Evaluation of Host Protein Biomarkers by ELISA From Whole Lysed Peripheral Blood for Development of Diagnostic Tests for Active Tuberculosis.

Tuberculosis (TB) remains a significant global health crisis and the number one cause of death for an infectious disease. The health consequences in high-burden countries are significant. Barriers to TB control and eradication are in part caused by difficulties in diagnosis. Improvements in diagnosis are required for organisations like the World Health Organisation (WHO) to meet their ambitious target of reducing the incidence of TB by 50% by the year 2025, which has become hard to reach due to the COVID-19 pandemic. Development of new tests for TB are key priorities of the WHO, as defined in their 2014 report for target product profiles (TPPs). Rapid triage and biomarker-based confirmatory tests would greatly enhance the diagnostic capability for identifying and diagnosing TB-infected individuals. Protein-based test methods e.g. lateral flow devices (LFDs) have a significant advantage over other technologies with regard to assay turnaround time (minutes as opposed to hours) field-ability, ease of use by relatively untrained staff and without the need for supporting laboratory infrastructure. Here we evaluate the diagnostic performance of nine biomarkers from our previously published biomarker qPCR validation study; CALCOCO2, CD274, CD52, GBP1, IFIT3, IFITM3, SAMD9L, SNX10 and TMEM49, as protein targets assayed by ELISA. This preliminary evaluation study was conducted to quantify the level of biomarker protein expression across latent, extra-pulmonary or pulmonary TB groups and negative controls, collected across the UK and India, in whole lysed blood samples (WLB). We also investigated associative correlations between the biomarkers and assessed their suitability for ongoing diagnostic test development, using receiver operating characteristic/area under the curve (ROC) analyses, singly and in panel combinations. The top performing single biomarkers for pulmonary TB versus controls were CALCOCO2, SAMD9L, GBP1, IFITM3, IFIT3 and SNX10. TMEM49 was also significantly differentially expressed but downregulated in TB groups. CD52 expression was not highly differentially expressed across most of the groups but may provide additional patient stratification information and some limited use for incipient latent TB infection. These show therefore great potential for diagnostic test development either in minimal configuration panels for rapid triage or more complex formulations to capture the diversity of disease presentations.

Validation of Differentially Expressed Immune Biomarkers in Latent and Active Tuberculosis by Real-Time PCR.

Tuberculosis (TB) remains a major global threat and diagnosis of active TB ((ATB) both extra-pulmonary (EPTB), pulmonary (PTB)) and latent TB (LTBI) infection remains challenging, particularly in high-burden countries which still rely heavily on conventional methods. Although molecular diagnostic methods are available, e.g., Cepheid GeneXpert, they are not universally available in all high TB burden countries. There is intense focus on immune biomarkers for use in TB diagnosis, which could provide alternative low-cost, rapid diagnostic solutions. In our previous gene expression studies, we identified peripheral blood leukocyte (PBL) mRNA biomarkers in a non-human primate TB aerosol-challenge model. Here, we describe a study to further validate select mRNA biomarkers from this prior study in new cohorts of patients and controls, as a prerequisite for further development. Whole blood mRNA was purified from ATB patients recruited in the UK and India, LTBI and two groups of controls from the UK (i) a low TB incidence region (CNTRLA) and (ii) individuals variably-domiciled in the UK and Asia ((CNTRLB), the latter TB high incidence regions). Seventy-two mRNA biomarker gene targets were analyzed by qPCR using the Roche Lightcycler 480 qPCR platform and data analyzed using GeneSpring™ 14.9 bioinformatics software. Differential expression of fifty-three biomarkers was confirmed between MTB infected, LTBI groups and controls, seventeen of which were significant using analysis of variance (ANOVA): CALCOCO2, CD52, GBP1, GBP2, GBP5, HLA-B, IFIT3, IFITM3, IRF1, LOC400759 (GBP1P1), NCF1C, PF4V1, SAMD9L, S100A11, TAF10, TAPBP, and TRIM25. These were analyzed using receiver operating characteristic (ROC) curve analysis. Single biomarkers and biomarker combinations were further assessed using simple arithmetic algorithms. Minimal combination biomarker panels were delineated for primary diagnosis of ATB (both PTB and EPTB), LTBI and identifying LTBI individuals at high risk of progression which showed good performance characteristics. These were assessed for suitability for progression against the standards for new TB diagnostic tests delineated in the published World Health Organization (WHO) technology product profiles (TPPs).

Healthcare Associated Infections in a Resource Limited Setting.

INTRODUCTION: Health Care associated Infections (HAI) are the most common complications affecting the hospitalized patients. HAI are more common in developing and under developed countries. However, there are no systematic surveillance programs in these countries. AIM: To find out the burden, predisposing factors and multidrug resistant organisms causing HAI in a resource limited setting. MATERIALS AND METHODS: This prospective observational study was done at Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER). Patients aged 13 years or more with stay of more than 48 hours in a 16 bedded Medical Intensive Care Unit (MICU) between November 2011 and April 2013 were included in the study. Patients were prospectively followed up till discharge or death for the development of HAI. Device associated HAI like Ventilator Associated Pneumonia (VAP), Catheter Related-Blood Stream Infection (CR-BSI) and Catheter Associated-Urinary Tract Infections (CA-UTI) were studied. Standard laboratory methods were used for identification of microorganisms causing HAI and to test their antibiotic sensitivity. RESULTS: A total of 346 patients were included in the study with median age of 38 years. Common indications for admission to Medical Intensive Care Unit (MICU) were poisoning (31.5%); neurological illness (23.4%) like Guillian-Barre syndrome, tetanus, meningitis, encephalitis; respiratory illness (14.5%) like pneumonia, acute respiratory distress syndrome and tropical infections (7.2%) like malaria, scrub typhus, leptospirosis. Fifty percent (174/346) patients developed one or more HAI with VAP being the most common. The rates of HAI per 1000 device days for VAP, CR-BSI, CA-UTI were 72.56, 3.98 and 12.4, respectively. Acinetobacter baumannii was the most common organism associated with HAI. Multidrug resistance was seen in 74% of the isolates. CONCLUSION: The burden of HAI, especially with MDR organisms, in resource constrained setting like ours is alarming. There is urgent need for infection control and monitoring system to reduce HAI.

Efficacy of intraoperative vancomycin in irrigating solutions on aqueous contamination during phacoemulsification.

PURPOSE: To study the efficacy of adding vancomycin in irrigating solutions, in comparison to topical antibiotic given preoperatively for a day, during phacoemulsification, in reducing the anterior chamber (AC) contamination. SETTINGS AND DESIGN: This was a prospective, interventional, hospital-based study. MATERIALS AND METHODS: This was a study involving 400 eyes of 400 paitens, undergoing routine phacoemulsification between January 2004 and June 2006. The patients were non-randomly assigned to two groups: Group 1 included 180 patients, who received topical ciprofloxacin eye-drops (four-hourly) for a day preoperatively and Group 2 included 220 patients, who underwent phacoemulsification with vancomycin (20 microg/ml) in the irrigating solution. Anterior chamber aspirate obtained at the end of the surgery was sent for microbial workup. The number of positive cultures in both the groups was determined. STATISTICAL ANALYSIS: This was performed using Chi-square test. RESULTS: Aqueous samples showed microbial growth in 38 (21.1%) out of 180 eyes in Group 1 and in 17 (7.7%) out of 220 eyes in Group 2 ( P = 0.001). Coagulase-negative staphylococcus was the most common organism in both the groups. Aqueous samples from four eyes in group 1 showed multiple organisms, while none of the sample from group 2 showed more than one organism. None of the eyes in either group showed fungal contamination. One patient in Group 1 developed endophthalmitis, and the causative organism was Alcaligenes faecalis. All patients were followed up for a minimum of six months (range: 6 to 14 months and mean: 9.3 months). CONCLUSION: Addition of vancomycin in irrigating solutions is more efficacious in reducing AC contamination in comparison to topical antibiotic administered a day preoperatively.