Prothrombotic state in HIV: A study on protein C, protein S, homocysteine and correlation with CD4 counts

Introduction: Human immunodeficiency virus (HIV) may result in variable haematological manifestations. Thrombotic events are more common among HIV-infected persons than the general population, possibly due to the increased inflammatory/hypercoagulable state and presence of concurrent comorbidities. Aims and Objectives: (1) Screen for coagulation abnormalities in HIV-infected patients. (2) Detect certain prothrombotic factors such as deficiency of protein C and protein S and elevation of homocysteine as possible precursors of coagulation defects in HIV patients. (3) Correlation of coagulation abnormalities with CD4 counts. Methods: A pilot study of 1-year duration conducted in the Department of Pathology in collaboration with ART centre, KGMU Lucknow. All diagnosed HIV-seropositive patients (n = 30) who were not taking Vitamin K, antithrombotic and antiplatelet drugs including aspirin, oral contraceptives and not having known protein C/S deficiency were included in the present study as cases. Apart from this, 30 age- and sex-matched healthy individuals were also included in the present study. Assessment of the bleeding time, prothrombin time and activated partial thromboplastin time, complete blood count was done. Protein C and S were measured by calorimetric assay. Serum homocysteine was measured by the semi-automated method. CD4 count was done by flow cytometry. Results: The findings of the present study suggest a relationship between HIV, its complications and thrombosis. The HIV-seropositive patients have reduced levels of haemoglobin, CD4 counts, platelet counts, mean platelet volume, protein C and S activity as compared to the healthy individuals. Thrombophilic abnormality in the form of hyperhomocysteinaemia is more frequent in HIV-infected patients. All these parameters have a definite correlation with CD4 count.

Persistence of Corynebacterium diphtheriae in Delhi & National Capital Region (NCR).

Despite the introduction of mass immunization, diphtheria continues to play a major role as a potentially lethal infectious disease in many countries. Delay in the specific therapy of diphtheria may result in death and, therefore, accurate diagnosis of diphtheria is imperative. This study was carried out at National Centre for Disease Control (NCDC), Delhi, India, on samples of suspected diphtheria cases referred from various government hospitals of Delhi and neighbouring areas during 2012-2014. Primary identification of Corynebacterium diphtheriae was done by standard culture, staining and biochemical tests followed by toxigenicity testing by Elek’s test on samples positive for C. diphtheriae. The results showed persistence of toxigenic C. diphtheriae in our community indicating the possibility of inadequate immunization coverage.

Screening and isolation of halophilic bacteria producing industrially important enzymes

Halophiles are excellent sources of enzymes that are not only salt stable but also can withstand and carry out reactions efficiently under extreme conditions. The aim of the study was to isolate and study the diversity among halophilic bacteria producing enzymes of industrial value. Screening of halophiles from various saline habitats of India led to isolation of 108 halophilic bacteria producing industrially important hydrolases (amylases, lipases and proteases). Characterization of 21 potential isolates by morphological, biochemical and 16S rRNA gene analysis found them related to Marinobacter, Virgibacillus, Halobacillus, Geomicrobium, Chromohalobacter, Oceanobacillus, Bacillus, Halomonas and Staphylococcus genera. They belonged to moderately halophilic group of bacteria exhibiting salt requirement in the range of 3-20%. There is significant diversity among halophiles from saline habitats of India. Preliminary characterization of crude hydrolases established them to be active and stable under more than one extreme condition of high salt, pH, temperature and presence of organic solvents. It is concluded that these halophilic isolates are not only diverse in phylogeny but also in their enzyme characteristics. Their enzymes may be potentially useful for catalysis under harsh operational conditions encountered in industrial processes. The solvent stability among halophilic enzymes seems a generic novel feature making them potentially useful in non-aqueous enzymology.

Pandemic Influenza A (H1N1) 2009 in India: Duration of virus shedding in patients under antiviral treatment.

Background: National Centre for Disease Control (NCDC), Delhi, is a national nodal centre for surveillance of pandemic Influenza A (H1N1) in India. The present study was undertaken to see the period of infectivity in positive cases undergoing antiviral therapy. Objective: To assess the duration of virus shedding by real-time polymerase chain reaction (real-time PCR) in some of the positive patients taking Oseltamivir treatment. Materials and Methods: Clinical samples (throat swabs, nasal swabs and nasopharyngeal swabs) collected by the clinicians from patients quarantined in government hospitals in different parts of India are being sent to the designated reference laboratory at Delhi for screening presence of pandemic Influenza virus. The samples are tested by Real-Time PCR using CDC recommended reagents and protocol for confirmation of the H1N1 novel influenza virus. In 150 of the positive cases, we requested the clinicians to send samples for 5 consecutive days after administration of antiviral therapy, to see the trend of therapy response on viral shedding. Samples for more than 5 days were received from patients till they showed no amplification for any of the three target genes (Influenza A, Swine Influenza A or Swine H1). Results and Conclusion: In 99.33% (149/150) cases, the influenza infection resolved within 10 days. Sixty-four percent (96/150) of the positive patients turned negative within 5 days of the start of antiviral treatment. Only one patient belonging to high risk group showed prolonged virus shedding (19 days).

Newborn with tail - a genetic throwback.

The birth of a child with a caudal appendage resembling a tail generates an unusual interest and anxiety. True human tail is a rare event; less than 40 cases have been reported in the literature so far. It is defined as a caudal, vestigial, midline protrusion of muscle and adipose tissue with skin covering. We are reporting a case of true tail in a baby, a rare event in human.

Characterization of RPO B gene for detection of rifampicin drug resistance by SSCP and sequence analysis.

Purpose: Because of the emergence of multidrug-resistant tuberculosis in recent times, the rapid detection of resistance to the first-line anti-tuberculosis drug rifampicin was felt worldwide. Accordingly, this study was conducted to evaluate the diagnostic potential of polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) for checking its utility as a rapid screening test for determination of rifampicin drug resistance. Materials and Methods: A total of 34 isolates of Mycobacterium tuberculosis ( M. tuberculosis ) (22 rifampicin resistant, 11 rifampicin sensitive and one control H37Rv) strains were analysed by PCR-SSCP and DNA sequencing within the 157-bp region of the rpo B gene (Ala 500 -Val 550 ). Results: Rifampicin resistance was detected successfully by PCR-SSCP in 20/22(90.90%) of rifampicin-resistant strains showing a total of nine different mutations in seven codon positions: codon 513 (CAA→CCA), 516 (GAC→GTC), 507 (GGC→GAC), 526 (CAC→GAC, TAC), 531 (TCG→TTG, TGG), 522 (TCG→TGG) and 533 (GTG→CCG). Two rifampicin-resistant strains showed an identical PCR-SSCP pattern with the wild type H37Rv; 77.27% rifampicin-resistant strains showed a single point mutation and 9.09% had no mutation. Three rifampicin-resistant strains showed characteristic double mutations at codon positions 526 and 531. Sensitivity and specificity were calculated as 90.90% and 100%. Conclusions: Rifampicin-resistant genotypes were mainly found in codon positions 516, 526 and 531. PCR-SSCP seems to be an efficacious method of predicting rifampicin resistance and substantially reduces the time required for susceptibility testing from 4 to 6 weeks to a few weeks.

Congenital CMV infection; diagnosis in symptomatic infants.

Background: Samples from babies exhibiting clinical symptoms suggestive of congenital infection are referred regularly to NICD, New Delhi,, from Government Hospitals located in Delhi and a home for abandoned children (Palna), for the diagnosis of etiological agents like toxoplasma, rubella, CMV and herpes. Blood samples of mothers of most of the affected babies are also received. Objective: Evaluation of rapid and accurate technique for the diagnosis of congenital CMV infection. Materials and Methods: One hundred and twenty five blood samples suggestive of symptomatic congenital CMV infection were selected from samples received at NICD during the period June 2005-March 2007. A request to collect and send the urine samples of the selected babies was sent to the respective hospitals. Serum samples of the babies were tested for CMV-IgM antibodies using µ-capture ELISA. Mothers' serum samples were subjected to CMV-IgM and IgG class antibodies assay by commercial ELISA kits. DNA isolation and amplification was performed in urine samples and some of the serum samples using a commercial PCR kit for detection of HCMV. Blood and urine samples from 20 normal babies were included in the study. Results: Twenty Seven serum samples (21.6%) of infants, of the 125 tested, were positive for CMV-IgM antibodies. Twenty five samples (20%) showed amplification of CMV -DNA. All 25 samples positive for PCR were positive for CMV IgM antibodies. Sera of 73 mothers, out of 75 tested (97.3%), were positive for CMV IgG antibodies. However, none of them was positive for CMV IgM antibodies. Mothers of all 27 positive babies were positive for CMV-IgG antibodies. Serum and urine samples from 20 normal babies were negative for ELISA and PCR. Conclusion: µ-capture ELISA technique was found to be more sensitive than PCR (92.6%) for detection of congenital CMV infection. ELISA is also rapid, less cumbersome and cost effective for diagnosis of CMV infection.

Diagnostic potential of IS6110, 38kDa, 65kDa and 85B sequence-based polymerase chain reaction in the diagnosis of Mycobacterium tuberculosis in clinical samples.

PURPOSE: The correlation between the presence of specific gene sequence of M. tuberculosis and specific diagnosis of clinical tuberculosis is not known. This study compared the results of polymerase chain reaction (PCR) amplification of M. tuberculosis specific DNA sequences (IS6110, 65kDa, 38kDa and mRNA coding for 85B protein) from different clinical samples of pulmonary and extrapulmonary tuberculosis. METHODS: One hundred and seventy-two clinical samples from suspected tuberculosis patients were tested for smear examination, culture (LJ and rapid BACTEC 460 TB system) and PCR. PCR was performed with specific primers for the targets: IS6110, 65 kDa, 38 kDa and 85 B. RESULTS: Each PCR test was found to have a much higher positivity than conventional test and BACTEC culture (P < 0.05). Smear positive samples (56) and the samples (36) showing positive results by conventional methods (smear and LJ medium culture) and BACTEC were found to be positive by all PCR protocols. No significant difference was found between the four PCR protocols (P> 0.05). The primer specific for amplifying the 123bp IS6110 fragment gave the highest positivity (83%), followed by 65kDa, 38kDa and 85B RT-PCR in descending order. CONCLUSIONS: These data suggest that the presence of IS6110 correlates more closely with the diagnosis of clinical tuberculosis than that of 65kDa, 38kDa and 85B proteins.

Comparison of various microbiological tests including polymerase chain reaction for the diagnosis of osteoarticular tuberculosis.

PURPOSE: To evaluate the utility of the polymerase chain reaction (PCR) test for diagnosing osteoarticular tuberculosis (TB). METHODS: Clinical samples (synovial tissue and synovial fluid) obtained from 23 cases of suspected osteoarticular tuberculosis were subjected to Ziehl Neelsen (ZN) smear examination, radiometric BACTEC culture and PCR test for tuberculosis by amplifying 65 kDa antigen coding region of Mycobacterium tuberculosis (M.tb) genome. RESULTS: PCR test was found to be much sensitive than the ZN smear examination and BACTEC culture (p<0.05) in the diagnosis of osteoarticular TB. In synovial fluid samples, PCR was positive in 73.9%, ZN smear examination in 17.39% and BACTEC culture in 39.13% of cases. The positivities were relatively lower with synovial tissue samples, the corresponding figures being 60.8, 8.6 and 26.08% respectively. Moreover, on combining the results of synovial fluid and tissues, the corresponding figures further increased to 78.2, 21.7 and 43.3% respectively. Further, sensitivity and specificity for PCR employing BACTEC culture as the "gold standard" was 100% respectively. Using BACTEC culture, the earliest positivity was seen in three days using synovial tissue specimen and 13 days with synovial fluid, the average detection times being 23.2 days and 32.6 days respectively. On the other hand, PCR test gave a positive result within 24 hours. CONCLUSIONS: PCR test was shown to be much more sensitive than ZN smear examination and BACTEC culture test for diagnosing osteoarticular tuberculosis.

Seroprevalence and incidence of rubella in and around Delhi (1988-2002).

National Institute of Communicable Diseases (NICD) has been engaged in rubella testing for serodiagnosis of the infection and screening for immunity status. The compiled and evaluated data of the work done on rubella testing for the past fifteen years has been presented here to show the trend and changing scenario of the disease in Delhi. Blood samples were from 7424 patients referred to NICD, Delhi for serodiagnosis of congenital Rubella syndrome (CRS) in malformed babies, in utero rubella infection in women and immunity status of pregnant women and women with bad obstetric history. They were tested for rubella IgG and/or rubella IgM antibodies using commercially available reagents and kits. The data from the 15 years of testing was then compiled and evaluated. From the available data it was seen that immunity status against rubella in childbearing age group of women increased steadily from 49% in 1988 to 87% in 2002. Reported cases of CRS at NICD are also on the decline over the time period. There is periodic indication of high incidence of rubella in the year 1988; 1991 and 1998 as the reported cases of acute rubella infection in childbearing age group is high during these years.

Comparison of the conventional diagnostic modalities, bactec culture and polymerase chain reaction test for diagnosis of tuberculosis.

PURPOSE: To evaluate the performance of 65 kDa antigen based PCR assay in clinical samples obtained from pulmonary and extrapulmonary cases of tuberculosis. METHODS: One hundred and fifty six samples were processed for detection of Mycobacterium tuberculosis by ZN smear examination, LJ medium culture, BACTEC radiometric culture and PCR tests. RESULTS: A significant difference was seen in the sensitivities of different tests, the figures being 74.4% for PCR test, 33.79% for ZN smear examination, 48.9% for LJ culture and 55.8% for BACTEC culture (P< 0.05). However, there was no significant difference (P>0.05) as far as specificity of different tests was concerned. PCR test sensitivity in pulmonary and extrapulmonary clinical samples were 72.7% and 75.9% respectively and found to be significantly higher (P< 0.05) when compared with those of other tests. The mean detection time for M.tuberculosis was 24.03 days by LJ medium culture, 12.89 days by BACTEC culture and less than one day by PCR test. CONCLUSIONS: PCR is a rapid and sensitive method for the early diagnosis of pulmonary and extrapulmonary tuberculosis.

Sensitivity index of antimicrobial agents: A new treatment criteria proposed for rational use of antimicrobials.

The treatment guidelines are generally decided on the basis of either percent resistant (%R) or percent sensitive (%S) bacterial population tested with a given antimicrobial that vary geographically and represent only a part of total bacterial population existing in response to the antimicrobial used. The isolates with intermediate sensitivity (%I) are either not reported or clubbed with resistant isolates though the two may differ in clinical response. Sensitivity Index (SI) of an antimicrobial is sensitive to change in any of the three co-existing bacterial population and may be a better criterion for rational use of antimicrobial.

Anthropometric profile and perinatal outcome of babies born to young women (< 18 years).

This study was conducted to ascertain the sociodemographic profile, maternal characteristics, neonatal anthropometry and perinatal outcome in adolescent pregnancy (18 years or less). 128 consecutive primiparous women more than 18 years of age served as the control group. There were 4556 deliveries during the study period. Young adolescents accounted for 1.25% of total pregnancies. The proportion of shorter (<145 cm), lighter (<45 Kg), and anemic (Hb. <9 g/dL) women was significantly higher in the study group. Incidence of premature delivery in the young adolescents was significantly higher. Mean birth weight, length, head circumference and chest circumference of full-term babies of adolescent mothers were significantly lower.

Antimicrobial susceptibility testing in India - A status survey.

Resistance to a variety of antimicrobial agents is emerging in bacterial pathogens throughout the world. Since the accuracy of the antimicrobial susceptibility data is associated with the performance standard of the test, strict adherence to the standard procedures is essential. The Kirby-Bauer disc diffusion susceptibility test, performed in accordance to NCCLS method gives reliable results and hence predicts clinical efficacy of the antibiotic tested. To assess the standard of performance of the antimicrobial susceptibility test, a survey was conducted by National Institute of Biologicals during 1999-2000. The findings indicated an urgent need of setting up a national quality control laboratory to provide the performance standards, reference Q.C. strains and quality antibiotic discs to ensure reproducible and reliable results.

Antiallergic/antiasthmatic effect of novel antiallergic hexapeptide-95/220 in various experimental models.

The effects of newly synthesized antiallergic hexapeptide 95/220 was investigated on various allergic and asthmatic test models. This newly developed peptide was found to be more potent than clinically used drug disodium cromoglycate (DSCG). Hexapeptide 95/220 inhibited immediate hypersensitivity reactions such as passive cutaneous anaphylaxis (PCA) and mast cell degranulation in rats, antigen-induced bronchoconstriction in actively sensitized guinea pigs in dose dependent manner like DSCG. Antigen-induced contraction of guinea pig ileum was also markedly inhibited by this newly developed hexapeptide in the same fashion as ketotifen and DSCG did but at comparatively lower dose. Egg albumin-induced histamine release was also blocked by this hexapeptide from chopped lung tissues of sensitized guinea pigs. These results suggest that hexapeptide' 95/220 has potent inhibitory effect on immediate hypersensitivity reactions thereby inhibiting mediator release from mast cell. Moreover, this newly synthesized peptide is orally active and effective at lower doses as compared to standard drugs.

Community studies on prevalence of HBsAg in two urban populations of southern India.

OBJECTIVE: To find out prevalence of HBsAg in general population, especially in under-five children. SETTING: Bangalore and Rajahmundry towns in southern India. METHODS: Localities were chosen as the sampling units in each town. About 10-20 households were randomly selected from each locality. Only the youngest but apparently healthy person present in the household was interviewed for age, sex and history of jaundice any time in life. Mothers were interviewed to collect data for children below 15 years of age. Blood samples were collected from these persons on filter paper strips (18-mm diameter disc, Whatman filter paper No. 3) by finger prick method. The samples were tested for HBsAg by Micro ELISA (Ortho-Clinical Diagnostics). RESULTS: Overall, 3.3% (95% CI, 2.0-4.5) of 737 persons in Rajahmundry and 4.2% (95% CI, 2.8-5.5) of 816 persons in Bangalore were found carriers of HBsAg. Age-specific or sex specific carrier rates were similar in Rajahmundry as well as in Bangalore. Most of the carriers (96%) denied having jaundice ever in life. CONCLUSIONS: The results from this community based study are in agreement with the historical data from hospital based studies that about 3-5% of persons may be carriers of HBsAg and that the pool of chronic carriers of hepatitis B virus in India is built up in childhood and is then maintained in older children and adults. The results highlight the need of completing hepatitis B immunization during the infancy.

Epidemiological characteristics of poliomyelitis in Delhi, 1997.

OBJECTIVE: To describe the epidemiological characteristics of poliomyelitis in Delhi in 1997 after four consecutive statewide immunization campaigns with oral polio vaccine (OPV). METHODS: Stool samples were collected from 158 cases of acute flaccid paralysis (AFP) along with their age, sex, residential address, immunization history and dates of onset of paralysis, reporting and investigation. The samples were processed for isolation of polioviruses. In addition, historical data on vaccination coverage surveys and OPV testing were reviewed. These data were analyzed to understand the epidemiological patterns of poliomyelitis in Delhi. RESULTS: Of 158 cases of AFP, about 23% were investigated within 2 days of onset of paralysis. Two samples each were collected from 97 (61%) cases, and one each from the remaining cases. Detection of 158 cases of AFP gave an incidence of 1.34 per 100,000 population. About 36% (57/158) of AFP cases excreted poliovirus, mostly (53/158) wild poliovirus. Of the wild poliovirus isolates, 72% (38/53) and 25% (13/53) were serotypes P1 and P3 respectively; 2 isolates were P2. Almost 95% (146/154) of AFP cases and all the laboratory confirmed cases (excreting wild poliovirus) occurred in children below 5 years of age. Only one-third of AFP (55/158) or laboratory confirmed cases (18/53) had received 3 or more doses of OPV before onset of paralysis. About one-fourth of cases in both the categories were totally unvaccinated. AFP cases occurred round the year but peaked in November-December. Peaks were always observed during July-August in the past. The cases were widely scattered without any obvious clustering in any locality. CONCLUSIONS: Poliomyelitis has declined substantially in Delhi. The study underscores the need for further efforts to improve vaccine coverage levels, AFP surveillance, and cold chain maintenance to achieve the complete interruption of transmission.

Widespread outbreaks of measles in rural Uttar Pradesh, India, 1996: high risk areas and groups.

OBJECTIVE: To describe outbreaks of measles which affected many districts in Uttar Pradesh (UP) during 1996. DESIGN: Outbreak investigations. SETTING: The state of Uttar Pradesh, India. METHODS: The reported data on measles morbidity, mortality and vaccine coverage from 1991 through 1996 were reviewed. Reported vaccine coverage levels were compared with the results of coverage surveys carried out in UP from 1992 through 1996. Line lists on measles cases were analyzed to ascertain the age, immunization status, geographical distribution, and age and sex-specific fatality ratios during the outbreaks. A community survey was organized in 7 affected villages to estimate vaccine effectiveness. RESULTS: Fifty one of 68 districts in UP reported 6922 measles cases and 281 deaths in 1996. The majority of cases and deaths occurred in June and July which are usually low transmission months. Overall cases fatality ratio (CFR) was 4.1%. CFRs were significantly higher in females and young children. The median age of cases was found to be below 5 years. There was heavy clustering of cases and deaths in rural areas. About 85% of the cases and virtually all the measles associated deaths occurred in unvaccinated children. Published documents on statewide coverage surveys revealed that the measles vaccine coverage levels ranged between 26% and 36% during 1992-96. Large gaps were found between reported coverage and survey results. Nevertheless, epidemiological studies indicated a vaccine effectiveness of more than 90%. CONCLUSIONS: The outbreaks occurred due to poor vaccine coverage levels and an inefficient surveillance system which failed to generate early warning signals. The study highlights the urgent need to raise the vaccine coverage levels rapidly in all districts to achieve measles control and prevent future outbreaks in UP.