Hospital-based norovirus surveillance in children <5 years of age from 2017 to 2019 in India.

After the introduction of the rotavirus vaccine into the Universal Immunization Program in India in 2016, relatively few studies have assessed the prevalence and epidemiological patterns of acute gastroenteritis (AGE) among hospitalized children ≤5 years of age. We used a uniform protocol to recruit children with AGE as well as standardized testing and typing protocols. Stool specimens from children with AGE younger than 5 years of age admitted to six hospitals in three cities in India were collected from January 2017 through December 2019. Norovirus was detected by real-time reverse transcription-polymerase chain reaction (RT-qPCR) followed by typing positive specimens by conventional RT-PCR and Sanger sequencing. Norovirus was detected in 322 (14.8%) of 2182 specimens with the highest rate in 2018 (17.6%, 146/829), followed by 2019 (14.4%, 122/849) and 2017 (10.7%, 54/504). Rotavirus vaccine status was known for 91.6% of the children of which 70.4% were vaccinated and 29.6% not. Norovirus positivity in rotavirus-vaccinated children was 16.3% and 12% in unvaccinated children. GII.4 Sydney[P16] (39.3%), GII.4 Sydney[P31] (18.7%), GII.2[P16] (10%), GI.3[P13] (6.8%), GII.3[P16] (5.9%), and GII.13[P16] (5%) accounted for 85.8% (188/219) of the typed strains. Our data highlight the importance of norovirus in Indian children hospitalized with AGE.

Diagnostic value of anti-VZV IgG in neurological diseases among varicella unvaccinated individuals.

Varicella zoster virus (VZV) is a neurotropic alphaherpesvirus that causes neurological manifestations either as a complication of primary infection or reactivation. VZV induced neurological diseases have a good prognosis when confirmed early and treated with anti-viral therapy. Myelitis, encephalitis, ventriculitis or meningitis can occur without a telltale rash in immunocompetent and immunocompromised individuals making the diagnosis difficult. We analyzed CSF and serum samples from 30 unvaccinated study participants (17 male and 13 female) to determine the presence of VZV DNA by PCR in CSF and to estimate serum and CSF anti-VZV IgG and albumin levels in participants with neurological manifestations with/without rash. Anti-VZV IgG was detected in CSF (n = 22, [73%]) and serum (n = 29, [97%]) of pediatric and adult participants. Anti-VZV IgG were detected in CSF of participants with varied clinical presentation altered sensorium (n = 8, [36%]), meningitis (n = 4, [18%]), acute febrile illness (n = 3, [14%], encephalopathy/meningoencephalitis (n = 2, [9%]), irritability (n = 2, [9%]) and each patient from cerebrovascular stroke, demyelinating disorder and febrile seizure (n = 1, [4.5%]). VZV DNA was detected from one participant and CSF serum albumin levels were elevated in 53% of study participants. VZV DNA is present up to 1-2 weeks post onset of disease, after which anti-VZV antibody may be the only indicator of disease and therefore both VZV DNA and anti-VZV IgG need to be tested for in CSF. As VZV DNA and VZV IgG antibody are both good indicators of VZV reactivation, routine testing would result in reduced morbidity and mortality by early detection of disease and antiviral treatment.

Satellite Epidemic of Covid-19 Associated Mucormycosis in India: A Multi-Site Observational Study.

BACKGROUND: Sudden upsurge in cases of COVID-19 Associated Mucormycosis (CAM) following the second wave of the COVID-19 pandemic was recorded in India. This study describes the clinical characteristics, management and outcomes of CAM cases, and factors associated with mortality. METHODS: Microbiologically confirmed CAM cases were enrolled from April 2021 to September 2021 from ten diverse geographical locations in India. Data were collected using a structured questionnaire and entered into a web portal designed specifically for this investigation. Bivariate analyses and logistic regression were conducted using R version 4.0.2. RESULTS: A total of 336 CAM patients were enrolled; the majority were male (n = 232, 69.1%), literate (n = 261, 77.7%), and employed (n = 224, 66.7%). The commonest presenting symptoms in our cohort of patients were oro-facial and ophthalmological in nature. The median (Interquartile Range; IQR) interval between COVID diagnosis and admission due to mucormycosis was 31 (18, 47) days, whereas the median duration of symptoms of CAM before hospitalization was 10 (5, 20) days. All CAM cases received antifungal treatment, and debridement (either surgical or endoscopic or both) was carried out in the majority of them (326, 97.02%). Twenty-three (6.9%) of the enrolled CAM cases expired. The odds of death in CAM patients increased with an increase in HbA1c level (aOR: 1.34, 95%CI: 1.05, 1.72) following adjustment for age, gender, education and employment status. CONCLUSION: A longer vigil of around 4-6 weeks post-COVID-19 diagnosis is suggested for earlier diagnosis of CAM. Better glycemic control may avert mortality in admitted CAM cases.

Evolutionary analysis of rotavirus G1P[8] strains from Chennai, South India.

Rotaviruses by virtue of its segmented genome generate numerous genotypes. G1P[8] is the most common genotype reported globally. We intend to identify the evolutionary differences among G1P[8] strains from the study with vaccine strains. Stool samples collected from children <5 years were screened for rotavirus antigen by enzyme linked immunosorbent assay. The samples that tested positive for rotavirus were subjected to VP7 and VP4 semi-nested RT-PCR. Sanger sequencing was performed in randomly chosen VP7 and VP4 rotavirus strains. Phylogenetic analysis showed less homology between study strains and vaccine strains and they were placed in different lineages. The VP7 and VP4 proteins of rotavirus were analyzed by two different platforms to identify the amino acid substitutions in the epitope regions. Nine amino acid substitutions with respect to Rotarix®, RotaTeq® and Rotasiil®-V66A, A/T68S, Q72R, N94S, D100E, T113I, S123N, M217T, and I281T were observed in VP7. There were five amino acid substitutions-S145G, N/D195G, N113D, N/I78T, E150D in VP4 (VP8 portion) with respect to Rotarix® and RotaTeq® vaccine strains. M217T substitution in VP7 (epitope 7-2) and N113D, D195G substitution in VP4 (epitope 8-3, 8-1) confer changes in polarity/electrical charge with respect to vaccine strains, thus indicating the need for continued surveillance.

Detection of parvovirus B19 in selected high-risk patient groups & their phylogenetic & selection analysis.

Background & objectives: Human parvovirus B19V (B19V) is known to be associated with erythema infectiosum commonly in children, aplastic crisis, especially in persons with underlying haemolytic disorders, hydrops fetalis in pregnancies and arthritis. This cross-sectional study was aimed to determine the presence of B19V infection in childhood febrile illnesses, association of B19V with arthropathies and in adult patients with end-stage renal disease (ESRD) on dialysis. The genetic diversity among the sequences was also analysed. Methods: A nested polymerase chain reaction (nPCR) assay was used for B19V DNA targeting VP1/VP2 region and used for testing 618 patients and 100 healthy controls. Phylogenetic analysis on nucleotide and amino acid sequences was carried out to compare our sequences with other Indian strains and global strains. Results: Among 618 samples tested, seven (1.13%) were found positive. The phylogenetic analysis revealed that all the seven sequences belonged to genotype 1 and showed low genetic diversity. The clustering pattern of seven sequences was similar both by nucleotide and by predicted amino acid sequences. The fixed effects likelihood analysis showed no positive or negatively selected sites. Interpretation & conclusions: Seven samples (4 from non-traumatic arthropathies, 2 from patients with ESRD and 1 from febrile illness patient) were found positive by nPCR. When our seven sequences were compared with global strains, the closest neighbour was other Indian strains followed by the Tunisian strains.

Serological, molecular and clinical correlates of dengue from a tertiary care centre in Chennai, India.

Dengue disease is caused by dengue viruses 1-4 and has been ranked by the World Health Organisation (WHO) as the fastest spreading vector-borne viral disease. Dengue is often underreported and misdiagnosed due to a wide spectrum of clinical manifestations. Diagnosis of dengue is based on clinical case definitions and laboratory methods. Newer case definitions of dengue have been formulated by clinical studies in order to improve case detection. Owing to its epidemic potential, mortality and morbidity, there is a need for a rapid and accurate diagnostic assay for dengue in order to help the clinician in the early detection of cases and to prevent disease progression. A duplex real time PCR targeting the 3'UTR region for rapid and simultaneous detection of all dengue viruses serotypes (1-4) was standardized based on published literature. About 150 patients with acute undifferentiated febrile illness classified based on the 2009 WHO dengue case definition were tested using the duplex real time dengue PCR. Sequencing based PCR was performed on selected PCR positive samples for partial nucleotide sequence of the CprM gene and a phylogenetic tree was constructed. Statistical analysis was done using the MedCalc software. Out of the 126 patients classified as dengue disease positive, according to the 2009 WHO dengue case definition, 54% had "probable dengue", 43% had "dengue with warning signs" and 3% had "severe dengue". The performance of the duplex real time PCR was assessed among the various clinical groups of dengue and it was found that in the "dengue with warning signs group" PCR had a positive predictive value of 85.29% (range - 68.94% to 95.05%) when compared with dengue NS1 ELISA. The average time for PCR positivity was found to be four days from the onset of illness. The cycling threshold values obtained from real time PCR were used as a semi quantitative measure of viremia. Accordingly, there was a relatively low CT value among the "warning signs dengue group" when compared to the "probable dengue group". The use of the duplex PCR is suggested in the early diagnosis of dengue, especially in the 'warning signs' group of patients as they showed a higher positivity rate. Also, the use of the resultant CT value as a semi-quantitative measure of viremia will assist the clinician in early diagnosis and prevention of disease development.

Development & standardization of an in-house IgM indirect ELISA for the detection of parvovirus B19 infections.

BACKGROUND & OBJECTIVES: Parvovirus B19 infections occur worldwide; the infection is acquired early in childhood but could occur later. B19 is reported to cause infection in childhood febrile illnesses, and arthropathies in adults and children and in end-stage renal disease (ESRD) seen in adults. This study was designed to develop an in-house IgM indirect ELISA for serological screening among patients and controls, and to compare ELISA results with those of nested polymerase chain reaction (nPCR) assay. METHODS: An in-house IgM indirect ELISA was standardized using peptide sequence of VP1/VP2 region of parvovirus B19. A total of 201 children and adult with febrile illnesses, 216 individuals with non-traumatic arthropathies, 201 cases of chronic anaemia associated with ESRD and 100 healthy controls were tested. Serum was separated from the blood and subsequently used for DNA extraction. The nested polymerase chain reaction (nPCR) for the detection of B19V DNA was performed using primers targeting the overlapping region of VP1/VP2 capsid protein genes. RESULTS: A total of 618 samples were tested for parvovirus B19 by an in-house IgM indirect ELISA. Among these samples, six were positive by in-house ELISA. The inter-rater agreement between ELISA and PCR assays was calculated using kappa coefficient analysis. The value of κ was 0.77 and the strength of agreement was 'good' (P<0.001). INTERPRETATION & CONCLUSIONS: The in-house IgM indirect ELISA was found to be simple with high sensitivity and specificity when compared with nPCR and could be used as an alternative to expensive commercial kits in resource-poor settings.

Rapid and Long-Term Immunity Elicited by DNA-Encoded Antibody Prophylaxis and DNA Vaccination Against Chikungunya Virus.

BACKGROUND: Vaccination and passive antibody therapies are critical for controlling infectious diseases. Passive antibody administration has limitations, including the necessity for purification and multiple injections for efficacy. Vaccination is associated with a lag phase before generation of immunity. Novel approaches reported here utilize the benefits of both methods for the rapid generation of effective immunity. METHODS: A novel antibody-based prophylaxis/therapy entailing the electroporation-mediated delivery of synthetic DNA plasmids encoding biologically active anti-chikungunya virus (CHIKV) envelope monoclonal antibody (dMAb) was designed and evaluated for antiviral efficacy, as well as for the ability to overcome shortcomings inherent with conventional active vaccination and passive immunotherapy. RESULTS: One intramuscular injection of dMAb produced antibodies in vivo more rapidly than active vaccination with an anti-CHIKV DNA vaccine. This dMAb neutralized diverse CHIKV clinical isolates and protected mice from viral challenge. Combination of dMAb and the CHIKV DNA vaccine afforded rapid and long-lived protection. CONCLUSIONS: A DNA-based dMAb strategy induced rapid protection against an emerging viral infection. This method can be combined with DNA vaccination as a novel strategy to provide both short- and long-term protection against this emerging infectious disease. These studies have implications for pathogen treatment and control strategies.

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.

Ensuring Transfusion Safety: Screening Blood Donors for Human Parvovirus B19.

Ensuring the safety of blood and blood products is a vital aspect of healthcare. The potential for transmission of pathogens through blood and blood products makes transfusion safety a significant concern. Despite advancements in testing methodologies, donated blood products still pose a risk for infection transmission. Human parvovirus B19 (B19V) is a small, single-stranded, non-enveloped DNA virus transmissible parenterally by blood transfusion. B19V causes a wide range of clinical manifestations, which is generally harmless in healthy individuals. B19V infection may cause severe complications, such as aplastic crises, as it affects erythrocyte progenitor cells in individuals with increased erythrocyte turnover. Additionally, B19V can be transmitted from pregnant women to their foetus, potentially causing hydrops fetalis and foetal death. The potential for transmission through blood and blood products makes B19V a significant concern for transfusion safety. In response to the growing recognition of B19V's impact on transfusion safety, various international health organisations have introduced guidelines to minimise its transmission through blood and plasma products. However, the implementation of these guidelines varies globally, with some regions, such as India, still lacking formal protocols for B19V monitoring. This review article explores the existing methodologies for screening blood donors for B19V, assesses the associated transfusion risks, and considers the implications for public health and clinical practice. By emphasising advancements in diagnostic techniques and the challenges of their implementation, this article provides a comprehensive overview of efforts to reduce the transmission of B19V through blood transfusions, thereby ensuring safer blood supplies and improved patient outcomes.

Expanded spectrum of varicella disease and the need for vaccination in India.

Varicella vaccine was first licensed in Japan and South Korea in 1989 for use in healthy children and was introduced in US in 1995. So far, 29 countries have adopted varicella vaccine in their universal immunization program (UIP). No Asian country, India included, has adopted the varicella vaccine as part of their UIP. The extra-cutaneous sites for VZV diseases are central nervous system and gastrointestinal tract, the expanded disease spectrum includes vasculopathy, myelitis, inflammatory bowel disease, perforated ulcers, and gastritis. The actual disease burden of varicella is not known as most of the infected individuals may not visit the physician. The amplifiable VZV DNA will not always be detectable in cerebrospinal fluid (CSF) samples in protracted illnesses such as vasculopathies, but demonstrable anti-VZV IgG in CSF has diagnostic value. The World Health Organization (WHO) position paper 2014 recommends two doses of varicella and zoster vaccines in targeted population. In India, varicella vaccine is not included in the UIP due to the cost and the belief that lifelong immunity occurs following primary infection. The expanded spectrum of VZV disease and the mounting body of evidence, however, suggest the need for both varicella and zoster vaccines in routine immunization schedule.

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.

Increased parvovirus B19 seropositivity in healthy blood donors in India.

A core component of every blood program is the supply of safe blood and blood products. The elevated risk of transmission through these products is due to parvovirus B19 (B19V) resistance to the virus inactivation procedures. Our study aimed to screen asymptomatic blood donors for B19V at a tertiary care hospital in Chennai, Tamil Nadu, between September 2020 and June 2021. Sera from 106 healthy blood donors who tested negative for Human immunodeficiency virus (HIV), Hepatitis B surface antigen (HBsAg), Hepatitis C virus (HCV), syphilis, and malaria were tested for anti-B19V IgM and IgG using a qualitative indirect enzyme-linked immunosorbent assay (ELISA). In the study population, 23.5% (n = 25) of donors tested IgM positive, 38.6% (n = 41) tested IgG positive, and 7.5% (n = 8) tested positive for both IgM and IgG. A proportion of 61.3% (n = 65) of the blood donors tested IgG negative, suggesting they had no past B19V infection. B19V DNA was not detected in any of the subjects. The high seroprevalence of IgM indicates that blood donors may have been recently exposed to B19V, potentially posing a risk to immunocompromised individuals and those with hematological stress. Further longitudinal studies with a larger sample size are recommended to better understand the risk of B19V transfusion transmission.

Assessment of aerobic bacterial contamination of computer keyboards in a tropical setting.

BACKGROUND: Computers are widely used in healthcare for improved and effective care. Previous published reports have shown microorganisms colonising computer keyboards in some clinical areas. OBJECTIVES: This study was undertaken to measure, compare and characterize the aerobic microorganisms in computer keyboards of hospital and non-hospital settings. METHODOLOGY: Samples were collected from commonly used keys of computers in hospital and non-hospital settings using moistened sterile swabs, inoculated in liquid and solid media, and incubated aerobically at 37 degrees C for 24-48 h. Growth was identified as per standard microbiological procedures. Antibiotic susceptibility was determined for pathogenic strains by Kirby-Bauer method. RESULTS: Growth was seen in all 80 samples (40 from each setting). Staphylococcus aureus was isolated from both settings (hospital: 6 MRSA, 11 MSSA; non-hospital: 4 MRSA, 9 MSSA). Gram-negative bacilli were isolated more frequently from hospital (33%). Statistical analysis showed homogeneity among isolates from computer keyboards in both settings, except for Pseudomonas. CONCLUSION: Isolation of microorganisms from "high-touch" surfaces such as computer keyboards is indicative of the need for awareness on cleaning of such surfaces or disinfection and adequate hand hygiene.

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.

Persistence of G10P[11] neonatal rotavirus infections in southern India.

BACKGROUND: Neonatal rotavirus infections are predominantly caused by distinct genotypes restricted to this age-group and are mostly asymptomatic. METHOD: Stool samples from neonates admitted for >48 h in neonatal intensive care units (NICUs) in Vellore (2014-2015) and Chennai (2015-2016) in southern India, and from neonates born at hospitals in Vellore but not admitted to NICUs (2015-2016) were tested for rotavirus by ELISA and genotyped by hemi-nested RT-PCR. RESULTS: Of 791 neonates, 150 and 336 were recruited from Vellore and Chennai NICUs, and 305 were born in five hospitals in Vellore. Positivity rates in the three settings were 49.3% (74/150), 29.5% (99/336) and 54% (164/305), respectively. G10P[11] was the commonly identified genotype in 87.8% (65/74), 94.9% (94/99) and 98.2% (161/164) of the neonates in Vellore and Chennai NICUs, and those born at Vellore hospitals, respectively. Neonates delivered by lower segment cesarian section (LSCS) at Vellore hospitals, not admitted to NICUs, had a significantly higher odds of acquiring rotavirus infection compared to those delivered vaginally [p = 0.002, OR = 2.4 (1.4-4.3)]. CONCLUSIONS: This report demonstrates the persistence of G10P[11] strain in Vellore and Chennai, indicating widespread neonatal G10P[11] strain in southern India and their persistence over two decades, leading to interesting questions about strain stability.

Gut microbiota and its mysteries.

Gut microbiota are microorganisms that inhabit the gut; they coexist peacefully with the host, thereby contributing to the health and well-being of individuals. Bacteroidetes and Firmicutes largely dominate the gut microbial flora. The intestinal flora promotes intestinal mucosal integrity, provides essential nutrients such as vitamins and enzymes, protects the body against pathogens and produces antimicrobial peptides such as defensins, C-type lectins, cathelicidins, they also play an active role in the innate and adaptive immune system. Gut microbial flora plays an active role in the synthesis of short-chain fatty acids such as butyrate, propionate and acetate. Gut microbiota also plays a significant role in the cognitive and behavioural functions of the host. A balanced gut microbiota shifts to dysbiosis, due to intake of high fat or sugar or other factors like sedentary lifestyle. The dysbiosis of the gut results in increased permeability, endotoxaemic, insulin resistant, systemic inflammation, adiposity and metabolic disorders such as type 2 diabetes mellitus, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, irritable bowel disorder, colorectal cancer, etc. A prudent lifestyle modification, added on with use of probiotics and prebiotic restore the normal flora of the gut, especially in patients with Clostridium difficle-associated diarrhoea, inflammatory bowel syndrome, liver disease and colon cancer. Faecal microbial transplant is an important therapeutic tool in many illness related with the gut. Thereby, understanding the gut microbial signatures in various diseases yields various novel therapeutic targets. Human gut microbiota has a prognostic, diagnostic and therapeutic potential which is recognised worldwide.

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.