Diagnostic performance of real time PCR for the detection of Mycobacterium tuberculosis in cerebrospinal fluid samples.

PURPOSE: We aimed this study to standardize real time - polymerase chain reaction (RT-PCR) for the detection of Mycobacterium tuberculosis (Mtb) in cerebrospinal fluid (CSF) samples and compare its diagnostic performance with GeneXpert (Xpert), Mycobacteria Growth Indicator Tube (MGIT) and Multiplex PCR (MPCR) for tuberculous meningitis (TBM). METHODOLOGY: A total of 217 CSF samples were obtained from patients with suspected TBM during the study period between January 2019 and December 2021. The optimal cycle threshold (CT) of RT-PCR was determined by comparing different gene targets of Mtb (IS6110, 16SrRNA, HSP65 and Ag85B). Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) was determined for RT-PCR, Xpert, MGIT960 and MPCR. Diagnostic accuracy of these assays was compared by using clinical diagnosis as reference standard. RESULTS: IS6110RT-PCR was found to be highly sensitive as compared to other gene targets. Sensitivities of IS6110RT-PCR, MPCR, Xpert and MGIT against a reference standard of definite, probable and possible TBM were 36.7%, 21.1%, 16.7% and 6.7%, respectively; specificities were 97.6%, 100%, 100% and 100%, respectively. Xpert, RT-PCR, MPCR and MGIT960 detected 6.91% (n = 15), 5.99% (n = 13), 5.99% (n = 13) and 2.76% (n = 6) of definite TBM, respectively. RT-PCR detected 6.45% (n = 14) and 2.76% (n = 6) of possible TBM and probable TBM, respectively and MPCR detected 1.38% (n = 3) of possible and probable TBM each. CONCLUSION: IS6110RT-PCR is highly sensitive for primary screening of suspected TB cases, which may help clinicians to start appropriate patient's treatment with clinical suspicion of TBM.

Thyroid Function Abnormalities and Outcomes in Hospitalized Patients with COVID-19 Infection: A Cross-Sectional Study.

Thyroid gland can be affected by the COVID-19 infection. The pattern of thyroid function abnormality reported in COVID-19 is variable; in addition, some drugs used in COVID-19 patients like glucocorticoids and heparin can affect the thyroid function tests (TFT). We conducted an observational, cross-sectional study of thyroid function abnormalities with thyroid autoimmune profile in COVID-19 patients with varying severity from November 2020 to June 2021. Serum FT4, FT3, TSH, anti-TPO, and anti-Tg antibodies were measured before the initiation of treatment with steroids and anti-coagulants. A total of 271 COVID-19 patients were included in the study, of which 27 were asymptomatic and remaining 158, 39, and 47 were classified to mild, moderate and severe categories, respectively, according to MoHFW, India criteria. Their mean age was 49±17 years and 64.9% were males. Abnormal TFT was present in 37.2% (101/271) patients. Low FT3, low FT4, and low TSH were present in 21.03%, 15.9% and 4.5% of patients, respectively. Pattern corresponding to sick euthyroid syndrome was the most common. Both mean FT3 and FT3/FT4 ratio decreased with increasing severity of COVID-19 illness (p=0.001). In multivariate analysis, low FT3 was associated with increased risk of mortality (OR 12.36, 95% CI: 1.23-124.19; p=0.033). Thyroid autoantibodies were positive in 58 (27.14%) patients; but it was not associated with any thyroid dysfunction. Thyroid function abnormality is common among COVID-19 patients. Both low FT3 and FT3/FT4 ratio are indicators of disease severity while low FT3 is a prognostic marker of COVID-19 associated mortality.

Molecular diagnosis, genetic diversity and drug sensitivity patterns of Mycobacterium tuberculosis strains isolated from tuberculous meningitis patients at a tertiary care hospital in South India.

Tuberculous meningitis (TBM) is the most severe form of Mycobacterium tuberculosis (Mtb) infection in humans and is a public health concern worldwide. We evaluated the performance of GeneXpert MTB/RIF (GeneXpert) for the diagnosis of TBM. In addition, genetic diversity and drug susceptibility profiling of Mtb strains isolated from TBM patients were also investigated. A total of 293 TBM suspected cerebrospinal fluid (CSF) samples were collected and subjected to GeneXpert and Mycobacterial Growth Indicator Tube (MGIT 960) culture, respectively. Sensitivity and specificity of GeneXpert was 72.7% and 98.5%, respectively by using MGIT 960 as a gold standard (GeneXpert (n = 20, 6.8%) vs MGIT 960 (n = 22, 7.5%)). All Mtb positive cultures were subjected to 24-locus Mycobacterial Interspersed Repetitive Unit Variable Number Tandem Repeat (MIRU-VNTR) typing, Line probe assay (LPA) and MGIT 960- Drug Susceptibility Testing (DST). The rpoB gene was amplified and sequenced for selected isolates. Among our TBM patients, East African Indian (EAI) lineage (n = 16, 72.7%) was most predominant followed by Beijing (n = 3, 13.6%), S-family (n = 2, 9.1%) and Delhi/CAS (n = 1, 4.5%). Three Mtb strains were found to be Isoniazid (INH) resistant by MGIT 960; however LPA revealed that two strains were INH resistant and one strain was multi drug resistant (MDR) (Resistant to Isoniazid and Rifampicin (RIF)). We identified rifampicin resistant isolate with the mutation D516F in rifampicin resistance-determining region (RRDR) and observed discordant results between LPA, GeneXpert and MGIT 960. In addition, GeneXpert showing false RIF resistance was identified (no mutation in RRDR). We conclude that GeneXpert is useful for the diagnostic confirmation of TBM; however a GeneXpert negative sample should be subjected to MGIT 960 culture or LPA to rule out TBM. EAI lineage was the most predominant among TBM patients in South India and associated with drug resistance. The discordance between GeneXpert, MGIT 960 and LPA with respect to rifampicin resistance has to be ruled out to avoid TB treatment failure or relapse.

Inadequate inhaler technique, an everlasting problem, is associated with poor disease control - A cross sectional study.

INTRODUCTION: Dry powder inhalers (DPI) have been in use in the treatment of chronic respiratory diseases for decades. DPIs require proper inhaler technique to ensure appropriate dose delivery to the lungs which in turn provides disease control and hence reduces the economic burden due to frequent acute attacks and hospital visits. Inadequate inhaler technique remains an everlasting problem among patients with chronic respiratory disease. Hence the aim is to assess the inhaler technique in patients using DPI and to determine the factors associated with inhaler technique. MATERIAL AND METHODS: A cross-sectional study was conducted and 385 patients with asthma or chronic obstructive pulmonary disease (COPD) were recruited. Patient-related and disease-related factors were noted. Severity of the disease were assessed using asthma control test/COPD assessment test questionnaire and spirometer. The investigator assessed the inhaler technique of the patient against standard checklist. RESULTS: Nearly 46.2% of the patients performed incorrect inhaler technique. Multivariate analysis showed factors like young age [Odd's ratio (OR) 4.13, CI 1.31-17.8], well controlled disease (OR 2, CI 1.1-3.65), and the patients who learnt the technique from a medical personnel (OR 3.67, CI 1.46-9.24) had better inhaler technique. CONCLUSION: This study shows that the proper use of inhaler is still an unattained goal and significance of correct use has to be reiterated.

Dynamic modulation of DC-SIGN and FcΥR2A receptors expression on platelets in dengue.

Platelet activation has been reported to play a major role in inflammatory response and thrombocytopenia during dengue viral infection. Cells expressing FcϒR2Aand DC-SIGN receptors are reported to be involved in dengue virulence. The present study is designed to assess the expression level of these two receptors on platelet surface collected from dengue patients and to study its association in patients with platelet RNA positive for dengue virus. This was an analytical cross-sectional study carried out in JIPMER hospital, Puducherry. Forty-four patients with dengue infection as cases and 44 patients with non dengue acute other febrile illness(OFI) as controls were recruited. Peripheral venous blood was withdrawn on day of admission, day 3 post admission and day of discharge and serological tests for NS1 dengue antigen and anti IgM antibody were analyzed for diagnosis of dengue infection. Platelet rich plasma was assessed for DC SIGN, FcϒR2A levels and platelets separated from dengue patients were subjected to RNA extraction and detection of presence of viral RNA. The study observed a decreased expression of DC-SIGN and FcϒR2A on platelets in dengue patients compared to OFI group on all the time points. Further, cells expressing DC-SIGN and FcϒR2A were found to be decreased on platelets in dengue patients who were positive for NS1 antigen. DC-SIGN and FcϒR2A expression was also found to be notably decreased in patients positive for platelet DENV RNA when compared with patients negative for platelet DENV RNA. Our results suggest that DC-SIGN and FcϒR2A, which are receptors for viral capture and immune mediated clearance respectively, might be down regulated on platelets in patients with dengue infection. The decreased receptor expression diminishes platelet activation and subsequently has protective action on the host from the ongoing conflict between immune system and dengue virus.

Validation of pneumococcal iron acquisition (piaA) gene for accurate identification of Streptococcus pneumoniae.

PURPOSE: The pneumococcal iron acquisition (piaA) gene is found to be highly specific and hence proposed as a diagnostic marker for identification of pneumococci. The objective of the present study was to evaluate the piaA gene as a genetic marker for the identification of pneumococci. METHODS: Twenty isolates were initially sequenced for lytA gene using published primers. PiaA-PCR (piaA polymerase chain reaction) was performed using in-house primers and protocol. Based on the sensitivity and specificity results, a final sample of 30 pneumococcal isolates and 11 non-pneumococcal isolates confirmed with lytA- sequencing were selected. Statistical analyses were performed using OpenEpi v3.01 and GraphPad Quickcalc at P < 0.05 as the level of statistical significance. RESULTS: Of the initial 20 samples tested, piaA PCR was positive in only 71.43% (10/14) of the pneumococcal isolates but was 100% specific (0/6 non-pneumococcal isolates) P = 0.011. When the PCR was performed on 41 samples, the sensitivity increased to 73.33% (95% of confidence interval [CI] = 55.55-85.82) and specificity remained the same P < 0.001. The level of agreement between the PCR and lytA-sequencing was found to be moderate (κ = 0.694; 95% CI = 0.432-0.955). CONCLUSIONS: PiaA-PCR can be used as a specific marker for the identification of pneumococcus, though it is less sensitive. As the level of agreement was moderate, further analyses on a large number of samples can give conclusive evidence for its use as a diagnostic marker for pneumococcus.

Rapid and Accurate Diagnosis of Acute Pyogenic Meningitis Due to Streptococcus Pneumoniae, Haemophilus influenzae Type b and Neisseria meningitidis Using A Multiplex PCR Assay.

INTRODUCTION: Acute bacterial meningitis is one of the major causes of morbidity and mortality in children and geriatric population, especially in developing countries. Methods of identification are standard culture and other phenotypic tests in many resource poor settings. AIM: To use molecular methods for the improvement of aetiological diagnosis of acute pyogenic meningitis in patients. MATERIALS AND METHODS: CSF samples of 125 patients were included for the study. Gram staining and culture were performed according to standard procedures. Antigen was detected using commercial latex agglutination test kit. Multiplex PCR was performed using previously published primers and protocols. Fischer's exact test was used for finding association between presence of the disease and clinical/biochemical parameters, considering two tailed p<0.05 as statistically significant. Sensitivity, specificity, positive and negative predictive values were calculated using Graphpad QuicCalc software. RESULTS: A total of 39 cases (31.2%) were confirmed to be of acute pyogenic meningitis based on biochemical methods. Only 10/39 was positive for the three organisms tested. Multiplex PCR was able to detect one additional isolate each of Streptococcus pneumoniae and Haemophilus influenzae type b. When compared with multiplex PCR as the gold standard, culture and latex agglutination tests had same sensitivity (80%), specificity (100%), PPV (100%) and NPV (97.8%), whereas Gram stain had poor sensitivity (40%) and good specificity (95.6%). Detection rates were higher in multiplex PCR for the two organisms Streptococcus pneumoniae and Haemophilus influenzae type b. CONCLUSION: Multiplex PCR was more sensitive than culture or antigen detection, and employing this assay can significantly increase the speed and accuracy of identification of the pathogen.

Effect of Vitamin D Supplementation on Vascular Functions and Oxidative Stress in Type 2 Diabetic Patients with Vitamin D Deficiency.

BACKGROUND: Vitamin D levels are reported to have an inverse liaison with the risk of cardiovascular diseases. Hence, we aimed to evaluate the effect of Vitamin D supplementation on changes in vascular functions and oxidative stress in type 2 diabetic patients with Vitamin D deficiency. SUBJECTS AND METHODS: One hundred and three patients with type 2 diabetes attending endocrinology outpatients department in a tertiary care hospital were screened for Vitamin D deficiency. Patients with serum 25-hydroxy Vitamin D levels <20 ng/ml were considered as deficient and were administered 60,000 IU of oral Vitamin D3 weekly for 8 weeks. In these patients, parameters of vascular functions (carotid-femoral pulse wave velocity, brachial-ankle pulse wave velocity, and arterial stiffness index) and oxidative stress (serum malondialdehyde levels and total antioxidant status) were measured at baseline and after 8 weeks of oral Vitamin D supplementation. RESULTS: Among 103 patients with type 2 diabetes, 75 (72.82%) were found to have Vitamin D deficiency. Amidst these patients, carotid-femoral pulse wave velocity (991.6 ± 161.82 vs. 899.29 ± 151.86, P < 0.001), right brachial-ankle pulse wave velocity (1446.16 ± 204.33 vs. 1350.8 ± 178.39, P < 0.001), and left brachial-ankle pulse wave velocity (1493.81 ± 219.65 vs. 1367.61 ± 220.64, P < 0.001) showed a significant reduction following Vitamin D supplementation. Further, these patients were found to have significant fall in serum malondialdehyde levels with rise in total antioxidant status ensuing Vitamin D supplementation. CONCLUSION: The present study shows that oral Vitamin D supplementation of 60,000 IU/week for 8 weeks significantly improves vascular functions and reduces oxidative stress in type 2 diabetic patients with Vitamin D deficiency.

Inhibition of Platelet Aggregation by the Leaf Extract of Carica papaya During Dengue Infection: An In Vitro Study.

Dengue cases were reported to undergo platelet activation and thrombocytopenia by a poorly understood mechanism. Recent studies suggested that Carica papaya leaf extract could recover the platelet count in dengue cases. However, no studies have attempted to unravel the mechanism of the plant extract in platelet recovery. Since there are no available drugs to treat dengue and considering the significance of C. papaya in dengue treatment, the current study aimed to evaluate two research questions: First one is to study if the C. papaya leaf extract exerts its action directly on platelets and second one is to understand if the extract can specifically inhibit the platelet aggregation during dengue viral infection. Sixty subjects with dengue positive and 60 healthy subjects were recruited in the study. Platelet-rich plasma (PRP) and platelet-poor plasma were prepared from both the dengue-infected and healthy control blood samples. Effect of the leaf extract obtained from C. papaya leaves was assessed on plasma obtained as well as platelets collected from both healthy and dengue-infected individuals. Platelet aggregation was significantly reduced when leaf extract preincubated with dengue plasma was added into control PRP, whereas no change in aggregation when leaf extract incubated-control plasma was added into control PRP. Upon direct addition of C. papaya leaf extract, both dengue PRP and control PRP showed a significant reduction in platelet aggregation. Within the dengue group, PRP from severe and nonsevere cases showed a significant decrease in aggregation without any difference between them. From the study, it is evident that C. papaya leaf extract can directly act on platelet. The present study, the first of its kind, found that the leaf extract possesses a dengue-specific neutralizing effect on dengue viral-infected plasma that may exert a protective role on platelets.

Clinical Utility of Fractional exhaled Nitric Oxide (FeNO) as a Biomarker to Predict Severity of Disease and Response to Inhaled Corticosteroid (ICS) in Asthma Patients.

INTRODUCTION: Bronchial asthma is a common chronic inflammatory airway disease diagnosed and is based on symptomatic history and Pulmonary Function Tests (PFT). Fractional exhaled Nitric Oxide (FeNO) is exclusively a non-invasive biomarker of on-going eosinophilic airway inflammation which remains unpredictable only with PFTs. FeNO measurement is recommended in predicting asthma severity and Inhaled Corticosteroid (ICS) response but further research is required to understand its clinical utility and agreement with current recommendations in a specific population. AIM: To estimate FeNO levels in Tamilian patients with mild-to-moderate persistent asthma and to correlate with disease severity and ICS response. MATERIALS AND METHODS: The study was a prospective cohort with a single group of 102 persistent asthma patients under standard ICS regimen for 8 weeks (follow-up period). PFT and FeNO were measured using portable spirometry and chemiluminescence based exhaled breath analyser, at baseline and during follow-up visits. Based on PFT and FeNO parameters, the study population was sub-grouped with respect to asthma severity (as mild, moderate and moderately severe), FeNO cut-off (> or < 50ppb) and ICS response classification (good vs poor ICS responders). RESULTS: Significant decrease in mean FeNO levels were found in mild, moderate and moderately severe asthmatic groups following ICS treatment (90.15±27.36, 75.74±31.98 and 77.18±32.79 ppb) compared to similar baseline FeNO levels (103.03±34.08, 91.38±37.60 and 97.90±43.84 ppb) in all the above groups. Similarly, significant decrease in mean FeNO levels was found - FeNO>50ppb, good and poor ICS responders groups, in post- ICS treatment (89.63±24.04, 77.90±31.12 and 86.49±32.57 ppb) compared to baseline levels (110.183±1.23, 97.12±42.04 and 99.68±34.71 ppb). CONCLUSION: The observed baseline FeNO values in all groups as stated above did not show significant difference to differentiate asthma severity or ICS responders groups. The present study results do not support the predictive association of baseline FeNO levels with asthma severity and future ICS response, but the decrements in FeNO levels on ICS treatment, supports its clinical utility in monitoring of ongoing airway inflammation and understanding treatment response rate.

Association of Intracellular T(H)1-T(H)2 Balance in CD4+ T-cells and MIP-1alpha in CD8+ T-cells with Disease Severity in Adults with Dengue

BACKGROUND: We tested the hypothesis that dengue haemorrhagic fever (DHF) is associated with a T(H)1-skewed immune response as opposed to dengue fever (DF). METHODS: We estimated intracellular (in T-cells) and serum levels of designate T(H)1/T(H)2 cytokines [interferon-gamma (IFN-gamma), interleukin-4 (IL-4), and tumor necrosis factor-alpha] and macrophage inflammatory protein-1alpha (MIP-1alpha) at admission, 48 h, and day 5 in 20 adults with dengue (DF=10, DHF=10) and 10 dengue-naive healthy controls. RESULTS: At admission, intracellular IFN-gamma/IL-4 ratio in CD4+ T-cells and proportion of MIP-1alpha-positive CD8+ T-cells were significantly higher in patients with DHF [7.21 (5.36~10.81) vs. 3.04 (1.75~4.02); p=0.011 and 6.2% (3.2~8.2%) vs. 2.4% (2.0~3.6%); p=0.023]. The latter showed a significant positive correlation with IFN-gamma/IL-4 ratio in CD4+ T-cells (Spearman's rho=0.64; p=0.003), percentage-change in haematocrit (rho=0.47; p=0.048), and serum alanine aminotransferase level (rho=0.61; p=0.009). CONCLUSION: We conclude that DHF is associated with a T(H)1-skewed immune response. Further, MIP-1alpha in CD8+ T-cells is an important immunologic correlate of disease severity in dengue.