Comparative Evaluation of Bioavailability, Efficacy and Safety of MICROCORE NESC® with Calcium Carbonate and Calcium Citrate Malate in Osteopenic and Osteoporotic Patients: A Randomized Clinical Trial.

Introduction: Osteoporosis is a skeletal disease characterized by loss of bone mass, reduced bone strength and increased bone fragility predisposing to fractures. This study was planned to evaluate the efficacy, safety and relative bioavailability of Microcore NESC® (Natural Egg Shell Calcium) in osteopenia and osteoporotic patients. Methods: This was a randomized, open label, parallel group interventional clinical trial which included 60 study participants with osteopenia and osteoporosis who were randomized into three groups (20 each). Group 1-Microcore NESC®, Group 2-Shelcal and Group 3-CCM with 12 weeks treatment period. The participants were evaluated for relative oral bioavailability, bone mineral density (BMD), serum osteocalcin, change in VAS pain scale and quality of life-Questionnaires. Results: There was significant improvement in the BMD T scores-post-treatment with MICROCORE NESC® and Shelcal. Higher percentage of improvement in calcium absorption as depicted by an increase in serum calcium levels (10.23%) in the MICROCORE NESC®-treated group when compared to Shelcal (7.7%) and CCM (7.2%). The relative bioavailability of MICROCORE NESC® with respect to Shelcal was 93%. Discussion: MICROCORE NESC®, has shown a better oral relative bio availability of calcium (93%), better improvement of BMD T score compared to Shelcal and CCM. The general health status has improved to very good/excellent in 83% of patients in MICROCORE NESC®-treated group. Thus, MICROCORE NESC® can be considered a better and safe calcium supplement, as there are very few side effects observed without any clinically significant abnormalities in lab parameters.

Paediatric pulmonary disease-are we diagnosing it right?

Background: It has been reported that differential diagnosis of bacterial or viral pneumonia and tuberculosis (TB) in infants and young children is complex. This could be due to the difficulty in microbiological confirmation in this age group. In this study, we aimed to assess the utility of a real-time multiplex PCR for diagnosis of respiratory pathogens in children with pulmonary TB. Methods: A total of 185 respiratory samples [bronchoalveolar lavage (15), gastric aspirates (98), induced sputum (21), and sputum (51)] from children aged 3-12 years, attending tertiary care hospitals, Chennai, India, were included in the study. The samples were processed by N acetyl L cysteine (NALC) NAOH treatment and subjected to microbiological investigations for Mycobacterium tuberculosis (MTB) diagnosis that involved smear microscopy, Xpert® MTB/RIF testing, and liquid culture. In addition, DNA extraction from the processed sputum was carried out and was subjected to a multiplex real-time PCR comprising a panel of bacterial and fungal pathogens. Results: Out of the 185 samples tested, a total of 20 samples were positive for MTB by either one or more identification methods (smear, culture, and GeneXpert). Out of these 20 MTB-positive samples, 15 were positive for one or more bacterial or fungal pathogens, with different cycle threshold values. Among patients with negative MTB test results (n = 165), 145 (87%) tested positive for one or more than one bacterial or fungal pathogens. Conclusion: The results suggest that tuberculosis could coexist with other respiratory pathogens causing pneumonia. However, a large-scale prospective study from different geographical settings that uses such simultaneous detection methods for diagnosis of childhood tuberculosis and pneumonia will help in assessing the utility of these tests in rapid diagnosis of respiratory infections.

Assessing the utility of Truenat in extrapulmonary tuberculosis diagnosis - A NRL's experience.

Background: Diagnosis of extra pulmonary TB (EPTB) remains a big challenge. While data on utility of Xpert testing in EPTB diagnosis is enormous, there is limited data on Truenat MTB testing. Aim: In this study we aimed to evaluate the usefulness of Truenat in EPTB diagnosis. Materials and methods: The study included patients suspected and/or treated for EPTB located from Chennai district during the year 2021-2022. All processed EPTB samples were subjected to smear microscopy, culture and Truenat MTB testing. Results: Of the 195 samples tested, 38 (19.4%) samples were positive for EPTB by any one of the diagnostic methods (smear, culture, microscopy). Out of these 38, 16 (42.1 %) were positive for MTB by Truenat and negative by Culture, 12 (31.5%) were positive by culture but negative by Truenat and 8 (21%) were positive by both Truenat and/or smear and culture. The sensitivity and specificity of the test was calculated with the composite reference standard (Culture (exclusion of colonies as positives), clinical conditions, and smear) and was found to be 60% and 100% respectively. Conclusion: Truenat MTB test is a cost-effective rapid molecular test that can be used only for the diagnosis of presumptive EPTB and not on follow-up samples.

Operational Challenges in Conducting a Subnational TB Prevalence Survey in India: Lessons Learned for Resource-Limited, High-Burden Settings.

Estimating the burden of TB at the subnational level is critical to planning and prioritizing resources for TB control activities according to the local epidemiological situation. We report the experiences and operational challenges of implementing a TB prevalence survey at the subnational level in India. Information was collected from research reports that gathered data from periodic meetings, informal discussions with study teams, letters of communication, and various site visit reports. During the implementation of the survey, several challenges were encountered, including frequent turnover in human resources, lack of survey participation and community engagement, breakdown of X-ray machines, laboratory issues that delayed sputum sample testing, delays in X-ray reading, and network and Internet connectivity issues that impeded data management. To help ensure the survey was implemented in a timely manner, we developed several solutions, including planning ahead to anticipate challenges, ensuring timely communication, having a high commitment from all stakeholders, having strong team motivation, providing repetitive hands-on training, and involving local leaders to increase community engagement. This experience may help future states and countries that plan to conduct TB prevalence surveys to address these anticipated challenges and develop alternative strategies well in advance.

Molecular Characterisation of M. kansasii Isolates by Whole-Genome Sequencing.

M. kansasii is the most common non-tuberculous mycobacteria, known to be causing pulmonary and extrapulmonary diseases in humans. Based on molecular methods, M. kansasii has been previously classified into seven different subtypes. Now, based on whole-genome sequence analysis, a new species designation was proposed, in which M. kansasii species was designated subtype 1 and is of pathogenic significance in both immunocompetent and immunocompromised patients. The aim of the study is to examine the distribution of subtypes, based on whole-genome sequence analysis, and identify the genetic determinants of drug resistance for the isolates. Whole-genome sequencing was performed using 12 isolates for which phenotypic DST results were available. A phylogenetic tree was constructed by alignment of each of the 12 isolates and the additional strains, as well as the M. kansasii reference strain, using the MAFFT algorithm. Based on this analysis, all 12 isolates were classified as subtype I. Drug-resistant mutations were identified by analysing the isolates with known drug-resistant loci of MTB and NTM. Although we had mutations in the drug-resistant genes, the significance of those mutations could not be explored due to the minimal availability of data available to compare. Further large-scale studies targeting the phenotypic and genotypic drug-resistance pattern, along with whole-genome analysis, will facilitate a better understanding of the resistance mechanisms involved in M. kansasii.

Comprehensive assessment of invalid and indeterminate results in Truenat MTB-RIF testing across sites under the national TB elimination program of India.

Introduction: Truenat MTB-RIF assay (Truenat), a nucleic acid amplification test (NAAT), is a real-time polymerase chain reaction (RT-PCR) chip-based assay that can detect Mycobacterium tuberculosis (Mtb) and rifampicin (RIF) drug resistance using portable, battery-operated devices. The National TB Elimination Program (NTEP) in India introduced this novel tool at the district and subdistrict level in 2020. This study aimed to assess the level and causes of inconclusive results (invalid results, errors, and indeterminate results) in MTB and RIF testing at NTEP sites and the root causes of these in the programmatic setting. Methods: Truenat testing data from 1,690 functional Truenat sites under the NTEP from April to June 2021 were analyzed to assess the rates of errors, invalid MTB results, and indeterminate RIF results. Following this analysis, 12 Truenat sites were selected based on site performance in Truenat testing, diversity of climatic conditions, and geographical terrain. These sites were visited to assess the root causes of their high and low rates of inconclusive results using a structured checklist. Results: A total of 327,649 Truenat tests performed for MTB and RIF testing were analyzed. The rate of invalid MTB results was 5.2% [95% confidence interval (CI): 5.11-5.26; n = 16,998] and the rate of errors was 2.5% (95% CI: 2.46-2.57; n = 8,240) in Truenat MTB chip testing. For Mtb-positive samples tested using the Truenat RIF chip for detection of RIF resistance (n = 40,926), the rate of indeterminate results was 15.3% (95% CI: 14.97-15.67; n = 6,267) and the rate of errors was 1.6% (95% CI: 1.53-1.78; n = 675). There was a 40.1% retesting gap for Mtb testing and a 78.2% gap for inconclusive RR results. Among the inconclusive results retested, 27.9% (95% CI: 27.23-28.66; n = 4,222) were Mtb-positive, and 9.2% (95% CI: 7.84-10.76; n = 139) were detected as RR. Conclusion: The main causes affecting Truenat testing performance include suboptimal adherence to standard operating procedures (SOPs), inadequate training, improper storage of testing kits, inadequate sputum quality, lack of quality control, and delays in the rectification of machine issues. Root cause analysis identified that strengthening of training, external quality control, and supervision could improve the rate of inconclusive results. Ensuring hands-on training of technicians for Truenat testing and retesting of samples with inconclusive results are major recommendations while planning for Truenat scale-up. The recommendations from the study were consolidated into technical guidance documents and videos and disseminated to laboratory staff working at the tiered network of TB laboratories under the NTEP in order to improve Truenat MTB-RIF testing performance.

Genotype MTBDRsl version 2 and phenotypic drug resistance detection of Mycobacterium tuberculosis for fluoroquinolones and aminoglycosides.

Background: Information on genotypic with comparison of phenotypic drug sensitivity test of anti-tuberculosis (TB) has been reported in several studies, which have variable results. The present study aimed to assess the Genotype MTBDRsl version 2.0/Line probe assay (LPA) for the detection of fluoroquinolones (FQ) and aminoglycosides (AMGs) resistance mutations among drug-resistant Mycobacterium TB (MTB) strains and also to compare the patterns of genotypic mutations of gyrA/B, rrs, and eis with mycobacteria growth indicator tube (MGIT 960). Methods: A total of 1416 samples were subjected to Genotype MTBDRsl version 2.0 assay. One hundred and twenty sputum smear positive MTB isolates and 37 sputum smear negative MTB isolates confirmed multiple drug resistance resistant to FQ and AMG by the Genotype MTBDRsl version 2.0 were subjected to phenotypic drug susceptibility testing (DST) were analyzed. Results: The association sensitivity, specificity, positive predictive value, negative predictive value, and accuracy for the resistance detection between MGIT (DST) and the Genotype MTBDRsl version 2.0 assay was significant (P < 0.01) of moxifloxacin (MFX) concentration. Sensitivity and specificity value for kanamycin (KAN) resistance was 76% and 89%; 47% and 94% for capreomycin (CAP); and 60% and 76% for low-level KAN, respectively. Conclusion: Our results indicate that MFX (0.25and 1 µg/mL), KAN (2.5 µg/mL), and CAP (2.5 µg/mL) significantly (P < 0.01) and support the World Health Organization guidance to test FQ and AMG by genotypic test.

Newer TB diagnostics: An update.

In recent years, nucleic-acid amplification tests (NAATs), which are highly specific and sensitive, have helped to transform the TB diagnostic landscape. According to the WHO 2021 Guidelines on Diagnostics, the NAATs used in TB diagnosis at the point of care (POC) include Xpert MTB/RIF a cartridge-based test manufactured by Cepheid, and Truenat a chip-based test manufactured by Molbio. Other POC tests that are expected to be implemented in near future include Xpert Omni and Xpert MTB/XDR. The use of line probe assay is involved at the level of reference labs for the detection of MTB and its resistance to first-line (Isoniazid and Rifampicin) and second-line (fluoroquinolones and second-line injectables) drugs. When the currently available NAATs detect mutations for drug resistance at a particular region of MTB sequence, the Whole genome sequencing (WGS) platform demonstrates the exceptional potential for reliable and comprehensive resistance prediction for MTB isolates, by multiple gene regions or whole genome sequence analysis allowing for accurate clinical decisions.

Standardization of a stool concentration method for Mycobacterium tuberculosis detection in the pediatric population.

Background: The inability of young children to expectorate sputum and paucibacillary status of Mycobacterium tuberculosis (MTB) increases its diagnostic complexity. In this study, we aimed to standardize a stool concentration method for the detection of MTB and its drug resistance by line probe assay (LPA). Methods: The stool from 10 healthy children spiked with H37Rv in five different dilutions (1:1, 1:10, 1:100, 1:1000, and 1:10,000), and stool from 10 confirmed TB and 54 clinically diagnosed TB children were subjected to an in-house stool concentration protocol. All the processed filtrates were subjected to smear microscopy, solid culture, Xpert ultra testing, and LPA. Results: Of 10 control samples, growth was seen in four samples (neat 1:1). In smear microscopy, bacilli could be seen in eight samples (1:1 and 1:10). Xpert ultra testing could detect MTB in eight samples in all dilutions with different loads. LPA could detect MTB in all samples and dilutions. In microbiologically confirmed children, seven out of 10 stool samples tested were positive. Out of 54 children with clinically diagnosed TB, 4 (7.4%) could be confirmed by microbiological diagnosis. Conclusion: The protocol standardized in this study proves to be better working in the molecular detection of MTB.

Characterization of probes associated with rifampicin resistance in M.tuberculosis detected by GenXpert from a national reference laboratory at Chennai.

With increasing use of Xpert MTB/RIF a point of care molecular test for simultaneous detection of TB and resistance to rifampicin, a growing number of rifampicin resistant cases are being detected and notified. Insights into the variation and frequencies in the probe mutations obtained through Xpert testing in the RRTB case will form the baseline information for further investigation on drug resistance. In this study we did a retrospective analysis of the GeneXpert data obtained from patient samples received at a National reference laboratory in Chennai between the years 2014 and 2020 to look at the probe distribution, the variation in the mutation and explore its significance. Probe E mutation was most commonly identified followed by Probe D, Probe A, Probe B and Probe C. Coexistence of multiple probe mutations in low bacillary load samples could be related to prolonged amplification cycle leading to delayed hybridization of probes. In such instances reporting false RR in xpert testing is possible. The probe mutations of RR should be monitored in depth with inclusion of codon specific targets for management of drug sensitive TB. In addition, heteroresistance needs to be further tested by alternative genotypic methods to avoid false resistance.

Drug susceptibility profiling of pulmonary Mycobacterium kansasii and its correlation with treatment outcome.

OBJECTIVES: With the introduction of newer molecular diagnostic tools to identify Mycobacterium tuberculosis, an increasing number of nontuberculous mycobacterium (NTM) is being identified. However, the drug resistance pattern of the NTM species identified is less explored. The objective of this study is to study the drug resistance patterns of Mycobacterium kansasii species isolated in a tuberculosis-endemic setting at South India. METHODS: A wide profile of NTM species were reported earlier from a prospective cohort of adults during 2017-2020. Out of this profile, a total of 22 M. kansasii species were subjected to drug susceptibility testing by two different methods: proportion sensitivity testing method and Sensititre testing method. RESULTS: Out of the 18 strains of M. kansasii subjected to Sensititre method of testing, the resistance pattern was demonstrated to be high for doxycycline (13) followed by rifampicin and trimethoprim/sulfamethoxazole (7). Out of the 22 strains subjected to proportion sensitivity testing method, 20 and 10 were resistant to isoniazid and ethambutol, respectively. CONCLUSION: There was a poor correlation between the treatment outcome and the resistance pattern of the antibiotics tested. With increasing numbers of NTM being reported, early and correct identification of NTM species is essential for the prompt initiation of appropriate treatment to achieve better outcome.

Nontuberculous mycobacterium: An emerging pathogen: Indian perspective.

Nontuberculous mycobacteria (NTM), considered as mere contaminants, are off late, being reported as potential pathogens through various studies. The infections due to NTM range from pulmonary to extra pulmonary including skin and soft-tissue infections, traumatic and surgical wound infections, and catheter and implant-associated infections. Although extrapulmonary infections are extensively explored, pulmonary infections are scarcely reported due to their misdiagnosis as tuberculosis caused by M. tuberculosis (MTB). Appropriate detection methods are essential in order to facilitate the differential diagnosis of NTM from MTB infections. We aimed to collate the data available on NTM diagnosis and its epidemiology in India in this review. While diagnosis of MTB itself is more challenging in India, for appropriate treatment of NTM, special training and attention is needed for differential diagnosis of the former from latter. Currently, in India, in addition to the available techniques for identification of NTM, line probe assay (Hains life sciences) is proving to be a promising tool for the detection of NTM (common mycobacteria/additional species kit) and their antimicrobial resistance (Genotype NTM-DR VER 1.0). In future, with the price of sequencing steadily coming down, with proper monitoring, whole-genome sequencing could be the test of choice to predict the species, drug resistance, outbreaks in hospitals, and transmission dynamics.

Management of Cervically Fractured Central Incisors: A Multidisciplinary Approach.

INTRODUCTION: Fracture of tooth structure at or below the gingival margin compromises rehabilitation and hampers esthetics and function. MANAGEMENT: Management of such cases by a post-core and crown restoration, or periodontal surgery or orthodontic extrusion alone may not always suffice in attaining a good result. CASE REPORT: A multi-disciplinary approach which includes all of the above mentioned procedures helps in long term success. CONCLUSION: Careful case evaluation, treatment planning and meticulous attention to detail are the keys to the best treatment outcome.

Molecular epidemiology of rotavirus in children and animals and characterization of an unusual G10P[15] strain associated with bovine diarrhea in south India.

Rotaviruses are enteric pathogens causing acute, watery, dehydrating diarrhea in various host species, including birds and mammals. This study collected data on the disease burden and strain prevalence of Group A rotavirus in animals and humans in Vellore and investigated interspecies transmission by comparison of circulating genotypes. Stool samples from children aged less than 5 years, admitted to the hospital between January 2003 and May 2006 for diarrhea and diarrheal samples from animals that were collected from a veterinary clinic and several dairy farms near Vellore between February 2007 and May 2008 were processed and subjected to RNA extraction and reverse-transcription PCR for genotyping of VP7 and VP4. Of 394 children with diarrhea, 158 (40%) were positive for rotavirus and the common G types identified were G1 (47, 29.7%), G2 (43, 27.2%), G9 (22, 13.9%), G10 (2, 1.2%), G12 (1, 0.6%) and mixed infections (27, 17.8%). The common P types were P[4] accounting for 57 (36%) samples, P[8] 57 (36%), P[11] 3 (1.8%) and P[6] 2 (1.2%). Of 627 animals, 35 (1 bullock, 2 goats, 32 cows) were found to be infected with rotavirus (5.5%). The common G types identified in order of frequency were G6 (17, 48.5%), G2 (10, 28%), G10 (4, 11%), G8 (2, 5.7%) and mixed infections (2, 5.7%). The common P types were P[6] accounting for 16 (46%) samples, P[4] 7 (20%), P[1] 3 (8.5%) and P[8] 3 (8.5%). An unusual P type P[15] was seen in one sample in combination with G10. The finding of G2 infections which are rarely identified in animals implies anthroponotic transmission since this genotype is predominantly associated with infection in humans.

Rotavirus shedding in symptomatic and asymptomatic children using reverse transcription-quantitative PCR.

Reverse transcription-real-time polymerase chain reaction (RT-qPCR) for the VP6 gene was used to study group A rotavirus shedding in children with symptomatic and asymptomatic rotavirus infection. Sequential stool samples (n = 345) from 10 children with rotavirus associated diarrhea and from five children (n = 161) with asymptomatic rotavirus infection were collected over a period of 2 months. A RT-qPCR assay on the samples using a rotavirus VP6 plasmid standard demonstrated high reproducibility, with an inter-assay coefficient of variation (CV) of 1.40-2.97% and an intra-assay CV of 0.03-3.03%. The median duration of shedding was longer in children with diarrhea compared to asymptomatic children (24 days vs. 18 days; P = 0.066). The median quantitation cycle (C(q)) at presentation in symptomatic children was 17.21 compared to 30.98 in asymptomatic children (P = 0.086). The temporal pattern in symptomatic children consisted of a high initial viral shedding coinciding with the duration of diarrhea, followed by a rapid fall, and then a small increase in secondary shedding 21 days later. Compared to children with rotavirus diarrhea, those with asymptomatic infection shed lower quantities of virus throughout the observation period. No difference was noted between the G and P genotypes of samples collected at onset of infection and during the shedding period. Shedding was intermittent in a subset of children in both groups. RT-qPCR is a useful method to characterize shedding patterns.

Astrovirus MLB1 is not associated with diarrhea in a cohort of Indian children.

Astroviruses are a known cause of human diarrhea. Recently the highly divergent astrovirus MLB1 (MLB1) was identified in a stool sample from a patient with diarrhea. It has subsequently been detected in stool from individuals with and without diarrhea. To determine whether MLB1 is associated with diarrhea, we conducted a case control study of MLB1. In parallel, the prevalence of the classic human astroviruses (HAstVs) was also determined in the same case control cohort. 400 cases and 400 paired controls from a longitudinal birth cohort in Vellore, India were analyzed by RT-PCR. While HAstVs were associated with diarrhea (p = 0.029) in this cohort, MLB1 was not; 14 of the controls and 4 cases were positive for MLB1. Furthermore, MLB1 viral load did not differ significantly between the cases and controls. The role of MLB1 in human health still remains unknown and future studies are needed.

Investigation of potential zoonotic transmission of cryptosporidiosis in southern India.

The common species and subgenotypes causing cryptosporidiosis were studied in 394 children and 627 animals with diarrhea in Vellore in southern India. Although no zoonotic strains were identified in 13 infected children, 1 of 12 infected animals had C. hominis, indicating the potential for cross-species transmission. This study also reports C. xiaoi for the first time in India.

Evaluation, using extracted human teeth, of Ricetral as a storage medium for avulsions--an in vitro study.

The prognosis of teeth replanted following avulsion is determined by the extra-alveolar time and storage medium used. This study was undertaken to determine the efficacy of an oral rehydration solution 'Ricetral', in retaining the vitality of periodontal ligament cells when used as a storage medium for avulsed teeth prior to replantation. The study consisted of a comparative evaluation between Ricetral and two currently recommended solutions, Hank's balanced salt solution (HBSS) and milk. Thirty extracted teeth were dried for 30min and soaked in the respective storage media for 45min. The periodontal ligament cells were isolated by an enzyme treatment with collagenase and trypsin. The cells were evaluated for vitality by trypan blue staining and number of vital cells counted in a hemocytometer. Statistical analysis revealed that cell vitality was high with Ricetral and HBSS, but poor with milk.

Pathotypes of diarrheagenic Escherichia coli in children attending a tertiary care hospital in South India.

The prevalence of diarrheagenic Escherichia coli (DEC) in children under 5 years was studied in children with diarrhea and controls in South India. Four polymerase chain reaction (PCR) "schemes" were used to detect genes of the 6 pathotypes of DEC. In 394 children with diarrhea, 203 (52%) DEC infections were found. Among the 198 controls, 126 (63%) DEC infections were found. Enteroaggregative E. coli was the most common pathotype by multiplex PCR both in cases (58, 14.7%) and controls (47, 23.7%), followed by enteropathogenic E. coli seen in 10% cases and 8% of controls. Enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC), and diffusely adherent E. coli (DAEC) were found in 4.1%, 2.0%, 1.0%, and 0.5% of cases, respectively. ETEC was found in 2.5% of controls, but EHEC, EIEC, and DAEC were not detected. Overall, no single assay worked well, but by discounting genes with a pathogenicity index of less than 1, it was possible to use the PCR assays to identify DEC in 75/394 (19%) cases and 12/198 (6.1%) controls, while mixed infection could be identified in 8/394 (2%) cases and 2/198 (1%) controls.