Epstein-Barr virus (EBV) DNAemia in saliva of viral encephalitis and nasopharyngeal carcinoma patients.

ABSTRACT: Epstein-Barr virus (EBV) is a lymphotropic virus that causes diseases ranging from a flu-like illness called infectious mononucleosis to nasopharyngeal carcinoma, Burkitt's lymphoma, and central nervous system (CNS) infection. Detection of EBV DNA is usually done using whole blood samples taken from the patients. We undertook the detection of EBV in blood, cerebrospinal fluid (CSF), and saliva by real-time quantitative PCR in two patients, one with a history of nasopharyngeal carcinoma, and the other having a case of viral encephalitis. EBV was detected only in saliva, whole blood in both patients, and CSF in the second case tested negative. This case series illustrates the importance of testing for EBV DNAemia in saliva by real-time polymerase chain reaction (PCR) to rule in a diagnosis of EBV.

Are we dealing with melioidosis under the mask of tuberculosis? - A case series.

Melioidosis is prevalent in South-East Asia. India is now become endemic to melioidosis. Melioidosis mimicks Tuberculosis (TB) and is often overlooked clinically. The spectrum of disease ranges from acute pulmonary infection to focal infection and septicemia. We report three cases of melioidosis, which was primarily suspected to be tuberculosis due to similarities in the clinical features. All patients were male and had risk factors such as type 2 diabetes mellitus as well as other risk factors such as chronic obstructive pulmonary disease (COPD), systemic hypertension, glucocorticoid therapy etc. All three patient samples were culture negative as well as negative for tests performed for the detection of tuberculosis. Conventional nested PCR targeting 251bp of 16S-23S spacer region of B. pseudomallei. was performed among individuals suspected to have extrapulmonary Tuberculosis. The presence of 251 bp was considered positive for B. pseudomallei. All three patients were treated with third generation cephalosporin and recovered due to timely diagnosis. Patients suspected for tuberculosis should be screened for B. pseudomallei, especially when AFB smear and MTB GeneXpert are negative. Often clinical samples may be culture negative for B. pseudomallei as patients are treated with antibiotics, therefore it is worthwhile performing PCR for B. pseudomallei to rule in a diagnosis of melioidosis and initiate appropriate antibiotics.

A shift in circulating rotaviral genotypes among hospitalized neonates.

In neonates, rotavirus (RV) infection is generally nosocomial. The control of rotaviral infection within hospital settings is challenging due to prolonged shedding of the virus and contamination of the surrounding environment. There are few studies that have reported asymptomatic infection within neonates. In this study, neonates were screened for RV infection and possible clinical manifestations that may play a role in RV acquisition were analysed. Stool samples were collected from 523 hospitalized neonates admitted for > 48 h in a low-cost and higher-cost tertiary centre. RV antigen was screened using ELISA and the samples which tested positive were confirmed by semi-nested RT-PCR. RV was detected in 34% of participants and genotypes identified included G12P[11] (44.4%), G10 P[11] (42.6%), G10G12P[11] (10.1%) and G3P[8] (2.9%). ICU admissions were associated with higher viral shedding (p < 0.05). Hospitalization in the low-cost facility ICU was associated with higher RV acquisition risk (p < 0.05). RV was detected in higher rates (36.9%) among neonates with gastrointestinal manifestations. G10P[11] was the predominant genotype for several years (1988-2016) among neonates within India. The preponderance of an emerging G12P[11] genotype and heterotypic distribution was documented. RV surveillance is important to identify emerging strains and establish the road ahead in managing RV infection.

Detection of cytomegalovirus disease by real-time quantitative PCR targeting immediate early gene (ppUL83) in different samples among post-renal-transplant recipients.

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.

Sequencing of Porphyromonas gingivalis from saliva in patients with periodontitis and type 2 diabetes mellitus.

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.

Hepatitis B virus X protein: The X factor in chronic hepatitis B virus disease progression.

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.

Next generation sequencing of oral microbiota in Type 2 diabetes mellitus prior to and after neem stick usage and correlation with serum monocyte chemoattractant-1.

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 141.6-980.5pg/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.

Maculopapular rash presentation of febrile illness in an adult with Varicella zoster virus infection.

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.

Serum monocyte chemoattractant protein-1 is a biomarker in patients with diabetes and periodontitis.

INTRODUCTION: The role of serum Monocyte Chemoattractant Protein-1 (MCP-1) as a biomarker of periodontitis is well documented; however, its role in diabetic patients with periodontitis is unknown. AIM: This study was conducted to determine the presence and concentration of serum MCP-1 in diabetic patients with and without periodontitis and correlate it glycemic status with periodontitis. MATERIALS AND METHODS: Adult diabetic patients were enrolled and grouped into group I, II, and III based on their glycemic status and serum MCP-1 estimated by ELISA. Linear regression and correlation tests were performed using R statistical software, Medcalc software to observe correlation between the serum MCP-1 and glycated hemoglobin level among different groups. RESULTS: Serum samples obtained from 37 patients tested positive for MCP-1. Mean serum MCP-1 concentration was highest (482.3 pg/ml) in group III, lowest (149.3 pg/ml) in group I, and intermediate 398.8 pg/ml in group II. Correlation and regression analysis was done between HbA1c and serum MCP-1. A significant positive correlation (P < 0.001) was observed. Serum MCP-1 increased by 37.278 pg/ml for every 1% rise in HbA1c, and the levels were raised in group II and group III than in group I irrespective of their glycemic status. With an HbA1c range of 6.5-6.9% (group II), the serum MCP-1 values cluster around 380-410 pg/ml. Elevated levels of serum MCP-1 (>500 pg/ml) in three subjects corresponded to HbA1c values more than 12.2% (group III). CONCLUSION: To our knowledge, this is the first study to document serum MCP-1 levels in diabetic patients with periodontitis. Glycemic status influences serum MCP-1, and lack of glycemic control contributes to increased serum MCP-1 levels. Serum MCP-1 may thus serve as a biomarker of inflammation and disease progression in diabetes with periodontitis.

Improved detection of Mycobacterium tuberculosis using two independent PCR targets in a tertiary care centre in South India.

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.