Approach towards surveillance-based diagnosis of acute respiratory illness in India: Expert recommendations.

BACKGROUND: Emerging infectious diseases, often zoonotic, demand a collaborative "One-Health" surveillance approach due to human activities. The need for standardized diagnostic and surveillance algorithms is emphasized to address the difficulty in clinical differentiation and curb antimicrobial resistance. OBJECTIVE: The present recommendations are comprehensive diagnostic and surveillance algorithm for ARIs, developed by the Indian Council of Medical Research (ICMR), which aims to enhance early detection and treatment with improved surveillance. This algorithm shall be serving as a blueprint for respiratory infections landscape in the country and early detection of surge of respiratory infections in the country. CONTENT: The ICMR has risen up to the threat of emerging and re-emerging infections. Here, we seek to recommend a structured approach for diagnosing respiratory illnesses. The recommendations emphasize the significance of prioritizing respiratory pathogens based on factors such as the frequency of occurrence (seasonal or geographical), disease severity, ease of diagnosis and public health importance. The proposed surveillance-based diagnostic algorithm for ARI relies on a combination of gold-standard conventional methods, innovative serological and molecular techniques, as well as radiological approaches, which collectively contribute to the detection of various causative agents. The diagnostic part of the integrated algorithm can be dealt at the local microbiology laboratory of the healthcare facility with the few positive and negative specimens shipped to linked viral disease research laboratories (VRDLs) and other ICMR designated laboratories for genome characterisation, cluster identification and identification of novel agents.

Association of acute toxic encephalopathy with litchi consumption in an outbreak in Muzaffarpur, India, 2014: a case-control study.

BACKGROUND: Outbreaks of unexplained illness frequently remain under-investigated. In India, outbreaks of an acute neurological illness with high mortality among children occur annually in Muzaffarpur, the country's largest litchi cultivation region. In 2014, we aimed to investigate the cause and risk factors for this illness. METHODS: In this hospital-based surveillance and nested age-matched case-control study, we did laboratory investigations to assess potential infectious and non-infectious causes of this acute neurological illness. Cases were children aged 15 years or younger who were admitted to two hospitals in Muzaffarpur with new-onset seizures or altered sensorium. Age-matched controls were residents of Muzaffarpur who were admitted to the same two hospitals for a non-neurologic illness within seven days of the date of admission of the case. Clinical specimens (blood, cerebrospinal fluid, and urine) and environmental specimens (litchis) were tested for evidence of infectious pathogens, pesticides, toxic metals, and other non-infectious causes, including presence of hypoglycin A or methylenecyclopropylglycine (MCPG), naturally-occurring fruit-based toxins that cause hypoglycaemia and metabolic derangement. Matched and unmatched (controlling for age) bivariate analyses were done and risk factors for illness were expressed as matched odds ratios and odds ratios (unmatched analyses). FINDINGS: Between May 26, and July 17, 2014, 390 patients meeting the case definition were admitted to the two referral hospitals in Muzaffarpur, of whom 122 (31%) died. On admission, 204 (62%) of 327 had blood glucose concentration of 70 mg/dL or less. 104 cases were compared with 104 age-matched hospital controls. Litchi consumption (matched odds ratio [mOR] 9·6 [95% CI 3·6 - 24]) and absence of an evening meal (2·2 [1·2-4·3]) in the 24 h preceding illness onset were associated with illness. The absence of an evening meal significantly modified the effect of eating litchis on illness (odds ratio [OR] 7·8 [95% CI 3·3-18·8], without evening meal; OR 3·6 [1·1-11·1] with an evening meal). Tests for infectious agents and pesticides were negative. Metabolites of hypoglycin A, MCPG, or both were detected in 48 [66%] of 73 urine specimens from case-patients and none from 15 controls; 72 (90%) of 80 case-patient specimens had abnormal plasma acylcarnitine profiles, consistent with severe disruption of fatty acid metabolism. In 36 litchi arils tested from Muzaffarpur, hypoglycin A concentrations ranged from 12·4 µg/g to 152·0 µg/g and MCPG ranged from 44·9 µg/g to 220·0 µg/g. INTERPRETATION: Our investigation suggests an outbreak of acute encephalopathy in Muzaffarpur associated with both hypoglycin A and MCPG toxicity. To prevent illness and reduce mortality in the region, we recommended minimising litchi consumption, ensuring receipt of an evening meal and implementing rapid glucose correction for suspected illness. A comprehensive investigative approach in Muzaffarpur led to timely public health recommendations, underscoring the importance of using systematic methods in other unexplained illness outbreaks. FUNDING: US Centers for Disease Control and Prevention.

Molecular epidemiology and evolutionary analysis of dengue virus type 2, circulating in Delhi, India.

Dengue virus type 2 (DENV-2) has been associated with severe dengue outbreaks in many countries including India. Its predominance was recorded nearly after a decade in the capital city, Delhi in 2013. The present study characterizes DENV-2 circulated during 2013-2014. Analysis based on envelope (E) gene showed the presence of two clades (I and II) of DENV-2, within the Cosmopolitan genotype. Analysis of time of most recent common ancestor revealed the existence of clade I for more than a decade (95 % HPD 13-16 years) however, clade II showed comparatively recent emergence (95 % HPD 5-13 years). Presence of different clades is of high significance as this may result in increased virus transmission and major outbreaks. Further, the presence of a unique amino acid substitution, Q325H was also observed in an isolate; 14/D2/Del/2013 (KT717981). This substitution falls in immune epitope (epitope id: 150268) and may have important role in host immune response.

Outbreaks of unexplained neurologic illness - Muzaffarpur, India, 2013-2014.

Outbreaks of an unexplained acute neurologic illness affecting young children and associated with high case-fatality rates have been reported in the Muzaffarpur district of Bihar state in India since 1995. The outbreaks generally peak in June and decline weeks later with the onset of monsoon rains. There have been multiple epidemiologic and laboratory investigations of this syndrome, leading to a wide spectrum of proposed causes for the illness, including infectious encephalitis and exposure to pesticides. An association between illness and litchi fruit has been postulated because Muzaffarpur is a litchi fruit-producing region. To better characterize clinical and epidemiologic features of the illness that might suggest its cause and how it can be prevented, the Indian National Centre for Disease Control (NCDC) and CDC investigated outbreaks in 2013 and 2014. Clinical and laboratory findings in 2013 suggested a noninflammatory encephalopathy, possibly caused by a toxin. A common laboratory finding was low blood glucose (<70 mg/dL) on admission, a finding associated with a poorer outcome; 44% of all cases were fatal. An ongoing 2014 investigation has found no evidence of any infectious etiology and supports the possibility that exposure to a toxin might be the cause. The outbreak period coincides with the month-long litchi harvesting season in Muzaffarpur. Although a specific etiology has not yet been determined, the 2014 investigation has identified the illness as a hypoglycemic encephalopathy and confirmed the importance of ongoing laboratory evaluation of environmental toxins to identify a potential causative agent, including markers for methylenecyclopropylglycine (MCPG), a compound found in litchi seeds known to cause hypoglycemia in animal studies. Current public health recommendations are focused on reducing mortality by urging affected families to seek prompt medical care, and ensuring rapid assessment and correction of hypoglycemia in ill children.

Northward movement of East Central South African genotype of Chikungunya virus causing an epidemic between 2006-2010 in India.

INTRODUCTION: Re-emergence of chikungunya virus in South India after a gap of 32 years in 2006 affected over a million people in the Indian subcontinent. We kept a close vigil over the emerging trend of this virus between 2006-2010 with a view to establish the identity of the circulating genotype(s) and to determine the route of virus transmission in different parts of India. METHODOLOGY: Nucleotide sequencing of the E1 gene region from 36 strains of chikungunya virus from three states in northern India was performed for this present study. Forty-four previously reported E1 sequences, retrieved from the global genome data base were used for making a phylogenetic tree. RESULTS: BLAST search revealed 99% homology of the northern Indian strains of the 2006-2010 outbreak with the Reunion Island isolates of 2006. Northern Indian strains of this study clustered with the East Central South African (ECSA) genotype. CONCLUSIONS: Findings indicate that the currently circulating strain of chikungunya virus in northern India had its origin from the 2006 epidemic strain of South India that moved toward northern India via the western central India between 2006-2010 in a phased manner with dominance of the ECSA genotype and not the Asian genotype.

Serological evidence of rickettsial infections in Delhi.

BACKGROUND & OBJECTIVES: Rickettsial infections remain under-diagnosed due to lack of diagnostic facilities in developing world. Here we present our experience at National Centre for Disease Control, Delhi, about a serosurvey done in Delhi for rickettsial disease with easy to perform low cost, low expertise Weil Felix test. METHODS: On the basis of cut-off titre obtained in healthy population, Weil Felix test results were interpreted along with clinical data. Entomological investigation was also carried out in select areas of Delhi. Rodents were trapped from houses and gardens and vector mites were collected. RESULTS: When serum samples were collected during initial 5 yr period from patients with fever of unknown origin, seropositivity was 8.2 per cent whereas when rickettsial infection was kept as one of the differential diagnosis by clinicians seropositivity increased to 33.3 per cent. Rickettsial infections detected were scrub typhus (48.2%) followed by spotted fever group (27.5%) and typhus group (6.8%) during 2005-2009. In preliminary entomological survey vector mite Leptotombidium deliense was found on rodents. INTERPRETATION & CONCLUSIONS: Our findings showed that results of Weil Felix test should not be disregarded, rather clinically compatible cases should be treated to save lives.

Continued persistence of a single genotype of dengue virus type-3 (DENV-3) in Delhi, India since its re-emergence over the last decade.

BACKGROUND/PURPOSE: The re-emergence of an epidemic strain of dengue virus type-3 (DENV-3) in Delhi in 2003 and its persistence in subsequent years marked a changing trend in dengue virus circulation in this part of India. Its evolving phylogeny over the past decade has not been studied in detail as yet. METHODS: Reverse transcription polymerase chain reaction and sequencing of the CprM gene junction of DENV-3 from different outbreaks since 2003 was carried out. Thirty CprM DENV-3 sequences from this study were compared with 46 other previously reported CprM DENV-3 sequences from India and other countries. Multiple sequence alignment and phylogenetic trees were constructed to determine the extent of genetic heterogeneity and trace the phylogeny of DENV-3. RESULTS: Thirty CprM DENV-3 sequences (Accession numbers AY706096-99, DQ645945-52, EU181201-14, and EU846234-36) were submitted to GenBank. The CprM junction was found to be AT rich (approximately 53%). Nucleotide sequence alignment revealed only nucleotide substitutions. Phylogenetic analysis indicated sustained evolution of a distinct Indian lineage of DENV-3 genotype III in Delhi. CONCLUSION: Active circulation of DENV-3 genotype III over the last decade in Delhi was evident and worrying. This genotype has been implicated in several outbreaks in South-East Asia and other parts of the world.

Phylogenetic studies reveal existence of multiple lineages of a single genotype of DENV-1 (genotype III) in India during 1956-2007.

BACKGROUND: Dengue virus type 1 (DENV-1) have been mostly circulating silently with dominant serotypes DENV-2 and DENV-3 in India. However recent times have marked an increase in DENV-1 circulation in yearly outbreaks. Many studies have not been carried out on this virus type, leaving a lacunae pertaining to the circulating genotypes, since its earliest report in India. In the present study, we sequenced CprM gene junction of 13 DENV-1 isolated from Delhi and Gwalior (North India) between 2001-2007 and one 1956 Vellore isolate as reference. For comparison, we retrieved 11 other Indian and 70 global reference sequences from NCBI database, making sure that Indian and global isolates from all decades are available for comparative analysis. RESULTS: The region was found to be AT rich with no insertion or deletion. Majority of the nucleotide substitutions were silent, except 3 non-conservative amino acid changes (I --> T, A --> T and L --> S at amino acid positions 59,114 and 155 respectively) in the Indian DENV-1 sequences, sequenced in this study. Except two 1997-98 Delhi isolates, which group in genotype I; all other Indian isolates group in genotype III. All Indian genotype III DENV-1 exhibited diversity among them, giving rise to at least 4 distinct lineages (India 1-4) showing proximity to isolates from diverse geographic locations. CONCLUSION: The extensive phylogenetic analysis revealed consistent existence of multiple lineages of DENV-1 genotype III during the last 5 decades in India.

Investigation of an outbreak of chikungunya in Malegaon Municipal areas of Nasik district, Maharashtra (India) and its control.

BACKGROUND & OBJECTIVES: An outbreak of chikungunya fever occurred in Malegaon town of Nasik district of Maharashtra state, India during February and March 2006. A total of 4530 fever cases were reported during this period including 1781 cases which were admitted in different hospitals of the town. An entomological and epidemiological investigation was carried out in the affected villages during the outbreak to study the possible causes of the outbreak and to isolate the virus responsible. METHODS: Entomological evaluation was done as per WHO guidelines. Sera samples were collected by venipuncture from clinically suspected chikungunya patients in hospitals and also during house-to-house survey in affected villages. IgM antibodies to dengue virus were detected using IgM capture ELISA (PANBIO) and by "Haemagglutination inhibition test" for detection of antibodies against Chikungunya virus. Acute sera samples were inoculated in cell lines for virus isolation. The isolates were confirmed by RT-PCR. RESULTS: On investigation, it was found that water storage containers like cement tanks, plastic containers or earthen pots placed in front of the individual houses were the potential breeding sites for Aedes aegypti. Entomological survey carried out in the most affected areas revealed high Aedes indices. House, container and breteau indices were found to be 27.2, 16.19 and 35.1, respectively. Out of the 13 acute sera samples collected, virus was isolated in 10 samples. The isolates were confirmed by RT-PCR and sequencing using primers from nsP1 gene of Chikungunya virus (CHIKV, Accession No. EF077609, EF077610). Of the 17 convalescent sera tested, significant level of HI antibodies to CHIKV was detected in five samples. One sample was positive for IgM antibodies against dengue virus. Based on clinico-epidemiological features and laboratory findings, the illness was confirmed to be of chikungunya viral disease. CONCLUSION: Control measures targeting the vector population and personal protective measures against the mosquito bites were instituted. Extensive IEC campaign with the involvement of community and religious leaders helped in containment of the disease.

Emergence of an independent lineage of dengue virus type 1 (DENV-1) and its co-circulation with predominant DENV-3 during the 2006 dengue fever outbreak in Delhi.

OBJECTIVES: The sudden emergence of dengue virus type 1 (DENV-1) and its co-circulation with predominant DENV-3 was the hallmark of the 2006 dengue fever outbreak in Delhi. Viruses that circulated between 1996 and 2005 in the City have been well characterized, but the genomic diversity in 2006 strains is not known. The present study was undertaken to reveal the emerging molecular genotype(s) and evolutionary trend of the viruses responsible for the dengue fever outbreak in Delhi during 2006. STUDY DESIGN: The CprM gene junction of the DENV isolates from the 2006 Delhi dengue fever outbreak were subjected to nucleotide sequencing. Comparative phylogenetic analysis was done using DENV-1 and DENV-3 sequences retrieved from the global database. RESULTS: Multiple sequence alignment revealed only substitutions, with no insertions or deletions. A dendrogram indicated emergence of a distinct lineage of DENV-1 (having similarity with the Comoros/Singapore 1993 and Delhi 1982 strains, but quite different from the Delhi 2005 lineage) and microevolution of the pre-circulating DENV-3. These findings point towards the circulation of two independent lineages of DENV-1 in Delhi during 2005 and 2006. CONCLUSIONS: It is feared that the introduction of an independent lineage of the outbreak-associated strain of DENV-1 and its co-circulation with the deeply-rooted strain of DENV-3 in Delhi may result in yet another, possibly more severe outbreak in the near future.

Knowledge, attitude and practices about animal bites and rabies in general community--a multi-centric study.

Rabies a disease as old as our civilization, continues to be the most feared of all communicable diseases. Despite the availability the state-of-the-art tools which ensure near cent percent protection against rabies, India is the largest contributant to rabies mortality in the world. A multicentric study was carried out from April 2001 to September 2002 with the objective of assessing the knowledge, attitudes, beliefs and practices (KAP) about animal bites and rabies in the general community. The proforma for interviewing the general community was developed and used after field testing. The study was carried out at six selected centres across the country viz. Delhi, Hyderabad, Raipur, Jamnagar, Coonoor and Rajahmundry and was co-ordinated by National Institute of Communicable Diseases (NICD), Delhi, after thorough briefing of designated nodal officers. A total of 1129 (male: female :: 48.5: 51.5) persons in the age group of 18 to 80 years were interviewed in this study. Of these about 751% of the individuals had attended school at some level and rest were illiterates. 68.7% people had heard about rabies. In 60.7% of cases the community associates rabies with dog bite only. Knowledge about appropriate wound toilet was found to be inadequate. Only 360 (31.9%0/) people felt that washing the wound with soap and water was the best option. Application of indigenous products like chillies (11.4%), turmeric (5.6%), lime (6.8%), kerosene oil (2.3%), herbal paste (4.2%) etc was suggested along with visit to occult medicine practitioner (1.5%) as part of the bite wound management. People were not aware of number of injections needed for treatment of animal bites. Multiple reasons viz negligence and ignorance 354 (31.4%), fear of multiple painful injections 365 (32.3%), expensive treatment 169 (15%) and long course requiring daily visits to anti-rabies clinics 73 (6.5%) were cited as reasons for non-compliance of treatment. KAP study suggests that there is need to create awareness amongst the masses regarding epidemiology of the disease and merits of prompt and appropriate post exposure treatment through enhanced IEC activities.

Safety and immunogenicity of the intradermal Thai red cross (2-2-2-0-1-1) post exposure vaccination regimen in the Indian population using purified chick embryo cell rabies vaccine.

PURPOSE: To test the immunogenicity of the WHO recommended "2-2-2-0-1-1" post-exposure rabies vaccination regimen in Indian subjects to determine the feasibility of replacing crude sheep brain nerve tissue rabies vaccine with modern tissue culture rabies vaccine at major anti-rabies treatment centers throughout India. METHODS: Purified chick embryo cell vaccine (PCECV) was administered in the dosage of 0.1 mL per site to 53 Indian subjects. RESULTS: All subjects produced rabies antibodies above 0.5 IU/mL by day 14 post-vaccination. Only minor adverse reactions including swelling (6.6%), erythema (5.4%) and pain (1.4%) were observed for which no treatment was required. CONCLUSIONS: This study demonstrated that PCECV is safe and highly immunogenic in Indian subjects when administered intradermally as 0.1 mL/site using the "2-2-2-0-1-1" post-exposure regimen.

Comparative evaluation of commonly used laboratory tests for post-mortem diagnosis of rabies.

Animal brain samples received at WHO Collaborating Centre laboratory at National Institute of Communicable Diseases (NICD) during the years 1991-2002 were tested by Seller's stain, Fluorescent Antibody Test (FAT) and Mouse Innoculation Test (MIT) as methods of rabies diagnosis. Negri bodies on Seller's staining could be detected in 52.5% of MIT positive brains, the concordance of this test with MIT was found to be 77.8%. FAT was positive in 91.5% of MIT positive brains, though it showed concordance of 95.7% with MIT results in the total samples. 12.2% of the samples were found positive by FAT of which 1/3rd also showed the presence of Negri bodies when MIT was negative i.e. showing that the virus is present in inactivated form. Thus emphasizing the need for timely and proper collection and transportation of specimens for testing. Seller's stain and FAT give reliable diagnosis of rabies in the brain samples in majority of the cases. MIT being time-intensive test, is of academic value only in decision making as regards initiation of Post Exposure Treatment (PET), it is recommended that in cases where Seller's stain and FAT have yielded negative results the decision to initiate PET should give due consideration to the nature and circumstances of the animal bite and other epidemiological features.

Human rabies in Delhi.

OBJECTIVE: Analysis of human rabies cases admitted in Infectious Diseases Hospital, Delhi during the course of one year was undertaken to understand the epidemiology of rabies. METHODS: The data was collected using standardized proforma and was analyzed on EPI Info 6.02 software version. A total of 49.8% of the human rabies came from Delhi and rest belonged to adjoining states of UP (30.3%), Haryana (18.3%), Bihar (0.8%), Punjab (0.4%) and MP (0.4%). Children in the age group 5-14 years fell victim to the disease in significantly higher numbers (36.7%) as compared to other age groups. Male Female ratio was 4:1. Biting animals involved were dog (96.7%), jackal (1.7%), cat (0.8%), monkey (0.4%) and mongoose (0.4%). Majority (78.8%) had Category III exposure. Hydrophobia, which is the pathogonomic feature of human rabies, was present in 95% of cases. Remaining (5%) gave history of animal bite and presented with features of aerophobia, photophobia along with fever, weakness in limbs, parasthesia, and/or paralysis. Significantly higher number (93.4%) did not receive any local wound treatment. Most (91.7%) cases never received any vaccination and remaining were inadequately vaccinated; only five had received 10-14 injections of Neural Tissue Vaccine (NTV) and one child who had multiple bites on face, received 5 doses of NTV, local wound treatment and intramuscular ARS on 3rd day. RESULT: The data strongly reveals that people who died due to rabies either did not receive any treatment or were inappropriately and inadequately treated. Hence, there is need to educate the community and the health care professionals about the importance of immediate and adequate post exposure treatment. The data also indicates that the epidemiology of the disease has not changed much over the decades. CONCLUSION: The disease can be prevented with the available tools and all we need to do is to implement them effectively.

Animal bites: the current management guidelines.

Rabies is a major public health problem in India. It is mainly transmitted by stray dogs, which form an overwhelming population in the country. Dogs are responsible for upto 95% of animal bites requiring antirabies treatment. In view of the exceptionally high fatality rate of human rabies, the prevention of infection after exposure is of utmost importance. With the availability of safe and effective tissue culture vaccines prevention of this dreaded disease is virtually assured by immediate and appropriate post exposure treatment. This is a three pronged approach including proper wound management, judicious use of antirabies serum and modern tissue culture vaccines. In India, Neural Tissue Vaccine is still used for post exposure treatment in public sector, though effective, this vaccine has serious side effects. The production and use of tissue culture vaccine should be encouraged with the aim to phase out neural tissue vaccine. WHO recommends use of intradermal route of inoculation of Tissue Culture Vaccine which makes the treatment very economical. However, this route as yet, is not approved by Drug Controller, Government of India (DCGI). There are no uniform guidelines for management of animal bite cases in India. In this article an attempt is made to discuss various aspects of animal bite management.

Immunogenicity of purified chick embryo cell anti-rabies vaccine in post-exposure treatment of animal bite cases.

The observations on immunogenicity of Purified Chick Embryo Cell (PCEC) anti rabies vaccination in post-exposure prophylaxis is reported. In total 207 serum samples collected from patients receiving 3 to 6 doses of PCEC were analysed for the presence of anti-rabies antibodies. The samples were collected from 10 days to 11 months after the last dose of vaccine. All the vaccinees (n=33) tested after 3 doses of PCEC showed protective titres (> or = 0.5 IU/ml) and those receiving 5-6 doses (n=161) showed 4-5 times higher than protective titres. The analysis pertains to specimens collected at one point of time only after the vaccination. However, in all 17 vaccinees where samples were collected 7-11 months after 3-6 doses of vaccine, the protective titres were sustained, these being 3-4 times higher than the protective titres in those receiving 5-6 vaccine doses. The results indicated that there was no need of routine anti-rabies antibody monitoring in healthy individuals receiving post-exposure prophylaxis in recommended doses of vaccine.