Distribution of hepatitis B virus genotype and cancer predicting precore and basal core promoter mutations.

BACKGROUND: Chronic hepatitis B (CHB) infection which is associated with an increased risk of developing liver disease including cirrhosis and hepatocellular carcinoma. Viral factors that may increase the risk for HCC development include HBV DNA level, genotypes, and naturally occurring mutations such as hepatitis B virus precore (PC) (G1896A) and basal core promoter (BCP) A1762T/G1764A double mutations. HBV genotypes and subgenotypes can significantly influence HBeAg seroconversion rates, viremia levels, mutational patterns that could significantly influence the heterogeneity in clinical manifestations and even response to antiviral therapy. METHOD: 94 CHB infected individuals with detectable serum HBV DNA levels were studied. HBsAg, HBeAg, anti-HBc IgM antibody estimations were done by ELISA. HBV DNA estimation was done. The HBV genotypes were determined by TSP-PCR and 10 samples randomly selected for DNA sequencing. PC and BCP mutations were determined by DNA sequence analysis of core region. RESULT: Of 94 study participant samples with detectable serum HBV DNA levels, 75 were successfully genotyped and sequenced for BCP/PC region. 30/75 (40%) harbored PC and BCP mutations. The total Double mutations of BCP at A1762T/G1764A nucleotide positions, and PC mutation at G1896A nucleotide position were seen in 29.3% and 21.3%, respectively. All 75 isolates were subtype D using TSP-PCR. However, by sequencing 2/10 were subtype A, while 8 were subtype D. CONCLUSION: Our study reinforces that D is the predominant genotype in Indian population. It reveals that Indian CHB subjects have increased prevalence of BCP & PC mutations, which possibly may lead to development of HCC.

Emergence of multidrug resistant enterococci at a tertiary care centre.

BACKGROUND: Enterococci have assumed great clinical importance because of their increasing resistance to various antimicrobial agents. Thus, knowledge about the antibiogram of these multidrug resistant isolates is of utmost importance in formulating an effective antibiotic policy to treat these infections and reducing the morbidity and mortality. Aim of this study was to assess the antimicrobial resistance pattern of enterococci and determine the prevalence of multidrug resistance among them. METHODS: This cross sectional study was carried out from August 2011 to February 2014, in which 200 non-repetitive clinical isolates of enterococci were included. Antimicrobial susceptibility testing was done by disc diffusion method. Minimum inhibitory concentration (MIC) of gentamicin, streptomycin, vancomycin, teicoplanin and linezolid was determined by E-test method. RESULTS: The prevalence of multidrug resistance among enterococcal isolates was found to be 63%. Varying levels of resistance was seen to various antibiotics. Most of the isolates were resistant to penicillin (95%), ampicillin (95%) and cotrimoxazole (90%). High level aminoglycoside resistance (HLAR) and glycopeptide resistance was seen in 39% and 14% isolates respectively. Only 4 isolates (2%) were found to be resistant to linezolid. CONCLUSION: The prevalence of multidrug resistance among enterococci was found to be 63%, the resistance being more common in Enterococcus faecium as compared to Enterococcus faecalis. The study highlights the emergence and increased prevalence of multidrug resistant enterococci which pose a serious therapeutic challenge.

Heterogeneous vancomycin-intermediate among methicillin resistant Staphylococcus aureus.

BACKGROUND: Hetero-resistance vancomycin intermediate Staphylococcus aureus (hVISA) is phenotype, which on in-vitro susceptibility test is vancomycin susceptible (VSSA) but has a minority population of vancomycin intermediate (VISA). hVISA is responsible for vancomycin treatment failure. Population Analysis Profile- Area under Curve (PAP-AUC) is a test for detection of hVISA; however, this test is unsuitable for clinical microbiology laboratory. Tests, such as Brain Heart Infusion Agar with 6 µg/ml vancomycin (BHIA6V), E test and Macromethod E Test (MET) are available; however reported to have variable results. METHODS: 58 clinical isolates of Methicillin resistant S aureus (MRSA) having MIC of vancomycin more than 1 µg/ml by E test and agar dilution were analyzed by PAP-AUC, BHIA6V and MET. RESULT: The prevalence of hVISA was 6.9%. hVISA isolates were having vancomycin E test MIC >2 µg/ml. Sensitivity of BHIA6V, MET and E test with MIC >2 µg/ml were 0.75, 0.67 and 1.0 respectively; however, positive predictive values (PPV) were 0.43, 0.4 and 0.27 respectively with PAP-AUC. PAP-AUC ratio correlated with MIC by E test and MET. CONCLUSIONS: There is need for screening MRSA isolates showing in-vitro vancomycin susceptibility ≤2 µg/ml by agar dilution method for detection of hVISA. PAP-AUC test is unsuitable for routine laboratory testing. BHIA6V, MET and E test can be used for screening, however have low PPV.

Detection of glycopeptide resistance genes in enterococci by multiplex PCR.

BACKGROUND: Vancomycin Resistant Enterococci (VRE) are a major cause of nosocomial infections. There are various phenotypic and genotypic methods of detection of glycopeptide resistance in enterococci. This study utilizes multiplex PCR for reliable detection of various glycopeptides resistance genes in VRE. METHOD: This study was conducted to detect and to assess the prevalence of vancomycin resistance among enterococci isolates. From October 2011 to June 2013, a total of 96 non-repetitive isolates of enterococci from various clinical samples were analyzed. VRE were identified by Kirby Bauer disc diffusion method with Clinical and Laboratory Standards Institute (CLSI) guidelines. Minimum inhibitory concentration (MIC) of all isolates for vancomycin and teicoplanin was determined by E-test. Multiplex PCR was carried out for all enterococci isolates using six sets of primers. RESULTS: Out of 96 isolates, 14 (14.6%) were found to be resistant to vancomycin by vancomycin E-test method (MIC ≥32 µg/ml). Out of these 14 isolates, 13 were also resistant to teicoplanin (MIC ≥16 µg/ml). VanA gene was detected in all the 14 isolates by Multiplex PCR. One of the PCR amplicons was sent for sequencing and the sequence received was submitted in the GenBank (GenBank accession no. KF181100). CONCLUSION: Prevalence of VRE in this study was 14.6%. Multiplex PCR is a robust, sensitive and specific technique, which can be used for rapid detection of various glycopeptide resistance genes. Rapid identification of patients infected or colonized with VRE is essential for implementation of appropriate control measures to prevent their spread.

Emergence of VIM-2 metallo-beta-lactamase producing Ralstonia pickettii clinical isolate in India.

A multidrug-resistant clinical isolate of Ralstonia pickettii from a woman was analysed. Modified Hodge test was positive for carbapenemase production. Conjugation experiment revealed the presence of conjugative plasmid of >140 Kb size typed as IncN type. This is the first report of emergence blaVIM-2 in R. pickettii in India.

Differences in vancomycin MIC among MRSA isolates by agar dilution and E test method.

In this study, the correlation between vancomycin minimum inhibitory concentration (MIC) obtained by the E test technique and the Clinical And Laboratory Standards Institute (CLSI) agar dilution method was evaluated. A total of 53 Methicillin Resistant Staphylococcus aureus (MRSA) strains were tested by both the methods in the present study. MICs of vancomycin obtained by the E test method were consistently higher (+0.5 to 2 log2 dilutions) than those obtained by the agar dilution method. Out of 53 MRSA isolates, 49 isolates showed higher MIC results by E test than by agar dilution method. Three isolates showed same MIC result by both methods. Since many studies have demonstrated increased clinical failure with MRSA isolates for which vancomycin MICs are increased (>1 µg/ml) but still within the susceptibility range (≤ 2 µg/ml), our findings suggest the requirement to re-look into the breakpoints for vancomycin for determining sensitivity of MRSA isolates. Guidelines should also specify the method to be used for determining the MIC.

Shooting up: the interface of microbial infections and drug abuse.

Illicit drug control has been on the global agenda for more than a century. Infections have long been recognized as one of the most serious complications of drug abuse. Drug users are susceptible to pulmonary, endovascular, skin and soft tissue, bone and joint, and sexually transmitted infections caused by a wide range of bacterial, viral, fungal and protozoal pathogens. In addition, injection drug users are at increased risk for parenterally acquired infections such as human immunodeficiency virus, hepatitis B virus, hepatitis C virus, tetanus and malaria. Factors related to drug use, such as unsterile injection practices, contaminated drug paraphernalia and drug adulterants, increase the exposure to microbial pathogens. Illicit drugs also affect several components of the complex immune system and thus modulate host immunity. In addition, lifestyle practices such as multiple sexual partners, overcrowded housing arrangements and malnutrition serve as co-factors in increasing the risk of infection. In this review we present an overview of the unique aspects of microbial pathogenesis, immune modulation and common infections associated with drug use. We have restricted the definition of drug abuse to the use of illegal drugs (such as opiates, marijuana, cocaine, heroin and amphetamines), not including alcohol and nicotine.

Correlation of CD4+ T cell Count with Total Lymphocyte Count, Haemoglobin and Erythrocyte Sedimentation Rate Levels in Human Immunodeficiency Virus Type-1 Disease.

BACKGROUND: Studies in human immunodeficiency virus (HIV) infected adults have demonstrated association of total lymphocyte count (TLC) <1200/mm (3) and subsequent disease progression or mortality. The association of other surrogate makers such as haemoglobin (Hb), and erythrocyte sedimentation rate (ESR) with CD4 count and disease progression has also been suggested. This study was carried out to determine the relationship of CD4-positive T lymphocyte counts with TLC, Hb and ESR in HIV-infected individuals. METHODS: The study population comprised of 215 antiretroviral treatment naïve HIV-1 infected adults. The CD4 positive T cell counts, TLC, Hb and ESR of study participants were measured. Spearman's rank order correlation and Receiver Operating Characteristic were used for statistical analyses. RESULT: The sensitivity, specificity, positive and negative likelihood ratios for cut-off value of TLC <1200/mm (3) for predicting CD4 counts <200 cells/mm (3) and <350 cells/mm (3) were 9.4 %, 100 %, not measurable and 1.1, and 6.1 %, 98.8 %, 5.13 and 0.95, respectively. The association of Hb (<10,11,12 g/dl and <10,12,14 g/dl for CD4 counts <200 cells/mm (3) and <350 cells/mm (3) , respectively), and ESR (<10, 20 and 30 mm fall after 1 hour) with these two CD4 counts cut-off values were suboptimal. CONCLUSION: This study reveals the poor association of TLC, Hb, and ESR with CD4 counts in HIV infected adults, thus highlighting the need to review the utility of these surrogate markers, for predicting CD4 counts in people living with HIV/AIDS.

Nodular regenerative hyperplasia of liver.

Nodular regenerative hyperplasia of the liver (NRHL) is a very rare cause of portal hypertension and liver failure. The condition is characterized by diffuse micronodular transformation of hepatic parenchyma without fibrous septa between the nodules. We present our experience with a 32-year-old woman who presented with recurrent episodes of upper gastrointestinal bleeding associated with massive splenomegaly who was subsequently found to have NRHL. This article considers the salient aspects of this rare condition, how it affects the patients and the options available in its management. A plea is made for the need for liver biopsy for all patients with portal hypertension especially those being considered for surgery.

Seroprevalence of Borrelia burgdorferi in North Eastern India.

BACKGROUND: There is paucity of data on Lyme disease in India. A seroprevalence study of B burgdorferi infection was carried out in North-Eastern states of India to assess the same. METHODS: Sera from 500 individuals of North-Eastern states of India were tested for IgG antibody by enzyme linked immunosorbent assay using commercial kits containing recombinant antigen. RESULT: Out of 500 persons, 65 (13%) were positive for B burgdorferi specific lgG Females showed higher positivity rate as compared to males (15.86% vs 10.95%). Higher prevalence rate was observed in the age group of 15-30 years in both sexes (11.48% in male and 18.69% in female). Arunachal Pradesh showed higher seroprevalence rate (17.8%) as compared to other North-Eastern states (8.46-9.6%). CONCLUSION: Seropositivity to B burgdorferi suggests infection by the organism and presence of Lyme disease in these areas. Further population and vector biology studies are required to find out the exact species involved in transmission of the organism.

Detection of Bacterial Pathogens in Cerebrospinal Fluid using Restriction Fragment Length Polymorphism.

BACKGROUND: Polymerase chain reaction (PCR) is useful for rapid microbial detection in body fluids with low microbial load. It is easier to use universal or broad range primers for the amplification of conserved stretches of DNA common to all bacteria like 16S rRNA gene, followed by restriction fragment length polymorphism (RFLP) of PCR products. METHODS: Forty samples of cerebrospinal fluid were collected. After DNA extraction, universal or broad range PCR was performed using two universal primers U1-5'-CCAGCAGCCGCGGTAATACG-3', corresponding to nucleotides 518 to 537 of the Escherichia coli 16S rRNA gene, and U2 - 5'-ATCGG(C/T)TACCTTGTTACGACTTC-3', corresponding to nucleotides 1513 to 1491 of the same gene. The PCR product was subjected to digestion by endonucleases- HaeIII, Mn11, BstB1 and Alu1. Restriction pattern obtained was compared with that of standard organisms to identify the pathogen. The results were compared with conventional methods. RESULT: Universal PCR could detect pathogens in 20% samples within 13-18 hours as compared to 16% by conventional methods. The analytical sensitivity was 10 Gram negative and 250 Gram positive organisms per 200 µl sample. Overall sensitivity was 83.3% and specificity was 91.2%. CONCLUSION: Universal PCR followed by RFLP of PCR product is a good alternative to conventional diagnosis of bacterial pathogens.

Hepatitis G Virus Infection in Healthy Individuals, Acute Viral Hepatitis and Persons at Risk for Parenteral Transmission.

BACKGROUND: Hepatitis G virus (HGV), transmitted mostly by parenteral route, has been under investigation for its role as an agent for viral hepatitis. This study was carried out to find out the prevalence of HGV in healthy individuals, multi-transfused patients with acute viral hepatitis and those under going dialysis. METHOD: The study included 200 healthy individuals and 180 patients, comprising acute viral hepatitis (100 cases), multi-transfused patients (50 cases) and patients undergoing dialysis (30 cases). HGV RNA and Hepatitis C virus (HCV) RNA was detected by reverse transcription and polymerase chain reaction (RT-PCR) in all. Viral marker studies for hepatitis A, B and E were carried out by ELISA in acute viral hepatitis cases. In healthy individuals, in patients with multiple transfusions or those undergoing dialysis, marker studies for HBV and HCV were carried out. RESULT: The prevalence of HGV in healthy individuals was 2.5% (5/200), in non A-E hepatitis 3% (3/100), in multi-transfused patients 4% (2/50) and in patients undergoing dialysis 6.67% (2/30). There was no significant difference in the prevalence rate of HGV infection in healthy individuals and in patients with non A-E hepatitis. CONCLUSION: Depending on prevalence rate, HGV could not be implicated as cause of acute viral hepatitis. Persons with parenteral risk factor (multiple blood transfusions and those undergoing dialysis) had higher prevalence rate as compared to healthy individuals.

Problems in Diagnosis of HIV Infection in Babies.

Serological diagnosis of human immunodeficiency virus (HIV) infection in babies born to HIV infected mothers is difficult because of presence of maternal anti-HIV antibody up to 18 months. Conventional enzyme-linked immunosorbent assay (ELISA) and western blot may be positive in un-infected cases. Various other modalities which have been adopted include detection of HIV specific IgA, IgM, IgE, detection of p24 antigen, viral culture and detection of HIV nucleic acid by polymerase chain reaction (PCR). Viral culture or PCR positivity within first 48 hours of life indicates intrauterine infection. An early diagnosis of HIV infection in babies born to HIV infected mothers is essential as definite antiretroviral therapy (ART) can be instituted and unnecessary toxicity of drug therapy avoided if found negative. Though viral culture and DNA-PCR has sensitivity of >95% after one month of age, some cases can not be diagnosed during this period. Other tests like viral RNA detection by reverse transcription polymerase chain reaction (RT-PCR) and combination of tests will be required.

Hepatitis G Virus: Prevalence in Blood Donors in Armed Forces.

BACKGROUND: A new RNA virus designated hepatitis G virus (HGV) was recently identified. Because HGV has less than 25% sequence or amino acid homology with hepatitis C virus (HCV) and other established Flaviviridae, it is considered to be a new genus in this growing family of hapatotropic viruses. Hepatitis G virus has been associated with hepatitis and is transmitted through parenteral and sexual route. MATERIAL AND METHODS: A study comprising 500 healthy voluntary blood donors (service personnel) was under taken to find out prevalence of HGV. HGV RNA was detected by reverse transcriptase - polymerase chain reaction (RT-PCR). Hepatitis B surface antigen (HbsAg) and antibody to HCV were detected by Enzyme Linked Immunosorbent Assay (ELISA). RESULTS: Thirteen donors (2.6%) were positive for HGV RNA. 17 donors (3.4%) were positive for antibody to hepatitis C virus (HCV) by ELISA. Co-infection of HGV with hepatitis B virus (HBV) was seen in 5 donors and with HCV infection in 2 donors. Co-infection of HGV, HBV and HCV was not seen in any donor. CONCLUSION: So far there is no conclusive evidence that HGV produces hepatitis. But presence of HGV in hepatitis cases casts a doubt on this finding. Prevalence rate in blood donors may be helpful in future studies when the exact role of HGV is known.

Incidence and Drug Susceptibility pattern of Mycobacterium tuberculosis in HIV infected Patien.

Incidence of HIV infection in patients with tuberculosis was found out by serological method (ELISA) with confirmation by Western Blot analysis. Out of 2116 tuberculosis patients tested for HIV, 150 cases were found to be positive for HIV infection (5.73%). Drug susceptibility to first line antitubercular drugs was carried out in 1378 isolates from HIV negative cases and 68 isolates from AIDS cases. The overall resistance pattern to one or more drugs was seen in 13.78% of isolates from HIV negative cases as compared to 7.2% isolates in AIDS cases. Multidrug resistance (MDR) was seen in 8.9% of isolates from HIV negative cases as compared to 4.4% of isolates from AIDS cases.

Epidemiological Differentials of Hepatitis B Carrier State in the Army : A Community Based Sero-epidemiological Study.

BACKGROUND: Most available studies on seroprevalence of Hepatitis B in the Armed Forces and also at the national level are based on hospital patients and blood donors. Hence, there was a perceived need to undertake a seroepidemiological study on an adequately large and representative random sample of the general cross section of Army personnel, with a view to obtain the exact picture of the frequency and distribution of HBV in the Army. METHODS: A community based cross sectional study with random samples from four groups were drawn, viz recruits from the Army Medical Corps (AMC) and other Arms and Services; AMC personnel and personnel from other Arms and Services who had served for more than 10 years. A structured pretested questionnaire was administered to all participants and blood samples were drawn aseptically subsequently, with separation of serum and testing by ELISA technique for HBsAg. Multivariate analysis using multiple logistic regression procedure was done after appropriate data entry. RESULTS: The overall seroprevalence was 7.9% (95%CI = 6.5% to 9.26%). The differential seroprevalence in the four groups being 7.72%, 7.92%, 8.28% and 7.75% respectively. There was statistically no significant difference as regards the seroprevalence levels [p > 0.05]. As regards serving medical personnel, the seroprevalence was observed to be higher among personnel involved in direct nursing care. On multiple logistic regression analysis, two risk factors emerged as independent and significant predictors of hepatitis B positivity. These were history of sexual exposure with commercial sex workers (CSWs) (OR = 3.06, p < 0.01) and history of having taken injections from civil sources (OR = 1.92, p < 0.001). CONCLUSION: The relatively high level of seroprevalence among recruits has led to certain recommendations on testing and further studies in specific groups, based on the findings of the study.

Potency Titration of Oral Polio Vaccine by Estimation of Live Virus Content Using Tissue Culture Technique.

Representative vaccines from 34 different batches of oral log10 (6.425) and a minimum titre of polio vaccine (OPV) were tested for potency by tissue culture technique. All 34 samples were found to be potent, a maximum titre of log10 (6.425) and a minimum titre of log10 (5.86) was obtained. In addition, 6 vaccines from the immunisation clinic (left over sample after immunisation) were also subjected to potency titration and their potency was found to be within normal limits. Adequate potency confirmed ideal storage condition of the vaccines in Armed Forces set up.

Early Diagnosis of Human Immunodeficiency Virus Infection by p24 Antigen Detection.

p24 antigen was estimated in human immunodeficiency virus (HIV) sero-negative individuals attending various sexually transmitted diseases (STD) clinics and also in sero-negative voluntary blood donors. A total of 300 STD cases and 500 voluntary blood donors, who also acted as controls, were included in this study. Antibody to HIV was detected by ELISA and was confirmed by western blot. In sero-negative individuals, p24 antigen detection was carried out by standard assay and immune-complex dissociation assay (ICD assay) using ELISA method and confirmation was done by neutralisation assay. In voluntary blood donors, 4 (0.8%) individuals were found to be HIV positive and no sero-negative individual was positive for p24 antigen. 41 out of 300 patients attending STD clinics were found to be positive for HIV and in 259 sero-negative patients, p24 antigen was detected in 6 (2.3%) cases by ICD assay whereas only 4 cases were detected by standard assay. By estimating p24 antigen an additional 2.3% HIV positive cases that were in window period were detected. Further, an ICD assay improves the detection of p24 positive individuals.

TO STUDY INCIDENCE OF CHLAMYDIAL GENITAL TRACT INFECTIONS USING ENZYME IMMUNO ASSAY-ANTIGEN DETECTION AND CELL CULTURE METHODS.

Endocervical swabs from 315 patients were screened for chlamydial infection by using Enzyme Immuno Assay technique for antigen detection. Of these, 190 patients were of infertility and 125 patients were with history suggestive of pelvic inflammatory disease (PID). 100 age matched controls were also screened for the detection of chlamydial antigen by using EIA. The overall incidence of chlamydial infection in this study group was 15.2%. 21 (11.05%) of the infertility patients and 27 (21.6%) of the pelvic inflammatory disease cases were found to be positive for chlamydial antigen. The prevalence rate was found to be high in the age group of 31-40 years in both study groups i.e. infertility group (14.7%) and PID group (50%). All the ELISA positive cases (48) and randomly selected (10) age matched controls were screened by tissue culture using McCoy cell line. In the tissue culture, 44 of the 48 samples were found to be positive and none of the controls groups were found positive. 4 samples showed discordant results possibly due to the presence of non-viable organism or inhibitory material present at the sample site. The sensitivity and specificity of ELISA with respect to tissue culture are 100% and 71% respectively. The positive predictive value and the negative predictive value of the ELISA are 91.6% and 100% respectively. The efficiency of the test was found to be 93.1%.