Case based measles surveillance in Pune: evidence to guide current and future measles control and elimination efforts in India.

BACKGROUND: According to WHO estimates, 35% of global measles deaths in 2011 occurred in India. In 2013, India committed to a goal of measles elimination by 2020. Laboratory supported case based measles surveillance is an essential component of measles elimination strategies. Results from a case-based measles surveillance system in Pune district (November 2009 through December 2011) are reported here with wider implications for measles elimination efforts in India. METHODS: Standard protocols were followed for case identification, investigation and classification. Suspected measles cases were confirmed through serology (IgM) or epidemiological linkage or clinical presentation. Data regarding age, sex, vaccination status were collected and annualized incidence rates for measles and rubella cases calculated. RESULTS: Of the 1011 suspected measles cases reported to the surveillance system, 76% were confirmed measles, 6% were confirmed rubella, and 17% were non-measles, non-rubella cases. Of the confirmed measles cases, 95% were less than 15 years of age. Annual measles incidence rate was more than 250 per million persons and nearly half were associated with outbreaks. Thirty-nine per cent of the confirmed measles cases were vaccinated with one dose of measles vaccine (MCV1). CONCLUSION: Surveillance demonstrated high measles incidence and frequent outbreaks in Pune where MCV1 coverage in infants was above 90%. Results indicate that even high coverage with a single dose of measles vaccine was insufficient to provide population protection and prevent measles outbreaks. An effective measles and rubella surveillance system provides essential information to plan, implement and evaluate measles immunization strategies and monitor progress towards measles elimination.

Progress toward laboratory containment of poliovirus after polio eradication.

BACKGROUND: The first steps (phase 1) toward laboratory containment of poliovirus after eradication are a national survey of biomedical facilities and a global inventory of such facilities retaining wild poliovirus (WPV) infectious and potentially infectious materials. METHODS: We reviewed published reports on national laboratory surveys and inventories of WPV materials from each of the 3 polio-free World Health Organization (WHO) regions (the European Region, completed in 2006; the Western Pacific Region, completed in 2008; and the Region of the Americas, completed in 2010), as well as reports on progress in polio-free countries of the remaining 3 regions (the African Region, the Eastern Mediterranean Region, and the WHO South-East Asia Region). RESULTS: Containment phase 1 activities are complete in 154 of 194 WHO Member States (79%), including all countries and areas of the polio-free regions and most polio-free countries in the remaining 3 regions. A reported 227 209 biomedical facilities were surveyed, with 532 facilities in 45 countries identified as retaining WPV-associated infectious or potentially infectious materials. CONCLUSIONS: Completion of containment phase 1 global activities is achievable within the time frame set by the Polio Eradication and Endgame Strategic Plan 2013-2018.

Improving molecular tools for global surveillance of measles virus.

BACKGROUND: The genetic characterization of wild-type measles viruses plays an important role in the description of viral transmission pathways and the verification of measles elimination. The 450 nucleotides that encode the carboxyl-terminus of the nucleoprotein (N-450) are routinely sequenced for genotype analysis. OBJECTIVES: The objectives of this study were to develop improved primers and controls for RT-PCR reactions used for genotyping of measles samples and to develop a method to provide a convenient, safe, and inexpensive means to distribute measles RNA for RT-PCR assays and practice panels. STUDY DESIGN: A newly designed, genetically defined synthetic RNA and RNA isolated from cells infected with currently circulating genotypes were used to compare the sensitivity of primer pairs in RT-PCR and nested PCR. FTA® cards loaded with lysates of measles infected cells were tested for their ability to preserve viral RNA and destroy virus infectivity. RESULTS: A new primer pair, MeV216/MeV214, was able to amplify N-450 from viruses representing 10 currently circulating genotypes and a genotype A vaccine strain and demonstrated 100-fold increased sensitivity compared to the previously used primer set. A nested PCR assay further increased the sensitivity of detection from patient samples. A synthetic positive control RNA was developed that produced PCR products that are distinguishable by size from PCR products amplified from clinical samples. FTA® cards completely inactivated measles virus and stabilized RNA for at least six months. CONCLUSIONS: These improved molecular tools will advance molecular characterization of circulating measles viruses globally and provide enhanced quality control measures.

Molecular characterization of wild-type measles viruses in Tamil Nadu, India, during 2005-2006: relationship of genotype D8 strains from Tamil Nadu to global strains.

Molecular characterization of measles viruses is a valuable tool for measuring the effectiveness of measles control and elimination programmes. WHO recommends that virological surveillance be conducted during all phases of measles control to document circulation of indigenous strains and trace future importation. This report describes the genetic characterization of wild type measles viruses from Tamil Nadu, India isolated between January 2005 and January 2006. In the study, 304 suspected measles cases (292 from 56 outbreaks and 12 sporadic cases) were investigated. Blood samples were collected from suspected measles outbreaks and 11 suspected sporadic cases and tested for the presence of measles and rubella specific IgM. Based on serological results, 53 outbreaks were confirmed as measles, 2 as a combination of measles and rubella, and 1 negative for both. Eight sporadic cases were confirmed as measles and one as rubella. Throat swab and urine samples were collected for virus isolation and 28 isolates were obtained. Sequencing and analysis showed that 3 isolates belonged to genotype D4 and 25 to genotype D8. Comparison of the genotype D8 sequences from Tamil Nadu with previously reported genotype D8 sequences from India and abroad showed six distinct clusters with Tamil Nadu strains forming two clusters. This study has established baseline molecular data and is the first report that describes genetic diversity of circulating measles strains in Tamil Nadu, a state in India. D8 has multiple lineages and this has been linked with importation of measles into the USA and UK.

South-East Asia Regional update on measles mortality reduction and elimination, 2003-2008.

In 2005, the World Health Assembly endorsed a global goal of 90% reduction in measles mortality from 2000 to 2010. Substantial progress has been made toward achieving this goal in countries of the South-East Asia Region (SEAR). More than 120 million children received a second dose of measles-containing vaccine during supplemental immunization activities conducted from 2000 to 2008; routine first-dose measles-containing vaccine coverage increased from 63% in 2000 to 75% by 2008; and measles surveillance is supported in all countries by the Measles-Rubella Laboratory Network. Overall, the estimated number of measles deaths decreased by 46% from 2000 to 2008, and all countries with the exception of India have already achieved the 90% mortality reduction target. Sustained political and financial commitment from SEAR countries is needed to achieve regional measles mortality reduction and elimination.

Molecular epidemiology of measles in India, 2005-2010.

Measles is a childhood disease that causes great morbidity and mortality in India and worldwide. Because measles surveillance in India is in its infancy, there is a paucity of countrywide data on circulating Measles virus genotypes. This study was conducted in 21 of 28 States and 2 of 7 Union Territories of India by MeaslesNetIndia, a national network of 27 centers and sentinel practitioners. MeaslesNetIndia investigated 52 measles outbreaks in geographically representative areas from 2005 through June 2010. All outbreaks were serologically confirmed by detection of antimeasles virus immunoglobulin M (IgM) antibodies in serum or oral fluid samples. Molecular studies, using World Health Organization (WHO)-recommended protocols obtained 203 N-gene, 40 H-gene, and 4 M-gene sequences during this period. Measles genotypes D4, D7, and D8 were found to be circulating in various parts of India during the study period. Further phylogenetic analysis revealed 4 lineages of Indian D8 genotypes: D8a, D8b, D8c, and D8d. This study generated a large, countrywide sequence database that can form the baseline for future molecular studies on measles virus transmission pathways in India. This study has created support and capabilities for countrywide measles molecular surveillance that must be carried forward.

Expansion of the global measles and rubella laboratory network 2005-09.

Enhancing measles surveillance with integration of epidemiologic and laboratory information is one of the key strategies for accelerated measles control and elimination. The World Health Organization (WHO) Global Measles and Rubella Laboratory Network (LabNet) has been developed since 2000 to currently include 690 laboratories serving 183 countries. The LabNet testing strategy follows well-validated, standardized procedures for confirming suspected cases and for monitoring measles and rubella virus transmission patterns. The strength of the LabNet is a strong quality assurance program that monitors the performance of all laboratories through annual proficiency testing and continuous assessment. In the 5-year period 2005-2009, the results of >1 million measles immunoglobulin M (IgM) tests have been reported by the LabNet and, in addition, sequence information on >7000 measles and 600 rubella viruses has been shared. Progress with the development of the LabNet during 2005-2009 is discussed.

Global distribution of measles genotypes and measles molecular epidemiology.

A critical component of laboratory surveillance for measles is the genetic characterization of circulating wild-type viruses. The World Health Organization (WHO) Measles and Rubella Laboratory Network (LabNet), provides for standardized testing in 183 countries and supports genetic characterization of currently circulating strains of measles viruses. The goal of this report is to describe the lessons learned from nearly 20 years of virologic surveillance for measles, to describe the global databases for measles sequences, and to provide regional updates about measles genotypes detected by recent surveillance activities. Virologic surveillance for measles is now well established in all of the WHO regions, and most countries have conducted at least some baseline surveillance. The WHO Global Genotype Database contains >7000 genotype reports, and the Measles Nucleotide Surveillance (MeaNS) contains >4000 entries. This sequence information has proven to be extremely useful for tracking global transmission patterns and for documenting the interruption of transmission in some countries. The future challenges will be to develop quality control programs for molecular methods and to continue to expand virologic surveillance activities in all regions.

Status of global virologic surveillance for rubella viruses.

The suspected measles case definition captures rubella cases. Therefore, measles surveillance will be improved in the course of the control and eventual elimination of rubella transmission. One aspect of rubella control, virologic surveillance, is reviewed here. A systematic nomenclature for rubella viruses (RVs) based on 13 genotypes has been established and is updated when warranted by increases in information about RVs. From 2005 through 2010, the genotypes of RVs most frequently reported were 1E, 1G, and 2B, and genotypes 1a, 1B, 1C, 1h, 1j, and 2C were less frequently reported. Virologic surveillance can support rubella control and elimination. Synopses of rubella virologic surveillance in various countries, regions, and globally are given, including characterization of viruses from imported cases in a country that has eliminated rubella and studies of endemic viruses circulating in countries without rubella control objectives. Current challenges are discussed.

Evaluation of three commercially available Japanese encephalitis virus IgM enzyme-linked immunosorbent assays.

We evaluated performance of three commercial Japanese encephalitis virus (JEV) IgM antibody capture enzyme-linked immunosorbent assay (MAC ELISA) kits with a panel of serological specimens collected during a surveillance project of acute encephalitis syndrome in India and acute meningitis and encephalitis syndrome in Bangladesh. The serum and cerebral spinal fluid specimens had been referred to the Centers for Disease Control and Prevention (CDC) for confirmatory testing. The CDC results and specimen classifications were considered the reference standard. All three commercial kits had high specificity (95-99.5%), but low sensitivities, ranging from 17-57%, with both serum and cerebrospinal fluid samples. Specific factors contributing to low sensitivity compared with the CDC ELISA could not be determined through further analysis of the limits and dilution end points of IgM detection.

Evaluation of IgM antibody capture enzyme-linked immunosorbent assay kits for detection of IgM against Japanese encephalitis virus in cerebrospinal fluid samples.

Infection with Japanese encephalitis virus (JEV) is a major public health problem in Asia. Detection of JEV-specific IgM in serum and cerebrospinal fluid (CSF) by the IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) is currently the most widely used diagnostic method to detect JEV infection. Because of the possible presence of IgM cross-reactivity with other flaviviruses in serum and the high ratio of inapparent-to-apparent JEV infections, a positive result in serum only suggests a recent infection and not necessarily an encephalitic illness caused by JEV. Consequently, detection of JEV-specific IgM in CSF assumes great diagnostic relevance. We evaluated two commercial JEV MAC-ELISA kits using 60 CSF samples obtained from patients with acute encephalitis syndrome. The Panbio and XCyton kits had sensitivities of 65-80% and 95% and specificities of 90% and 97.5%, respectively. Performance information on these commercial JEV MAC-ELISA kits for CSF should assist in laboratory-based JE surveillance programs.

First detection of measles genotype D7 from India.

National Institute of Virology, India has instituted molecular surveillance of measles strains in the country. In phased manner, three major cities Pune, Chennai and Bangalore were covered. Throat swab and urine from suspected measles cases from Chennai and Pune and freshly frozen brain tissues, CSF from suspected SSPE case from Bangalore were subjected to RNA extraction and Measles N&H gene RT-PCR as per WHO standard protocols. PCR positive products were sequenced. Sequence alignment and phylogenetic analysis was carried out using WHO standard reference sequences. Virus isolation was attempted using B95a cell line. Measles genotype D7 was detected from two classical measles cases (Chennai and Pune) and in a fulminant SSPE case (Bangalore). This is the first detection of measles genotype D7 from India.

Preliminary studies on induction of apoptosis by genistein on HepG2 cell line.

Consumption of soy products has been linked to lower the incidence of number of cancers. Genistein, one of the principal soy isoflavones, has been shown to inhibit the growth of a number of tumor cell lines in vitro. In this study, we investigate the effects of genistein on cell growth and apoptosis in human hepatocellular carcinoma HepG2 cell by looking for the formation of nuclear apoptotic bodies and DNA ladder formation. Additionally, flow cytometry analysis with propidium iodide staining has been conducted to detect the apoptotic cells. We found inhibition of cell growth and apoptotic nuclei, DNA fragmentation and increased apoptotic cells after treatment with genistein, indicating apoptotic cell deaths. From these results we observed that genistein inhibits the growth of HepG2 cells and induce apoptosis, however, further definitive studies are needed. These results may support the potentially effective chemopreventive and/or chemotherapeutic of genistein against liver cancer.

Fenugreek (Trigonella foenum graecum) seed extract prevents ethanol-induced toxicity and apoptosis in Chang liver cells.

The protective effect of a polyphenolic extract of fenugreek seeds (FPEt) against ethanol (EtOH)-induced toxicity was investigated in human Chang liver cells. Cells were incubated with either 30 mM EtOH alone or together in the presence of seed extract for 24 h. Assays were performed in treated cells to evaluate the ability of seeds to prevent the toxic effects of EtOH. EtOH treatment suppressed the growth of Chang liver cells and induced cytotoxicity, oxygen radical formation and mitochondrial dysfunction. Reduced glutathione (GSH) concentration was decreased significantly (P < 0.05) while oxidized glutathione (GSSG) concentration was significantly elevated in EtOH-treated cells as compared with normal cells. Incubation of FPEt along with EtOH significantly increased cell viability in a dose-dependent manner, caused a reduction in lactate dehydrogenase leakage and normalized GSH/GSSG ratio. The extract dose-dependently reduced thiobarbituric acid reactive substances formation. Apoptosis was observed in EtOH-treated cells while FPEt reduced apoptosis by decreasing the accumulation of sub-G1 phase cells. The cytoprotective effects of FPEt were comparable with those of a positive control silymarin, a known hepatoprotective agent. The findings suggest that the polyphenolic compounds of fenugreek seeds can be considered cytoprotective during EtOH-induced liver damage.

Serosurvey of rubella in five blocks of Tamil Nadu.

BACKGROUND & OBJECTIVE: Rubella, normally a mild, self-limiting disease characterized by rash, fever and lymphadenopathy, is a vaccine preventable disease. It carries little morbidity and apparently only minor complications in children. Infection during early pregnancy may lead to congenital rubella infection. Presence of rubella specific IgG in an unvaccinated population is a long term marker of previous rubella infection, which helps to assess the immune status of that population. Though many seroprevalence studies on rebella have been reported earlier from India, no study has been conducted in recent years. We undertook this study in 2003 in five blocks identified by the Integrated Child Development Scheme (ICDS), in the five districts of Tamil Nadu to assess the immune status to rubella in two age groups (1-5 yr boys and girls and 10-16 yr adolescent girls) before vaccination and draw strategies for future vaccination programme. METHODS: A total of 300 blood samples were collected by vein puncture from girls and boys of 1-5 yr age and adolescent girls of 10-16 yr age. Samples were tested for the presence of rubella specific IgG antibody by ELISA. RESULTS: Of the 300 samples tested, 145 (48.3%) were negative for rubella IgG antibodies. The seronegativity was 82.2 per cent in 1-5 yr and 13.5 per cent in the 10-16 yr age groups, the difference was statistically significant (P<0.001). INTERPRETATION & CONCLUSION: Large percentage of children, 82.2 per cent in the 1-5 yr age group and 13.5 per cent in 10-16 yr population were susceptible to rubella infection highlighting the fact that there was a risk of congenital rubella syndrome. There is a need to implement routine measles, mumps, rubella (MMR) immunization programme for under five children and mass scale one time immunization with monovalent rubella vaccine for adolescent girls.

Investigation of measles and rubella outbreaks in Tamil Nadu, India-2003.

The aims of the present study were to confirm measles outbreaks by detection of measles-specific IgM antibodies, isolation of measles virus, and genetic characterization to document the circulating genotypes in Tamil Nadu. Eight outbreaks were reported from six districts of Tamil Nadu, India during the period Jan-Dec 2003. Blood samples were collected for serology, urine, and throat swabs for virus isolation. Genotypic characterization of measles isolates was based on the sequence of the N gene. All the clinically suspected outbreaks (n = 8) were confirmed by serology; six out of the eight as measles and two as combination of measles and rubella highlighting the need to carry out rubella serology on measles-negative samples. Genetic characterization of three isolates obtained revealed one as genotype D4 and two as D8. Measles genotypes D4 and D8 were found to circulate in three districts of Tamil Nadu. It is necessary to be aware of the circulating genotypes within the geographical area. The information would be valuable to evaluate control measures and identify viral transmission and importation.

Influenza activity among the paediatric age group in Chennai.

BACKGROUND & OBJECTIVE: Respiratory viral infections have a major impact on public health. Acute respiratory infections largely caused by viruses, are the most common illnesses experienced by otherwise healthy adults and children. Among the respiratory viruses, influenza viruses are known to cause outbreaks globally. Information on the activity of influenza virus in our country is limited and none from Chennai. The present study was carried out to isolate and identify the influenza virus serotypes causing acute respiratory infection in children attending a tertiary care centre at Chennai. METHODS: During January to December 2002, 240 children with acute respiratory infection attending the out patient clinic of Institute of Child Health were included by convenient sampling. Throat swabs were collected from 4 to 5 cases every week. Isolation of influenza virus was attempted by inoculating the sample in Madin Darby Canine Kidney (MDCK) cell line. The isolates were typed by haemagglutination inhibition test and confirmed by immunoflourescence assay. RESULTS: Virus isolation was positive in 30 (12.5%) of the 240 samples. Influenza A/H3N2/Panama/ 2000/99 was the predominant serotype isolated accounting for 24 (80%) of the 30 isolates. Influenza B/Sichuan/379/99 was isolated in 4 (13.33%) and a combination of Influenza A/H3N2 and B/Sichuan in 2 (6.6%) of the isolates. INTERPRETATION & CONCLUSION: Isolation of influenza A and B viruses indicated a significant activity of these viruses in Chennai. Peak activity was observed during and after the first spell of rain. The predominance of A/H3N2/ Panama is an indication that the Indian scenario is similar to the global picture of influenza activity.

Dengue fever epidemic in Chennai--a study of clinical profile and outcome.

Children with dengue fever presenting to the Institute of Social Pediatrics, Government Stanley Hospital, during the months of October to December 2001, were prospectively followed up for clinical profile and outcome. Commonest clinical features were fever, vomiting, bleeding, body pain and hepatomegaly. Elevated liver enzymes and low platelet counts were common laboratory findings in dengue. Hepatomegaly, positive tourniquet test, elevated haematocrit and thrombocytopenia were more common in DHF and DSS group. Retro-orbital pain was slightly more in DHF and DSS groups and there was a tendency for DSS to present at an earlier age. There was no correlation between platelet counts and bleeding in classical dengue cases.