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Objective: The aim of this study was to determine the proportion of influenza-like illnesses (ILIs) caused by influenza A and influenza A H1N1 and to determine the proportion of influenza B in a smaller group of samples with ILIs and influenza A H1N1 negative by qualitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) using TaqMan-based assay at a tertiary health-care center in Chennai, Tamil Nadu. Material and Methods: Laboratory samples of participants from all age groups who had ILIs were included in this study. The study was conducted from January 2018 to January 2019 at a tertiary health-care center in Chennai, Tamil Nadu. The sample size of the study was 1755. This is a cross-sectional study. RNA extraction was performed using QIAamp Viral RNA Mini Kit (Qiagen, U.S.A) as per the manufacturer instructions. The assay is a TaqMan®-based real-time detection of circulating novel influenza A H1N1 and H3N2. Real-time PCR for influenza B virus was performed in influenza A H1N1-negative patients using artus Infl/H1 LC/RG RT-PCR kit (Cat 4523003, Qiagen, Germany). Samples that had a crossing threshold value 15–35 cycles were considered positive. Results: The majority of the participants were in the pediatric and young adult age group (n = 798) were positive for influenza A, of which 32.7% (n = 575) were positive for influenza A H1N1. Both influenza A H1N1 and influenza A other than H1N1 incidence started to rise in September and spiked between October and December. Among patients with persistent ILI, screening for influenza B was done in 48 samples. Among 48 samples, 18% (n = 8) had influenza B. Conclusion: The need for increased vaccination is demonstrated through the high influenza A H1N1 positivity rate among pediatric patients with ILIs. Detection of influenza B among influenza A H1N1-negative individuals demonstrates the need for influenza B screening. Incidence of influenza is highest in cooler months. The implementation of vaccination against influenza before the beginning of the cooler seasons could possibly reduce the burden of influenza on the health-care system. The importance of surveillance for the continued screening of influenza could be expanded in the private sector as a majority of the disease burden is observed in that sector.

Background: Porphyromonas gingivalis is a major periodontal pathogen. Saliva is the most easy, non-invasive microbiological sample for detection of periodontal pathogens. Aim and Objectives: A prospective study on 37 diabetic patients was grouped into well-controlled diabetes with/without periodontitis and uncontrolled diabetic with periodontitis. PCR and sequencing of P. gingivalis was performed in saliva samples. Materials and Methods: DNA was extracted from saliva using Triton X-100 and 16s rRNA gene (404 bp) was amplified by polymerase chain reaction. DNA sequencing was performed for two samples. Results:P. gingivalis was detected in 27.03% (n = 10), of which 30% (n = 9) were diabetic with periodontal disease and 14.3% (n = 1) were diabetic without periodontal disease. The percentage of poor oral hygiene was 50% and 20% in uncontrolled and controlled glycaemic patients, respectively. DNA sequencing of two samples showed 100% identity with the sequences in the GenBank database (Gen Bank accession no: KX640913-KX640914). Conclusion: Type 2 diabetes mellitus and periodontitis are interlinked. Early detection of P. gingivalis and appropriate treatment with doxycycline will also assist in controlling the glycaemic status.

Introduction: Hepatitis B virus (HBV) is the most common aetiological factor causing hepatocellular carcinoma (HCC). HBx gene plays an enigmatic role in HBV-related HCC. In this study we have analysed amino acid substitutions in HBx from HBV-infected individuals of different clinical stages. Materials and Methods: HBV-infected individuals (n = 93) were recruited in the study. DNA was extracted from plasma, amplified, and DNA sequencing was performed using specific primers targeting HBx gene (540 bp). Results: Among the study participants, 57% had chronic HBV infection, 30% had chronic liver disease (CLD) and 13% had HBV related HCC. Genotypes such as D1, D2, D3, A1, C2 and B2 were identified of which genotype D2 was predominant (78%). HBxC-terminal deletion was observed in four hepatitis B e antigen (HBeAg) negative participants with CLD. The frequency of aminoacid substitution in proapoptotic domain was higher in HBeAg negative participants including I127V (34%), K130M (34%), V131I (40%). The frequency of double mutation (K130M+V131I) and triple mutation (I127V+K130M+V131I) were found to be higher (32% and 36%) in HBeAg negative participants. Also, we identified L5M substitution (4.3%) in HBeAg positive participants with advanced liver disease. Conclusion: In HBx gene, aminoacid substitutions at positions 127, 130, 131 are associated with poor expression of HBeAg. We suggest screening for HBx aminoacid substitutions especially in patients with HBeAg negative chronic HBV infection to predict the clinical outcome and enable early treatment to prevent disease progression.

Renal transplantation is a treatment option for end-stage renal disease (ESRD). Cytomegalovirus (CMV) infection was analysed among symptomatic and asymptomatic post-renal-transplant recipients (PRTRs). A total of 30 PRTRs were enrolled. DNA was extracted and quantitative real-time PCR for CMV (CMV R-Gene, France) targeting ppUL83 gene was performed on whole blood, urine and saliva. The detection rate of CMV was found to be 27% (n = 8) in different samples, including whole blood, urine and saliva. Among 30 PRTRs, 53% (n = 16) of the PRTRs did not shed virus in saliva. About 7% of CMV was detected only in saliva among PRTRs who were symptomatic.

Aim: Co-occurrence of Epstein–Barr virus (EBV) with Cytomegalovirus (CMV) is associated with an increased risk of EBV-associated posttransplant lymphoproliferative disorder (PTLD). Quantitation of EBV by real-time polymerase chain reaction (PCR) can aid the clinicians in the initiation of preemptive measures to improve the survival of the graft. Methods: The study was conducted among postrenal transplant recipients (PRTRs) who were attending the nephrology department from 2011 to 2016. Real-time quantitative PCR for EBV was performed in whole blood. PRTRs were classified into asymptomatic with altered renal parameters (Group A) and symptomatic (Group B), which were further subcategorized into Group B1 (fever with anemia, leukopenia, thrombocytopenia, or altered liver enzymes (any two), Group B2 (Group B1 + end-organ disease or only end-organ disease), and Group B3 (graft dysfunction [GDF]). The posttransplant period was also defined. DNA was extracted (Qiagen, Hilden, Germany) from whole blood, and real-time PCR was performed using QuantiTect multiplex PCR kit. Unpaired t-tests and ANOVA were used to analyze the data. Results: A total of 89 PRTRs were enrolled, of which 39.3% (n = 35) had EBV DNAemia, 43.1% during very late, 41.1% in late and 28.6% in immediate post transplant periods. EBV DNAemia ranged from 324 to 32,436 copies/ml. EBV DNAemia was found in 84% (n = 75) of symptomatic (Group B) and 16% (n = 14) of asymptomatic (Group A). Among the PRTRs with GDF (Group B3), 44% (n = 11/25) had EBV DNAemia of 2893.9 ± 1869 copies/ml. EBV DNAemia was considerably higher in PRTRs without GDF (8700.2 ± 9675.6 copies/ml) than PRTRs with GDF and the difference was statistically significant (P = 0.004). EBV DNAemia with CMV DNAemia among PRTRs was found in 21.3% (n = 19). Conclusion: High EBV DNAemia may precede PTLD or GDF; therefore, regular screening of EBV DNAemia is warranted. CMV and EBV DNAemia may also co-exist in PRTRs. As CMV is an immunomodulating virus, it increases the risk of opportunistic infections, especially EBV.

Introduction Oral microbiome impacts health and disease. T2DM and periodontitis are associated. Neem ( Azadiracta indica ) has antibacterial activity against oral microbiota. Objectives To characterize oral microbiota (OMB) in saliva samples of T2DM patients by Next generation sequencing. To analyze MCP-1 levels among the T2DM patients before and after a month of neem stick usage as a toothbrush. Materials and methods Blood and saliva samples were collected from adult T2DM patients before and after the neem stick usage. Metagenomic sequencing was performed on saliva samples targeting V6 region of 16s rRNA. Serum MCP-1 levels were determined using a quantitative sandwich Human MCP-1 standard ABTS development kit (Peprotech, USA). Results The profile of oral microbiota of T2DM patients (n = 24) consists of Streptococcus (95.8%) counts ranging from 2644 to 27,214, Veillonella (72.2%, counts 25–19,709, Neisseria (87.5%) 453–33,445), Rothia (63.6%, 233–6734), Actinomycetes (25%, 161–3730), Fusobacterium (21%, 2252–21,334), and Pigmentiphaga (12.5% 3–16,644). Oral microbiota in healthy controls (n = 10), consists of Streptococcus (26.1%), Veillonella (21.9%), Neisseria (16.9%), Haemophilus (10.7%), Actinomycetes (2.6%), Rothia (3.1%), Oribacterium (1.7%). Post neem samples showed drastic reduction in the load of bacteria which was statistically significant. The mean serum MCP-1 before the use of neem stick was 265.18 ± 79.44 (range 141.6–980.5 pg/ml) and dropped to 33.6 ± 7.35 after a month of neem stick usage (P value > 0.001). Conclusion OMB of T2DM patients and healthy controls were similar, however bacterial loads were significantly higher in T2DM patients. Use of neem stick has a statistically significant reduction on bacterial loads and MCP-1 levels in T2DM patients.

Objectives: Since melioidosis mimics tuberculosis clinically and radiologically, there is a need for a rapid diagnostic method to help the clinician to initiate appropriate antimicrobial treatment in order to prevent mortality. Our objective was to standardize a nested PCR for B. pseudomallei and its detection in pulmonary and extra pulmonary samples from patients with suspected TB. Materials and Methods: Archived pulmonary and extra pulmonary samples which were negative for M. tuberculosis smear microscopy, culture and PCR were included in the study. DNA was extracted (QiAmp Blood DNA kit, Qiagen, Germany) and conventional nested PCR were carried out to detect the presence of 16S-23S spacer region of B. pseudomallei. The DNA was detected by 2% agarose gel electrophoresis and the presence of 251 bp was considered positive. Results: A total of 55 samples were tested, out of which 9 (16.3%) samples tested positive for Burkholderia pseudomallei using nested PCR, which included 5 extra pulmonary and 4 pulmonary samples. These patients belonged to Tamil Nadu 8 (88.8%) and West Bengal 1 (11.1%) both of which are rice growing regions. Among the nine patients who were positive for B. pseudomallei by nested PCR, 2 (22%) were receiving empirical anti-tubercular treatment (ATT). Also, these patients encountered co-morbid condition like renal failure, malignancy, diabetes and co-infection with HIV. Conclusion: We suggest that the patients with symptoms suggestive of both pulmonary and extra pulmonary tuberculosis should be routinely tested for Burkholderia pseudomallei by molecular methods for timely initiation of appropriate therapy and avoid unnecessary exposure to ATT. J Microbiol Infect Dis 2017; 7(1): 21-28

Porphyromonas gingivalis is a major periodontal pathogen. Saliva is the most easy, non-invasive microbiological sample for detection of periodontal pathogens.A prospective study on 37 diabetic patients was grouped into well-controlled diabetes with/without periodontitis and uncontrolled diabetic with periodontitis. PCR and sequencing of P. gingivalis was performed in saliva samples.DNA was extracted from saliva using Triton X-100 and 16s rRNA gene (404 bp) was amplified by polymerase chain reaction. DNA sequencing was performed for two samples.P. gingivalis was detected in 27.03% (n = 10), of which 30% (n = 9) were diabetic with periodontal disease and 14.3% (n = 1) were diabetic without periodontal disease. The percentage of poor oral hygiene was 50% and 20% in uncontrolled and controlled glycaemic patients, respectively. DNA sequencing of two samples showed 100% identity with the sequences in the GenBank database (Gen Bank accession no: KX640913-KX640914).Type 2 diabetes mellitus and periodontitis are interlinked. Early detection of P. gingivalis and appropriate treatment with doxycycline will also assist in controlling the glycaemic status.

Varicella zoster usually manifests as maculopapular rash (MPR), which later progresses to vesicle. It can also manifest as MPR without progression to the vesicle stage. This atypical manifestation is more common in adults and immunocompromised patients. A 30-year-old female presented with high-grade fever and rash over face and body for 5 days. She was diagnosed to have Varicella zoster virus (VZV) infection by positive VZV immunoglobulin M enzyme-linked immunosorbent assay and polymerase chain reaction. We present this case to increase awareness among clinicians on the atypical manifestations of VZV and prevent complications by early diagnosis.

Introduction: Re-emergence of Chikungunya is a major public health problem in the southern states of India. Objectives: This study was undertaken to investigate an outbreak of Chikungunya, in June-August 2008 using PCR and determine the prevalent genotypes of Chikungunya virus (CHIKV) associated with the outbreak. Materials and Methods: Samples of blood were collected (in heparinized vacutainer tubes) from suspected patients of CHIKV infection from both Government Taluk Hospital in Kerala and a tertiary care hospital in Chennai, Tamil Nadu. A one-step RT-PCR was carried out on a block thermo-cycler targeting the E2 gene that codes for the viral envelope protein. The amplicons were verified for 305 bp size by standard agarose gel electrophoresis. The PCR products were purified, sequenced, and compared with other CHIKV strains reported from different geographical regions. A phylogenetic tree was constructed using MEGA 4. Results: Altogether 118 samples were collected from patients who presented with sudden onset of fever and/or joint pain, myalgia, and headache. CHIKV infection was confirmed by RT-PCR in 14 patients and all these cases were from Kerala. The positivity correlated with the early stage of the disease as all these patients had fever of less than seven days duration. The study isolates have been allotted the GenBank accession nos. GQ272368-GQ272381. Phylogenetic analysis of recent CHIKV isolates by partial sequencing of E2 region shows that isolates are closely related to strains from neighboring states and the African type. Conclusion: RT-PCR is a useful technique for the early detection of CHIKV infection during outbreaks. Molecular characterization of the strains indicates that majority of the strains have originated from the Central/East African strains of CHIKV.

Hospital indoor air can be a source for transmitting nosocomial infections in resource limited settings. This study was undertaken over two months period (February – March 2010) in Chennai, India, to 1) characterise bacteria isolated from indoor air of healthcare facility; and 2) establish whether environmental and clinical isolates are similar by molecular typing methods. Daily visits were made to microbiology laboratory to determine clustering of cases of nosocomial infections. Patients with illnesses related to respiratory tract and skin and soft tissues were included. Clinical strains (from laboratory) with similar antibiogram patterns were systematically stocked. Indoor air samples were collected from such locations by exposed plate method for 30 minutes. Growth was identified by standard microbiological procedures. Phenotypically similar strains were further subjected to genotyping by polymerase chain reaction and pulsed field gel electrophoresis (PFGE) to confirm similarity. Coagulase-negative staphylococci (CNS) were only isolated from both environmental and clinical samples during the study period. Totally, 15 clinical and six environmental strains of CNS were isolated over two months. One airborne and one clinical strain, isolated subsequently from a patient in the same location, had similar phenotypic (biochemical and antibiogram) characteristics. Isolates were identified Corresponding Author Ms. Suchithra Sudharsanam Sri Ramachandra Medical College & Research Institute, Sri Ramachandra University, Porur, Chennai 600116, India. Email: suchisanam@gmail.com

Introduction: Tuberculosis (TB) causes significant morbidity and mortality worldwide as one of the leading infectious diseases. In India, more than 1.8 million new cases occur every year. Rapid and accurate diagnosis of TB would improve patient care and limit its transmission.This study aimed to evaluate a dual target polymerase chain reaction (PCR) diagnostic assay to detect Mycobacterium tuberculosis from pulmonary and extra-pulmonary samples at a tertiary care centre in South India. Methodology: Samples were collected from patients with a low index of suspicion of TB. Acid-fast smears were performed by Auramine O fluorescent microscopy and PCR was performed by using two site-specific primer pairs targeting IS6110 by nested PCR and TRC4 by conventional PCR. Amplified products for IS6110 and/or TRC4 were indicative of M. tuberculosis.Results: Among 114 (19 pulmonary and 95 extra-pulmonary) samples tested by PCR assay, 12 (11%) were positive for both IS6110 and TRC4, of which 11 (10%) were non-respiratory and one was (1%) respiratory in origin. PCR for TRC4 alone was positive for eight (7%) non-respiratory and two (2%) respiratory samples, while IS6110 alone tested positive for six (5%) non-respiratory samples and one (1%) respiratory sample. Of a total of 29 PCR positive samples, 17 (15 %) were acid-fast smear positive. Conclusion: Although the target site of IS6110 is specific for M. tuberculosis, some strains from South India may lack this region. Therefore, the use of an additional target site (TRC4) is required for improved detection of M. tuberculosis.

Results All enrolled patients had fever with rash without evidence of vesicles. Majority, (n=29) were adults and 18 were male. Of the 14 (38.9%) that were positive by IgM ELISA for VZV, 5 (35.7%) belonged to the age group of 19-30 years, 4 (28.5%) were 1.5 to < 2 lakh /cumm). All sera that were tested were negative for Dengue NS1 antigen, IgM and IgG antibody capture ELISA (Panbio, Australia).