribosylating and vacuolating protein, designated community-acquired respiratory distress syndrome (CARDS) toxin. CARDS toxin alone is sufficient to induce and mimic major inflammatory and histopathological phenotypes associated with M. pneumoniae infection in rodents and primates. In order to elicit its ADP-ribosylating and vacuolating activities, CARDS toxin must bind to host cell receptors, be internalized via clathrin-mediated pathways, and subsequently be transported to specific intracellular organelles. Here, we demonstrate how CARDS toxin utilizes its unique KELED sequence to exploit the retrograde pathway machinery to reach the endoplasmic reticulum (ER) and fulfill its cytopathic potential. The knowledge generated from these studies may provide important clues to understand the mode of action of CARDS toxin and develop interventions that reduce or eliminate M. pneumoniae-associated airway and extrapulmonary pathologies.

Predictors of unfavourable treatment outcome in patients with multidrug-resistant tuberculosis in India.

Setting: India has one of the highest global rates of multidrug-resistant tuberculosis (MDR-TB), which is associated with poor treatment outcomes. A better understanding of the risk factors for unfavourable outcomes is needed. Objectives: To describe 1) the demographic and clinical characteristics of MDR-TB patients registered in three states of India during 2009-2011, 2) treatment outcomes, and 3) factors associated with unfavourable outcomes. Design: A retrospective cohort study involving a record review of registered MDR-TB patients. Results: Of 788 patients, 68% were male, 70% were aged 15-44 years, 90% had failed previous anti-tuberculosis treatment or were retreatment smear-positive, 60% had a body mass index < 18.5 kg/m2 and 72% had additional resistance to streptomycin and/or ethambutol. The median time from sputum collection to the start of MDR-TB treatment was 128 days (IQR 103-173). Unfavourable outcomes occurred in 40% of the patients, mostly from death or loss to follow-up. Factors significantly associated with unfavourable outcomes included male sex, age ⩾ 45 years, being underweight and infection with the human immunodeficiency virus. Adverse drug reactions were reported in 24% of patients, with gastrointestinal disturbance, psychiatric morbidity and ototoxicity the most common. Conclusion: Long delays from sputum collection to treatment initiation using conventional methods, along with poor treatment outcomes, suggest the need to scale up rapid diagnostic tests and shorter regimens for MDR-TB.

Contexte : L'Inde a l'un des taux les plus élevés au monde de tuberculose multirésistante (TB-MDR), qui est associée à des résultats médiocres du traitement. Une meilleure compréhension des facteurs de risque de résultats défavorables est requise.Objectifs : Décrire : 1) les caractéristiques démographiques et cliniques des patients TB-MDR enregistrés dans trois états d'Inde de 2009 à 2011, 2) les résultats du traitement, et 3) les facteurs associés à des résultats défavorables.Schéma : Une étude de cohorte rétrospective impliquant une revue des dossiers des patients TB-MDR enregistrés.Résultats : Il y a eu 788 patients, dont 68% d'hommes, 70% âgés de 15­44 ans, 90% ayant eu un échec de leur traitement anti-tuberculose précédent ou ayant un frottis positif en retraitement, 60% ayant un index de masse corporelle < 18,5 kg/m2 et 72% ayant en plus une résistance à la streptomycine et/ou à l'éthambutol. Le délai médian entre le recueil de crachats et la mise en route du traitement de la TB-MDR a été de 128 jours (IQR 103­173). Les résultats ont été défavorables pour 40% des patients, en majorité des décès ou des pertes de vue. Les facteurs significativement associés à un résultat défavorable ont inclus le sexe masculin, l'âge ⩾ 45 ans, la maigreur et le fait d'être positif pour le virus de l'immunodéficience humaine. Des effets secondaires des médicaments ont été notés dans 24% des cas, avec des troubles gastro-intestinaux, des problèmes psychiatriques et une ototoxicité comme symptômes les plus fréquents.Conclusion : De longs délais entre le recueil de crachats et la mise en route du traitement basé sur des méthodes conventionnelles et des résultats médiocres du traitement signalent la nécessité d'intensifier la mise en œuvre des tests de diagnostic rapide et des protocoles de traitement court de la TB-MDR.

Marco de referencia: La tasa de tuberculosis multirresistente (TB-MDR) en la India es una de las tasas más altas en el mundo y se asocia con desenlaces terapéuticos desfavorables. Es preciso lograr un mejor conocimiento de los factores de riesgo que determinan la ineficacia del tratamiento.Objetivos: 1) Describir las características demográficas y clínicas de los pacientes con TB-MDR registrados en tres estados de la India del 2009 al 2011; 2) analizar los desenlaces terapéuticos; y 3) describir los factores asociados con los resultados desfavorables del tratamiento.Método: Un estudio retrospectivo de cohortes a partir del análisis de las historias clínicas de los pacientes registrados con diagnóstico de TB-MDR.Resultados: Se incluyeron en el estudio 788 pacientes; el 68% era de sexo masculino, en el 70% la edad estaba comprendida entre 15 años y 44 años, el 90% tenía antecedente de fracaso de un tratamiento antituberculoso o estaba en retratamiento con baciloscopia positiva, el índice de masa corporal era inferior a 18,5 en el 60% de los casos y el 72% presentaba resistencia adicional a estreptomicina, etambutol o ambos. La mediana del lapso entre la recogida de la muestra de esputo y el comienzo del tratamiento de la TB-MDR fue 128 días (intervalo intercuartil 103­173). Se observaron desenlaces desfavorables en 40% de los pacientes y consistieron en su mayoría en defunciones o pérdidas durante el seguimiento. Los factores que se asociaron de manera significativa con estos desenlaces fueron el sexo masculino, la edad ⩾ 45 años, el bajo peso y la serología positiva frente del virus de la inmunodeficiencia humana. Se notificaron reacciones adversas a los medicamentos en el 24% de los casos, de las cuales las más frecuentes fueron los trastornos gastrointestinales, las afecciones psiquiátricas y la ototoxicidad.Conclusión: La observación de plazos prolongados entre la recogida de las muestras de esputo y la iniciación del tratamiento cuando se utilizan los medios diagnósticos corrientes y de desenlaces terapéuticos desfavorables destaca la necesidad de ampliar la escala de aplicación de las pruebas rápidas de diagnóstico y la administración de pautas más cortas de tratamiento de la TB-MDR.

N-acetyltransferase gene polymorphisms & plasma isoniazid concentrations in patients with tuberculosis.

BACKGROUND & OBJECTIVES: Variations in the N-acetyltransferase (NAT2) gene among different populations could affect the metabolism and disposition of isoniazid (INH). This study was performed to genotype NAT2 gene polymorphisms in tuberculosis (TB) patients from Chennai, India, and compare plasma INH concentrations among the different genotypes. METHODS: Adult patients with TB treated in the Revised National TB Control Programme (RNTCP) in Chennai, Tamil Nadu, were genotyped for NAT2 gene polymorphism, and two-hour post-dosing INH concentrations were compared between the different genotypes. Plasma INH was determined by high-performance liquid chromatography. Genotyping of the NAT2 gene polymorphism was performed by real-time polymerase chain reaction method. RESULTS: Among the 326 patients genotyped, there were 189 (58%), 114 (35%) and 23 (7%) slow, intermediate and fast acetylators, respectively. The median two-hour INH concentrations in slow, intermediate and fast acetylators were 10.2, 8.1 and 4.1 µg/ml, respectively. The differences in INH concentrations among the three genotypes were significant (P<0.001). INTERPRETATION & CONCLUSIONS: Genotyping of TB patients from south India for NAT2 gene polymorphism revealed that 58 per cent of the study population comprised slow acetylators. Two-hour INH concentrations differed significantly among the three genotypes.

Anti-tuberculosis drug concentrations in tuberculosis patients with and without diabetes mellitus.

PURPOSE: The aim of the study was to compare plasma concentrations of rifampicin (RMP), isoniazid (INH) and pyrazinamide (PZA) between tuberculosis (TB) patients with and without diabetes mellitus (DM). METHODS: Two-hour post-dosing concentrations of RMP, INH and PZA were determined in adult TB patients that were studied with (n = 452) and without DM (n = 1460), treated with a thrice-weekly regimen in India. Drug concentrations were estimated by HPLC. RESULTS: The median (IQR) INH [6.6 (3.9-10.0) and 7.8 (4.6-11.3)] and PZA [31.0 (22.3-38.0) and 34.1 (24.6-42.7)] microgram per milliliter concentrations were significantly lower in diabetic than non-diabetic TB patients (p < 0.001 for both drugs). Blood glucose was negatively correlated with plasma INH (r = -0.09, p < 0.001) and PZA (r = -0.092, p < 0.001). Multiple linear regression analysis showed RMP, INH and PZA concentrations were influenced by age and drug doses, INH and PZA by DM, RMP by alcohol use and PZA by gender and category of ATT. DM reduced INH and PZA concentrations by 0.8 and 3.0 µg/ml, respectively. CONCLUSIONS: TB patients with DM had lower INH and PZA concentrations. Negative correlation between blood glucose and drug concentrations suggests delayed absorption/faster elimination of INH and PZA in the presence of elevated glucose.