Epidemiological Profile of Acute Viral Encephalitis.

OBJECTIVE: To study the etiology and clinico-epidemiological profile of acute viral encephalitis in children with acute encephalitis syndrome (AES). METHODS: An observational study including 100 patients fulfilling the criteria for AES was conducted in children of age group 1 mo - 16 y. Viral isolation was done on RD cells, HEp-2 cells and Vero cells from the cerebrospinal fluid samples of suspected viral encephalitis (VE) cases. An enzyme immunoassay for IgM antibodies was performed for measles, mumps, Varicella zoster virus (VZV), Herpes simplex virus 1 (HSV1) and Japanese encephalitis virus (JEV). Multiplex polymerase chain reaction (PCR) was done for Cytomegalovirus, Epstein Barr virus (EBV), HSV1 & 2, VZV, Enterovirus, Parecho virus, Human Herpes virus (HHV 6, 7) and Parvovirus B19. A micro neutralization test was performed for Enterovirus 71. RESULTS: Out of enrolled 100 patients, 73 were of probable viral encephalitis. HSV1 (31.50%) was the commonest virus followed by Adenovirus (10.95%), Parvovirus (2.73%), JE virus (1.36%), Enterovirus (1.36%), EBV (1.36%), and mixed infection with HSV & EBV (1.36%). HSV 1 caused significant morbidity in children. The common computed tomography (CT) findings were hypodensities in the fronto- parietal lobe followed by cerebral edema. CONCLUSIONS: The landscape of AES in India has changed in the previous decade, and both outbreak investigations and surveillance studies have increasingly reported non-JEV etiologies; because of these findings there is a need to explore additional strategies to prevent AES beyond vector control and JEV vaccination.

New Delhi metallo-ß-lactamase - type carbapenemases producing Escherichia coli isolates from hospitalized patients: A pilot study.

BACKGROUND & OBJECTIVES: Resistances to carbapenem group of antimicrobials among Escherichia coli due to production of carbapenemases, especially the New Delhi metallo-ß-lactamase (NDM) types, pose serious challenges in the treatment of infections in healthcare settings. This study was undertaken to detect NDM producing E. coli isolates from hospitalized patients with urinary tract infection (UTI). METHODS: A total of 30 non-repetitive isolates of E. coli from hospitalized patients with clinical suspicion of UTI were subjected to antimicrobial susceptibility testing. Screening for the production of extended-spectrum ß-lactamases (ESBL) was carried out by minimum inhibitory concentration (MIC) test strip ESBL followed by phenotypic confirmation by double-disc synergy test. Phenotypic confirmation of carbapenemase production was carried out by MIC test strip metallo-ß-lactamases. Molecular identification of the blaNDM gene was carried out by polymerase chain reaction (PCR) and sequencing of the amplified fragment. RESULTS: Seventeen of the 30 isolates were detected as ESBL producers, of which three were found to be carbapenemase producers. NDM genes were detected by PCR followed by gene sequencing in all three isolates positive for ESBL as well as carbapenemase. The amino acid sequence of the three isolates showed complete identity to the reference sequences of NDM-1, NDM-4 and NDM-8, respectively. INTERPRETATION & CONCLUSIONS: Our study showed the circulation of NDM variants among the clinical isolates of E. coli that were producers of ESBL as well as carbapenemase.

Seroprevalence of transplacentally acquired measles antibodies in HIV-exposed versus HIV-unexposed infants at six months of age.

BACKGROUND & OBJECTIVES: Measles infection is reported to be more severe, prolonged and associated with a higher complication rate in children with HIV infection. Reports indicate that infants born to HIV-infected women [HIV exposed infants (HEI)] may be more vulnerable to measles. The World Health Organization recommends measles vaccination starting at six months of age in these infants who may be HIV-infected themselves. However, in India, they are given measles vaccination at nine months of age like all other infants. In this study, the seroprevalence of transplacentally acquired measles antibodies was compared in HEI and unexposed infants (HUnI) at six months of age and the proportion of HEI undergoing seroconversion after immunization with measles vaccine was assessed. METHODS: In this prospective longitudinal study, measles IgG antibodies were estimated in serum of 49 HEI and 50 HUnI aged 6-7 months. Measles vaccine was then administered to HEI. Assessment for measles IgG antibodies was repeated 8-12 wk post-immunization. RESULTS: Measles IgG antibodies were detected in two of 49 (4.1%) HEI and 16 of 50 (32%) HUnI. HEI were 11 times more likely to lack measles antibodies as compared to HUnI (odds ratio=11.05, 95% confidence interval=2.989-40.908). Post-vaccination, seroprevalence of measles antibodies increased to 38.5 per cent (PInterpretation & conclusions: Most HEI lacked measles antibodies at six months age and were, therefore, more vulnerable to measles than HUnI. Seroconversion in response to a single dose of measles vaccine administered at six months age was low in these infants, signifying the need of additional dose(s) of measles/measles-containing vaccine.

DNA chip based sensor for amperometric detection of infectious pathogens.

Several infectious pathogens are found in human whose detection is essential for rapid cure of diseases. The most commonly found pathogen in human is Streptococcus pyogenes which leads to a wide range of infections from mild pharyngitis to rheumatic heart disease. An ultrasensitive DNA chip based sensor was developed for quick identification of pathogen S. pyogenes from patient throat swab samples. The amperometric response was measured after hybridization of specific probe with single stranded genomic DNA (ssG-DNA) from the patient samples. The DNA chip was characterized by FTIR, SEM and validated with suspected patient real samples. The sensitivity of the DNA chip based sensor was found 951.34(µA/cm2)/ng DNA and lower limit of detection (LOD) was 130fg/6µL samples. The DNA chip based sensor is highly specific and takes only 30min for identification of specific pathogen.

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.

Incidence, Etiology and Risk Factors Associated with Neonatal Healthcare-Associated Conjunctivitis: A Prospective Study from a Tertiary Care Hospital in India.

INTRODUCTION: Healthcare-associated conjunctivitis (HAC) can lead to serious sequelae including blindness. We conducted a one-year prospective study to determine the epidemiology of neonatal HAC at a tertiary-care hospital in India. METHODS: From the neonates fulfilling a set of predefined inclusion criteria, cases of HAC were diagnosed based on CDC guidelines. Conjunctival swabs, obtained from neonates with suggestive clinical signs, were processed using standard protocols. Twenty-eight potential risk factors were analyzed. RESULTS: We detected 24 cases of HAC among 591 enrolled neonates, with Escherichia coli being the most frequently isolated microorganism. On multivariate analysis, intubation at birth (p = 0.046) and orogastric feeding (p = 0.029) had a statistically significant association with neonatal HAC. Average hospitalization increased from 9.6 to 20.8 days for neonates diagnosed with HAC. CONCLUSION: A standardized case-definition and physician awareness of potential serious sequelae would help improve detection rates and timely institution of therapy. Hand hygiene could help control the menace of neonatal HAC.

Chromoblastomycosis Caused by Cladophialophora bantiana in a Renal Transplant Recipient From Delhi, India.

A 45-year-old accountant residing in Delhi, India, presented to our dermatology clinic with a small asymptomatic plaque on the little finger of his left hand of 3 months' duration. The onset of the lesion was insidious and gradually progressed to 4 cm across at the time of his first visit. The patient had undergone renal transplantation twice (the first procedure 3 months prior and the second 18 months prior). Since then, he had been receiving cyclosporine A (400 mg daily) and prednisolone (40 mg) daily in immunosuppessive doses. The patient denied any kind of cutaneous injury prior to the onset of the lesion and any similar lesions in the past.

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.

Carbon-Mercaptooctadecane/Carboxylated Multi-walled Carbon Nanotubes Composite Based Genosensor for Detection of Bacterial Meningitis.

Human brain bacterial meningitis is a life-threatening disease mainly caused by Neisseria meningitidis, lead to several complications including damage of brain or even death. The present available methods for diagnosis of meningitis have one or more limitations. A rmpM gene based genosensor was fabricated by immobilizing 5'-amino modified 19-mer single stranded DNA probe onto carbon-mercaptooctadecane/carboxylated multi-walled carbon nanotubes composite electrode and hybridized with 2.5-40 ng/6 µL of single stranded genomic DNA (ssG-DNA) of N. meningitidis from cerebrospinal fluid (CSF) of the suspected meningitis patients. The electrochemical response was measured by using cyclic voltammetry and differential pulse voltammetry (DPV) using 1 mM methylene blue as redox indicator in 30 min (including a response time of 1 min) at 25 °C. The sensitivity of the genosensor was 3.762 (µA/cm(2))/ng and limit of detection was 2 ng of ssG-DNA of N. meningitidis with DPV. The genosensor has specificity only to N. meningitidis and does not hybridize with the genomic DNA of any other possible pathogen in human CSF. The immobilization of the probe and hybridization of the ssG-DNA were characterized by using electrochemical impedance in presence of 5 mM potassium ferricyanide and scanning electron microscopy. The genosensor loses only 12 % of its original DPV current on storage at 4 °C for 6 months. Carbon composite based electrochemical array can be constructed to detect multiple bacterial meningitis suspected patient CSF samples during an outbreak of the disease.

Appearance of L90I and N205S Mutations in Effector Domain of NS1 Gene of pdm (09) H1N1 Virus from India during 2009-2013.

In the present study, full length sequencing of NS gene was done in 91 samples which were obtained from patients over the time period of five years from 2009 to 2013. The sequencing of NS gene was undertaken in order to determine the changes/mutations taking place in the NS gene of A H1N1 pdm (09) since its emergence in 2009. Analysis has shown that the majority of samples belong to New York (G1 type) strain with valine at position 123. Effector domain of NS1 protein displays the appearance of three mutations L90I, I123V, and N205S in almost all the samples from 2010 onwards. Phylogenetic analysis of available NS1 sequences from India has grouped all the sequences into four clusters with mean genetic distance ranging from 12% to 24% between the clusters. Variability in length of NS1 protein was seen in sequences from these clusters, 230-amino-acid-residue NS1 for all strains from year 2007 to 2008 and for 21 strains from year 2009 and 219-residue products for 37 strains from year 2009 and all strains from year 2010 to 2013. Mutations like K62R, K131Q, L147R, and A202P were observed for the first time in NS1 protein and their function remains to be determined.

nspA gene as a specific genetic marker for detection of Neisseria meningitidis causing bacterial meningitis.

Bacterial meningitis caused by Neisseria meningitidis which causes human brain meninges damage, is generally diagnosed from patient cerebrospinal fluid through microscopy, immunological assays, biochemical test, PCR, microarray and biosensors. However, these methods are expensive, time-consuming or non-confirmatory due to certain limitations. A quick PCR based method was developed for detection of bacterial meningitis caused by N. meningitidis using specific primers based on amplification of virulence nspA (Neisseria surface protein A) gene partial sequence (202 bp). The nspA gene amplicon could be used as a genetic marker for minimum detection of 10 ng genomic DNA (G-DNA) of N. meningitidis with high sensitivity only in 80 min, which is least time reported for the confirmation of the disease. However, the lower detection limit was found as low as 1.0 ng G-DNA, but with less sensitivity. The cross-reactivity of the genetic marker, was also studied with other possible pathogens. A comparison with the presently available detection methods and our method was also done using patient samples.

Gold-mercaptopropionic acid-polyethylenimine composite based DNA sensor for early detection of rheumatic heart disease.

The first gold-mercaptopropionic acid-polyethylenimine composite based electrochemical DNA biosensor was fabricated for the early detection of Streptococcus pyogenes infection in humans causing rheumatic heart disease (heart valve damage). No biosensor is available for the detection of rheumatic heart disease (RHD). Therefore, the mga gene based sensor was developed by the covalent immobilization of a 5'-carboxyl modified single stranded DNA probe onto the gold composite electrode. The immobilized probe was hybridized with the genomic DNA (G-DNA) of S. pyogenes from throat swabs and the electrochemical response was measured by cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance (EI). Covalent immobilization of the probe onto the gold composite and its hybridization with G-DNA was characterized by FTIR and SEM. The sensitivity of the sensor was 110.25 µA cm(-2) ng(-1) with DPV and the lower limit of detection was 10 pg per 6 µL. The sensor was validated with patient throat swab samples and results were compared with available methods. The sensor is highly specific to S. pyogenes and can prevent damage to heart valves by the early detection of the infection in only 30 min.

mga genosensor for early detection of human rheumatic heart disease.

The 5' amino-labeled DNA probe complementary to mga gene of Streptococcus pyogenes was immobilized on carboxylated multiwall carbon nanotubes electrode and hybridized with 0.1-100 ng/6 µl single-stranded genomic DNA (ssG-DNA) of S. pyogenes from throat swab of suspected rheumatic heart disease (RHD) patients. Electrochemical response was measured by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance (EI). The sensitivity of the sensor was 106.03 (µA/cm(2))/ng and limit of detection (LOD) was found 0.014 ng/6 µl with regression coefficient (R(2)) of 0.921 using DPV. The genosensor was characterized by FTIR and SEM, and electrode was found stable for 6 months on storage at 4 °C with 5-6 % loss in initial DPV current. mga genosensor is the first report on RHD sensor which can save life of several suspected patients by early diagnosis in 30 min.

rmpM genosensor for detection of human brain bacterial meningitis in cerebrospinal fluid.

Human brain bacterial meningitis is a life-threatening disease caused mainly by Neisseria meningitidis, lead to damage of the outer membrane covering (meninges) of brain or even death. The usual methods of diagnosis are either time-consuming or have some limitations. The specific rmpM (reduction-modifiable protein M) virulent gene based genosensor is more sensitive, specific, and can detect N. meningitidis directly from the patient cerebrospinal fluid in 30 min including 1-min response time. 5'-Thiol-labeled single-stranded DNA (ssDNA) probe was immobilized onto screen-printed gold electrode (SPGE) and hybridized with denatured (95 °C) single-stranded genomic DNA (ssG-DNA) for 10 min at 25 °C. The electrochemical response was measured by cyclic voltammetry, differential pulse voltammetry (DPV) and electrochemical impedance using redox indicators. The sensitivity of the genosensor was 9.5087 (µA/cm(2))/ng with DPV and limit of detection was 3 ng/6 µL ssG-DNA. The immobilization of the ssDNA probe and hybridization with ssG-DNA from N. meningitidis was characterized by atomic force microscopy and Fourier transform infrared spectroscopy. The rmpM genosensor was stable for 6 months at 4 °C with 10 % loss in initial DPV current. The advantage of rmpM genosensor is to detect bacterial meningitis simultaneously in multiple patients using SPGE array during an outbreak of the disease.

Omp85 genosensor for detection of human brain bacterial meningitis.

The 5'-thiolated DNA probe based on specific virulence gene, Omp85, was immobilized onto a screen-printed gold electrode followed by hybridization with 6-100 ng/6 µl (5.9 × 10(5)-9.3 × 10(6 )c.f.u.) of Neisseria meningitidis single stranded genomic DNA (ssG-DNA) for 10 min at 25 °C from the cerebrospinal fluid (CSF) of a meningitis patient. The Omp85 genosensor can detect as little as 6 ng ssG-DNA in 6 µl CSF of a human brain meningitis patient in 30 min including a response time of 1 min by cyclic voltammetry, differential pulse voltammetry (DPV) and electrochemical impedance. The sensitivity of the genosensor electrode was 2.6(µA/cm(2))/ng using DPV with regression coefficient (R(2)) 0.954. The genosensor was characterized using Fourier transform infrared spectroscopy and atomic force microscopy. Omp85 genosensor was stable for 12 months at 4 °C with 12 % loss in DPV current.

Atypical subacute sclerosing panencephalitis with short onset latency.

An 11-month-old boy presented with focal seizures, myoclonic jerks and altered sensorium of one month duration, with a history of measles at eight months of age. A diagnosis of Subacute sclerosing panencephalitis (SSPE) was made on the basis of typical EEG changes and presence of anti-measles antibody in cerebrospinal fluid. A differential diagnosis of SSPE should be considered in all forms of acute encephalopathy in infants for early diagnosis and management.

Glycoprotein B genotyping in congenital/perinatal Cytomegalovirus infection in symptomatic infants.

BACKGROUND: Molecular epidemiological studies on circulating strains of CMV in cogenital/perinatal infections have not been done earlier in this region. OBJECTIVE: To study the glycoprotein B genotypes in babies with symptomatic congenital/perinatal CMV infection and to assess the possible influence of genotype on the outcome of the infection. METHODS: Clinical samples (blood and urine) of symptomatic babies are sent to the Virology Department of NCDC, Delhi for the diagnosis of congenital infections. 375 clinical samples of infants (newborn - 6 months old) were included for the study. Serum samples were subjected to ELISA for detection of IgM antibodies against CMV. DNA isolation and amplification of CMV genomic DNA targeting gB gene fragment by nested PCR, was carried out in the samples. The amplified fragment including the cleavage site was subjected to RFLP using restriction enzymes Rsal and Hinf1. They were also verified by sequencing using Big Dye Terminator chemistry. RESULTS: 75 samples out of 375 tested were confirmed positive for CMV infection by serology and PCR. Both RFLP and sequencing of gB gene fragment showed that gB 1, 2 and 3 genotypes were in circulation. gB 3 was the most prevalent genotype in symptomatic infants. Hepatosplenomegaly was the most common feature in gB-3 genotype of CMV. gB2 congenital CMV infection was more commonly associated with long term sequelae.

Quick diagnosis of human brain meningitis using omp85 gene amplicon as a genetic marker.

The usual diagnosis of life-threatening human brain bacterial meningitis are expensive, time consuming or non-confirmatory. A quick PCR based diagnosis of meningitis in cerebrospinal fluids (CSF) using specific primers of virulent Omp85 gene of Neisseria meningitidis can detect as low as 1.0 ng of genomic DNA (G-DNA) in 80 min for confirmation of bacterial meningitis caused by N. meningitidis infection. The 257 bp amplicon of Omp85 gene does not show homology with other suspected pathogens in CSF and can be used as a specific genetic marker for diagnosis of the disease.

Emergence of pandemic 2009 influenza A H1N1, India.

BACKGROUND & OBJECTIVES: Pandemic H1N1 caused deluge of cases from 74 countries and prompted World Health Organization to raise warning to phase 6. The present study was conducted on throat and nasal swab samples received and tested at National Centre for Disease Control, Delhi, India during 2009-2010 to collect epidemiological and clinical information on positive cases. METHODS: Throat and nasopharyngeal swabs from category C influenza A H1N1 patients during May 2009-September 2010 along with their clinico-epidemiological details were collected from identified hospitals from Delhi and other States. Samples were tested by Real time reverse transcriptase PCR using primers and probes developed at CDC, Atlanta for four influenza target genes. RESULTS: A total of 33,751 samples, both throat and nasal swab samples from each patient were tested for H1N1 influenza virus, of which, 7943 (23.5%) were positive for pandemic influenza A H1N1 and 3759 (11.1%) were positive for influenza A (seasonal flu). Maximum number of positive cases (N=2792, 35.1%) were from 20-39 yr age group, comprising 1790 (22.5%) males and 1182 (14.8%) females. Only 2620 (33%) positive cases were close contact of influenza A H1N1 positive patient. Majority cases presented (N=2792, 35.1%) with fever 7005 (88.1%), followed by 6133 cases (77.2%) exhibiting fever and cough, 377 (4.7%) complained of fever, cough, nasal catarrh and 362 (4.5%) cases had fever with shortness of breath. INTERPRETATION & CONCLUSIONS: The study showed a peak of cases of pandemic influenza A H1N1 in December 2009 and indicated predominance of H1N1 positive cases among 20-39 yr age group and among males compared to females.