A multiplex SNaPshot assay for the detection of single nucleotide polymorphism associated with clinical severity of enterovirus infections.

Non-polio enterovirus infections are known to cause a variety of diseases and neurological complications. It is also known that the severity of these diseases largely differs among individuals with different genotypes and alleles. The Single Nucleotide Polymorphisms (SNPs) within specific genes have a considerable effect on the immune response to enteroviruses and on the outcome of disease, leading to variations in complications and infection susceptibility. Knowing the distribution of such SNPs can be valuable for individual case management and studying epidemiological parameters of enterovirus infections. In this feasibility study, a multiplex version of the primer extension-based technique called the SNaPshot Assay has been developed to examine SNPs in various relevant genes for predicting the clinical severity of enterovirus infections. It is already established that this technique is precise, consistent, scalable, and likely to exhibit high throughput. The multiplex SNaPshot can investigate multiple genetic susceptibility markers simultaneously, and the assay can be used to identify vulnerable populations, understand the epidemiology of infections, and manage the outbreaks of enteroviruses. Based on the literature, 15 SNPs were identified which are suspected for higher susceptibility to the worst outcomes after enterovirus infection and the assay was developed. Blood samples of 100 healthy volunteers were collected and tested for assay feasibility as well as to know the proportions of 15 selected SNPs. After the analysis, seven SNPs have been identified and suggested to be considered for future assays. Based on the pilot test results, it appears that positivity for any three out of the identified seven SNPs might indicate a higher risk, and future studies correlated with clinical studies among patients with and without severe diseases utilizing this assay will provide robust parameters to determine at-risk individuals more accurately.

Poliovirus nonpermissive CD155 knockout cells derived from RD cell line for handling poliovirus potentially infectious materials in virology laboratories.

Destruction of all poliovirus containing materials, safe and secure handling of retained polioviruses for vaccine production, and research will be obligatory to eliminate facility-associated risks. Polioviruses and poliovirus potentially infectious materials (PIM) including fecal or respiratory samples requiring containment have been defined in World Health Organization-Global Action Plan (GAP III) documents. Non-polio laboratories culturing viruses from PIM are most affected as cell cultures of human and monkey origin are also poliovirus permissive. CRISPR gene-editing technology was used to knockout the poliovirus receptor (PVR/CD155) gene in the rhabdomyosarcoma (RD) cell line. PVR knockout RD cell susceptibility was tested using known non-polio enterovirus (NPEV) types. A selected clone (RD-SJ40) was field evaluated for virus isolation from 626 stool samples of acute flaccid paralysis cases. Poliovirus nonpermissive cells derived from the RD cell line did not show CD155-specific cell-surface immunofluorescence. CD155 gene sequencing confirmed nucleotide base pair deletions within exon2 and exon3. The CD155 knockout RD-SJ40 cells did not support the growth of poliovirus from positive stool samples. All NPEV types were isolated in RD and RD-SJ40 cells. CRISPR correctly edited the CD155 gene of RD cells to render them poliovirus nonpermissive while susceptibility to NPEV remained unchanged. RD-SJ40 cells are safe for NPEV isolation from poliovirus PIM without derogating GAP III containment requirements.

Development of a RT-LAMP assay for detection of SARS-CoV-2.

Background & objectives: The pandemic of SARS-COV-2 began in Wuhan, China in December 2019 and has caused more than 101 million cases worldwide. Diagnostic technologies possessing sensitivity and specificity equivalent to real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) assays are needed to ramp up testing capacity in most countries. Newer platforms need to be technically less demanding, require minimum equipment and reduce turn-around time for reporting results. The objective of this study was to exploit loop-mediated isothermal amplification (LAMP) for the detection of SARS-CoV-2 and evaluate its performance by comparison with rRT-PCR. Methods: Reverse-transcription LAMP (RT-LAMP) assay primers were designed to detect envelop (E) and nucleocapsid (N) genes of SARS-CoV-2. Positive control RNA was prepared by in vitro transcription of E and N genes clones. RT-LAMP amplification reactions were incubated at 65°C for 30 min. Results were recorded visually. RT-LAMP results were evaluated by comparing the results obtained with a commercial rRT-PCR kit. Results: The RT-LAMP assay for E and N genes was carried out in separate tubes. RT-LAMP detected about 40 copies of SARS-CoV-2 RNA per reaction. A total of 253 throat swabs were tested using the RT-LAMP assay. The overall diagnostic sensitivity and specificity of the LAMP assay were 98.46 and 100 per cent, respectively, as compared to the rRT-PCR. Interpretation & conclusions: SARS-CoV-2 RT-LAMP assay was designed, standardized and evaluated. The assay showed diagnostic sensitivity and specificity equivalent to rRT-PCR assays. The assay will be useful to increase testing capacity for the detection of SARS-CoV-2 in the country.

Immunogenicity of Fractional Dose Inactivated Poliovirus Vaccine in India.

INTRODUCTION: Following the withdrawal of Sabin type 2 from trivalent oral poliovirus vaccine (tOPV) in 2016, the introduction of ≥1 dose of inactivated poliovirus vaccine (IPV) in routine immunization was recommended, either as 1 full dose (0.5mL, intramuscular) or 2 fractional doses of IPV (fIPV-0.1mL, intradermal). India opted for fIPV. We conducted a comparative assessment of IPV and fIPV. METHODS: This was a 4-arm, open-label, multicenter, randomized controlled trial. Infants were enrolled and vaccines administered according to the study design, and the blood was drawn at age 6, 14, and 18 weeks for neutralization testing against all 3 poliovirus types. RESULTS: Study enrolled 799 infants. The seroconversion against type 2 poliovirus with 2 fIPV doses was 85.8% (95% confidence interval [CI]: 80.1%-90.0%) when administered at age 6 and 14 weeks, 77.0% (95% CI: 70.5-82.5) when given at age 10 and 14 weeks, compared to 67.9% (95% CI: 60.4-74.6) following 1 full-dose IPV at age 14 weeks. CONCLUSION: The study demonstrated the superiority of 2 fIPV doses over 1 full-dose IPV in India. Doses of fIPV given at 6 and 14 weeks were more immunogenic than those given at 10 and 14 weeks. Clinical Trial Registry of India (CTRI). Clinical trial registration number was CTRI/2017/02/007793.

A rapid point of care CC16 kit for screening of occupational silica dust exposed workers for early detection of silicosis/silico-tuberculosis.

Silicosis is an irreversible, incurable and progressive occupational disease caused by prolonged exposure to crystalline-silica dust while working in the relevant industries. Conventionally diagnosis is done by chest radiology, often in an advanced stage as early symptoms often go unnoticed. Early detection and necessary intervention (secondary prevention) could be a realistic possible control strategy for controlling silicosis as no effective treatment is available to stop and/or reverse the pathological process. Additionally, these patients are also vulnerable to pulmonary tuberculosis, which often becomes difficult to treat and with uncertain treatment outcome. Considering India has a huge burden of silicosis and silico-tuberculosis, a rapid and inexpensive screening method was realized to be an urgent need for early detection of silicosis among silica dust exposed workers. Serum club cell protein 16 (CC16) is evidenced to be a useful proxy screening marker for early detection of silicosis as evidenced from the recent research work of ICMR-National Institute of Occupational Health (ICMR-NIOH), India. In this study a lateral-flow assay for semi-quantitative estimation of serum CC16 level was developed. The detection was performed using gold nanoparticles conjugated anti-CC16 monoclonal antibodies. A sum of 106 serum samples was tested to do the performance evaluation of the assay. A concentration of 6 ng/ml or less produced one band, 6.1-9 ng/ml produced two bands, while more than 9 ng/ml produced all the three bands at the test zone. The sensitivity of the assay was 100% while the specificity was 95%. This assay may be used as a sensitive tool for periodic screening of silica dust exposed vulnerable workers for early detection of silicosis in them.

SARS-CoV-2 detection in sewage samples: Standardization of method & preliminary observations.

Background & objectives: Since its first recognition in Wuhan, China, in December 2019, the SARS-CoV-2 has spread rapidly across the world. Though SARS-CoV-2 spreads mainly via the droplets of respiratory secretions, it was also detected in stool samples of patients, indicating active infection of the gastrointestinal tract. Presence of SARS-CoV-2 RNA in sewage samples was reported in February 2020, raising the possibility of using environmental water surveillance to monitor SARS-CoV-2 activity in infected areas. The aim of this study was to standardize the methodology for detection of SARS-CoV-2 from sewage and explore the feasibility of establishing supplementary surveillance for COVID-19. Methods: Sewage specimens were collected from six sites in Mumbai, India, using the grab sample method and processed using polyethylene glycol (PEG)-dextran phase separation method for virus concentration. Real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was used to detect the presence of SARS-CoV-2 RNA. Results: A total of 20 sewage samples collected from six different wards in Mumbai city, before the spread of SARS-CoV-2 infections and during May 11-22, 2020, were processed using the phase separation method. The WHO two-phase PEG-dextran method was modified during standardization. SARS-CoV-2 was found to concentrate in the middle phase only. All samples collected before March 16, 2020 were SARS-CoV-2 negative. Viral RNA was detected in sewage samples collected during the ongoing COVID-19 pandemic in all the six wards. Interpretation & conclusions: PEG-dextran phase separation method was effectively used to concentrate SARS-CoV-2 from domestic waste waters to detection levels. It would be feasible to initiate sewage surveillance for SARS-CoV-2 to generate data about the viral transmission in various epidemiologic settings.

Poliomyelitis seroprevalence in high risk populations of India before the trivalent-bivalent oral poliovirus vaccine switch in 2016.

INTRODUCTION: This study assessed the seroprevalence against all three polioviruses among the last cohort of infants aged 6-11 months who received tOPV before the tOPV-bOPV switch and had an opportunity to receive a full dose of inactivated poliovirus vaccine introduced in the routine immunization schedule. METHODS: Serum was tested for neutralizing antibodies against polioviruses among infants residing in three different risk- category states for poliovirus transmission in India viz., Bihar historically high-risk state for polio, Madhya Pradesh a State with low routine immunization coverage and Chhattisgarh with lower acute flaccid paralysis surveillance indicators. RESULTS: A total of 1113 serum samples were tested across the three states. The overall seroprevalence was 98.5% (97.7-99.2), 98.9% (98.3-99.5) and 94.4% (93.0-95.8) for poliovirus types 1, 2 and 3 respectively. The median antibody titers for corresponding serotypes were 575, 362 and 181. Infants who received five doses of tOPV showed respective seroprevalence rates of 98.7%, 98.7% and 93.7% against types 1, 2 and 3 polioviruses. There was no significant difference in seroprevalence across the group of IPV recipients. The median reciprocal titers across the groups of IPV recipient was significantly higher (p = 0.006) for poliovirus-3. CONCLUSION: The seroprevalence rates observed in the study are historically the highest in the series of serosurveys that India has conducted to assess the population immunity against polioviruses. Poliovirus 2 seroprevalence was very high at the time of the tOPV-bOPV switch in India effected in April 2016.

Detection of Immunoglobulin M and Immunoglobulin G Antibodies Against Orientia tsutsugamushi for Scrub Typhus Diagnosis and Serosurvey in Endemic Regions.

OBJECTIVE: To estimate the regional cutoff of optical density (OD) values for immuno-globulin M (IgM) antibodies against Orientia tsutsugamushi in serum and cerebrospinal fluid (CSF) for clinical diagnosis of scrub typhus and immunoglobulin G (IgG) antibodies in serum for sero-epidemiology in Gorakhpur, Uttar Pradesh, India. METHODS: We used data from a serological investigation of acute encephalitis syndrome patients (n=407) during the 2016 outbreak in Gorakhpur, India to determine the cutoff for OD values for IgM antibodies, and from community-based serosurveys (n=1991) to estimate the cutoff for OD values for IgG antibodies. RESULTS: We determined regionally relevant cutoff for OD values of 0.76 for IgM antibodies in serum and 0.22 in cerebrospinal fluid for scrub typhus diagnosis. For serosurveys, IgG antibody cutoff was 1.5. CONCLUSION: We have proposed locally relevant cutoffs for scrub typhus endemic regions, which may be useful for correctly classifying infected population.

Natural Clearance of Prolonged VDPV Infection in a Child With Primary Immunodeficiency Disorder.

The emergence of immunodeficiency-associated vaccine-derived polioviruses (iVDPV) from children with primary immunodeficiency disorders poses a threat to the eradication program. Herein, we report a patient with severe combined immunodeficiency (SCID), identified as a prolonged serotype 3 iVDPV (iVDPV3) excreter with 13 VDPV3 isolates and a maximum of 10.33% nucleotide divergence, who abruptly cleared infection after a period of 2 years. Occurrence of an episode of norovirus diarrhea associated with increased activated oligoclonal cytotoxic T cells, inverse CD4:CD8 ratio, significantly elevated pro-inflammatory cytokines, and subsequent clearance of the poliovirus suggests a possible link between inflammatory diarrheal illness and clearance of iVDPV. Our findings suggest that in the absence of B cells and sufficiently activated T/NK cells, macrophages and other T cells may produce auto-inflammatory conditions by TLR/RLR ligands expressed by previous/ongoing bacterial or viral infections to clear VDPV infection. The study highlights the need to screen all the patients with combined immunodeficiency for poliovirus excretion and intermittent follow-up of their immune parameters if found positive, in order to manage the risk of iVDPV excretion in the polio eradication endgame strategy.

Poliovirus Excretion in Children with Primary Immunodeficiency Disorders, India.

Prolonged excretion of poliovirus can occur in immunodeficient patients who receive oral polio vaccine, which may lead to propagation of highly divergent vaccine-derived polioviruses (VDPVs), posing a concern for global polio eradication. This study aimed to estimate the proportion of primary immunodeficient children with enterovirus infection and to identify the long-term polio/nonpolio enterovirus excreters in a tertiary care unit in Mumbai, India. During September 2014-April 2017, 151 patients received diagnoses of primary immunodeficiency (PID). We isolated 8 enteroviruses (3 polioviruses and 5 nonpolio enteroviruses) in cell culture of 105 fecal samples collected from 42 patients. Only 1 patient with severe combined immunodeficiency was identified as a long-term VDPV3 excreter (for 2 years after identification of infection). Our results show that the risk of enterovirus excretion among children in India with PID is low; however, systematic screening is necessary to identify long-term poliovirus excreters until the use of oral polio vaccine is stopped.

Comparison of IPV to tOPV week 39 boost of primary OPV vaccination in Indian infants: an open labelled randomized controlled trial.

BACKGROUND: The final endgame strategy of global polio eradication initiative includes switching from trivalent oral poliovirus vaccines (tOPV) to bivalent oral polio vaccine (bOPV), and introduction of inactivated poliovirus vaccine (IPV). This study compares IPV with tOPV week 39 boost in Indian infants. METHODS: Starting 28 March 2012, we enrolled 372 Indian infant-mother pairs from Kolkata, India in an open-label, block-randomized, controlled trial comparing a 39 week tOPV to an IPV boost among infants immunized with three doses of tOPV. The primary outcome was mucosal immunity to poliovirus as measured by fecal polio virus shedding after OPV challenge. The secondary outcome was humoral response as defined by >1:8 titers for neutralizing antibodies at week 40. Seroconversion was measured by change in level of antibody titers from week 18 to week 40. The analyses were performed by both intention-to-treat (ITT) and per-protocol (PP) comparing the occurrences of outcomes between the arms of the study. FINDINGS: Both the study arms provided equivalent mucosal immunity at 52 weeks with a total shedding prevalence of 28%. Vaccination with IPV resulted in significantly higher seroconversion rates for Polio type 2 (p = 0.03) and Polio type 3 (p < 0.01). CONCLUSIONS: This study indicates that an IPV boost at week 39 is equivalent to tOPV in intestinal immunity, and provides higher seroconversion compared to tOPV. The major limitation of the study was the additional OPV doses receive by infants during pulse polio immunization resulted in additional mucosal boosting, diminishing the impact of IPV or tOPV boost at week 39. However, IPV for OPV boost should prove to be a step forward in the global polio eradication initiative to reduce the problem of circulating vaccine-derived poliovirus (cVDPV).

Genomic characterization of two new enterovirus types, EV-A114 and EV-A121.

Enteroviruses cause a variety of illnesses of the gastrointestinal tract, central nervous system and cardiovascular system. Phylogenetic analysis of VP1 sequences has identified 106 different human enteroviruses classified into four enterovirus species within the genus Enterovirus of the family Picornaviridae. It is likely that not all enterovirus types have been discovered. Between September 2013 and October 2014, stool samples of 6274 apparently healthy children of up to 5 years of age residing in Gorakhpur district, Uttar Pradesh, India were screened for enteroviruses. Virus isolates obtained in RD and Hep-2c cells were identified by complete VP1 sequencing. Enteroviruses were isolated from 3042 samples. A total of 87 different enterovirus types were identified. Two isolates with 71 and 74 % nucleotide sequence similarity to all other known enteroviruses were recognized as novel types. In this paper we report identification and complete genome sequence analysis of these two isolates classified as EV-A114 and EV-A121.

Assay for identification of heterozygous single-nucleotide polymorphism (Ala67Thr) in human poliovirus receptor gene.

BACKGROUND & OBJECTIVES: It is important to understand the role of cell surface receptors in susceptibility to infectious diseases. CD155 a member of the immunoglobulin super family, serves as the poliovirus receptor (PVR). Heterozygous (Ala67Thr) polymorphism in CD155 has been suggested as a risk factor for paralytic outcome of poliovirus infection. The present study pertains to the development of a screening test to detect the single nucleotide (SNP) polymorphism in the CD155 gene. METHODS: New primers were designed for PCR, sequencing and SNP analysis of Exon2 of CD155 gene. DNAs extracted from either whole blood (n=75) or cells from oral cavity (n=75) were used for standardization and validation of the SNP assay. DNA sequencing was used as the gold standard method. RESULTS: A new SNP assay for detection of heterozygous Ala67Thr genotype was developed and validated by testing 150 DNA samples. Heterozygous CD155 was detected in 27.33 per cent (41/150) of DNA samples tested by both SNP detection assay and sequencing. INTERPRETATION & CONCLUSIONS: The SNP detection assay was successfully developed for identification of Ala67Thr polymorphism in human PVR/CD155 gene. The SNP assay will be useful for large scale screening of DNA samples.

Human Circulating Antibody-Producing B Cell as a Predictive Measure of Mucosal Immunity to Poliovirus.

BACKGROUND: The "gold standard" for assessing mucosal immunity after vaccination with poliovirus vaccines consists in measuring virus excretion in stool after challenge with oral poliovirus vaccine (OPV). This testing is time and resource intensive, and development of alternative methods is a priority for accelerating polio eradication. We therefore evaluated circulating antibody-secreting cells (ASCs) as a potential means to evaluate mucosal immunity to poliovirus vaccine. METHODS: 199 subjects, aged 10 years, and previously immunized repeatedly with OPV, were selected. Subjects were assigned to receive either a booster dose of inactivated poliovirus vaccine (IPV), bivalent OPV (bOPV), or no vaccine. Using a micro-modified whole blood-based ELISPOT assay designed for field setting, circulating poliovirus type-specific IgA- and IgG-ASCs, including gut homing α4ß7+ ASCs, were enumerated on days 0 and 7 after booster immunization. In addition, serum samples collected on days 0, 28 and 56 were tested for neutralizing antibody titers against poliovirus types 1, 2, and 3. Stool specimens were collected on day 28 (day of bOPV challenge), and on days 31, 35 and 42 and processed for poliovirus isolation. RESULTS: An IPV dose elicited blood IgA- and IgG-ASC responses in 84.8 to 94.9% of subjects, respectively. In comparison, a bOPV dose evoked corresponding blood ASC responses in 20.0 to 48.6% of subjects. A significant association was found between IgA- and IgG-ASC responses and serum neutralizing antibody titers for poliovirus type 1, 2, 3 (p<0.001). In the IPV group, α4ß7+ ASCs accounted for a substantial proportion of IgA-ASCs and the proportion of subjects with a positive α4ß7+ IgA-ASC response to poliovirus types 1, 2 and 3 was 62.7%, 89.8% and 45.8%, respectively. A significant association was observed between virus excretion and α4ß7+ IgA- and/or IgG-ASC responses to poliovirus type 3 among immunized children; however, only a weak association was found for type 1 poliovirus. DISCUSSION: Our results suggest that virus-specific blood ASCs, especially for type 3 poliovirus, can serve as surrogate of mucosal immunity after vaccination. Further studies are needed to evaluate the duration of such memory responses and to assess the programmatic utility of this whole blood-based mucosal ASC testing for the polio eradication program.

Real-time reverse transcription-polymerase chain reaction assays for identification of wild poliovirus 1 & 3.

BACKGROUND & OBJECTIVES: The poliovirus serotype identification and intratypic differentiation by real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay is suitable for serotype mixtures but not for intratypic mixtures of wild and vaccine poliovirus strains. This study was undertaken to develop wild poliovirus 1 and 3 (WPV1 and WPV3) specific rRT-PCR assays for use. METHODS: Specific primers and probes for rRT-PCR were designed based on VP1 sequences of WPV1 and WPV3 isolated in India since 2000. The specificity of the rRT-PCR assays was evaluated using WPV1 and WPV3 of different genetic lineages, non-polio enteroviruses (NPEVs) and mixtures of wild/wild and wild/Sabin vaccine strains. The sensitivity of the assays was determined by testing serial 10-fold dilutions of wild poliovirus 1 and 3 stock suspensions of known titre. RESULTS: No cross-reactivity with Sabin strains, intertypic wild poliovirus isolates or 27 types of NPEVs across all the four Enterovirus species was found for both the wild poliovirus 1 and 3 rRT-PCR assays. All WPV1 and WPV3 strains isolated since 2000 were successfully amplified. The rRT-PCR assays detected 10 4.40 CCID 50 /ml of WPV1 and 10 4.00 CCID 50 /ml of WPV3, respectively either as single isolate or mixture with Sabin vaccine strains or intertypic wild poliovirus. INTERPRETATION & CONCLUSIONS: rRT-PCR assays for WPV1 and WPV3 have been validated to detect all the genetic variations of the WPV1 and WPV3 isolated in India for the last decade. When used in combination with the current rRT-PCR assay testing was complete for confirmation of the presence of wild poliovirus in intratypic mixtures.

Immunogenicity of a new routine vaccination schedule for global poliomyelitis prevention: an open-label, randomised controlled trial.

BACKGROUND: Polio eradication needs a new routine immunisation schedule--three or four doses of bivalent type 1 and type 3 oral poliovirus vaccine (bOPV) and one dose of inactivated poliovirus vaccine (IPV), but no immunogenicity data are available for this schedule. We aimed to assess immunogenicity of this vaccine schedule. METHODS: We did an open-label, randomised controlled trial in four centres in India. After informed consent was obtained from a parent or legally acceptable representative, healthy newborn babies were randomly allocated to one of five groups: trivalent OPV (tOPV); tOPV plus IPV; bOPV; bOPV plus IPV; or bOPV plus two doses of IPV (2IPV). The key eligibility criteria were: full-term birth (≥37 weeks of gestation); birthweight ≥2·5 kg; and Apgar score of 9 or more. OPV was administered at birth, 6 weeks, 10 weeks, and 14 weeks; IPV was administered intramuscularly at 14 weeks. The primary study objective was to investigate immunogenicity of the new vaccine schedule, assessed by seroconversion against poliovirus types 1, 2, and 3 between birth and 18 weeks in the per-protocol population (all participants with valid serology results on cord blood and at 18 weeks). Neutralisation assays tested cord blood and sera collected at 14 weeks, 18 weeks, 19 weeks, and 22 weeks by investigators masked to group allocation. This trial was registered with the India Clinical Trials Registry, number CTRI/2013/06/003722. FINDINGS: Of 900 newborn babies enrolled between June 13 and Aug 29, 2013, 782 (87%) completed the per-protocol requirements. Between birth and age 18 weeks, seroconversion against poliovirus type 1 in the tOPV group occurred in 162 of 163 (99·4%, 95% CI 96·6-100), in 150 (98·0%, 94·4-99·6) of 153 in the tOPV plus IPV group, in 153 (98·7%, 95·4-99·8) of 155 in the bOPV group, in 155 (99·4%, 96·5-100) of 156 in the bOPV plus IPV group, and in 154 (99·4%, 96·5-100) of 155 in the bOPV plus 2IPV group. Seroconversion against poliovirus type 2 occurred in 157 (96·3%, 92·2-98·6) of 163 in the tOPV group, 153 (100%, 97·6-100·0) of 153 in the tOPV plus IPV group, 29 (18·7%, 12·9-25·7) of 155 in the bOPV group, 107 (68·6%, 60·7-75·8) of 156 in the bOPV plus IPV group, and in 121 (78·1%, 70·7-84·3) of 155 in the bOPV plus 2IPV group. Seroconversion against poliovirus type 3 was achieved in 147 (90·2%, 84·5-94·3) of 163 in the tOPV group, 152 (99·3%, 96·4-100) of 153 in the tOPV plus IPV group, 151 (97·4%, 93·5-99·3) of 155 in the bOPV group, 155 (99·4%, 96·5-100) of 156 in the bOPV plus IPV group, and 153 (98·7%, 95·4-99·8) of 155 in the bOPV plus 2IPV group. Superiority was achieved for vaccine regimens including IPV against poliovirus type 3 compared with those not including IPV (tOPV plus IPV vs tOPV alone, p=0·0008; and bOPV plus IPV vs bOPV alone, p=0·0153). 12 serious adverse events occurred (six in the tOPV group, one in the tOPV plus IPV group, three in the bOPV group, zero in the bOPV plus IPV group, and two in the bOPV plus 2IPV group), none of which was attributed to the trial intervention. INTERPRETATION: The new vaccination schedule improves immunogenicity against polioviruses, especially against poliovirus type 3. FUNDING: WHO, through a grant from Rotary International (grant number 59735).

Serum IgG and IgA levels in polio and non-polio acute flaccid paralysis cases in western Uttar Pradesh, India.

OBJECTIVE: IgG and IgA immunocompetence of children with wild poliovirus poliomyelitis and non-polio acute flaccid paralysis. METHODS: 932 cases of acute flaccid paralysis, reported in 2008-2009, were tested for presence of polio and non-polio enteroviruses according to the WHO standards. Serum IgA and IgG levels were determined by sandwich ELISA. RESULTS: Mean (SD) IgA levels [0.87 (0.62)g/L; n=28] of virologically confirmed poliomyelitis cases were lower than those of virus negative [1.21 (0.83)g/L; n=612] and non-polio Enterovirus positive [1.22 (0.79)g/L; n=240] cases of acute flaccid paralysis. No significant difference was observed in the concentration of IgG among these groups. CONCLUSIONS: IgA plays an important role in protection against poliomyelitis.

Genetic diversity of enterovirus A71, India.

We have identified circulation of 3 genogroups of enterovirus (EV) A71 in India. A new genogroup (proposed designation G) was discovered during this study. We isolated genogroups D and G in wide geographic areas but detected subgenogroup C1 only in 1 focus in western India. A systematic nationwide search for EV-A71 is warranted.

Assessing population immunity in a persistently high-risk area for wild poliovirus transmission in India: a serological study in Moradabad, Western Uttar Pradesh.

BACKGROUND: Moradabad district in Uttar Pradesh reported the highest number of paralytic polio cases in India during 2001-2007. We conducted a study in Moradabad in 2007 to assess seroprevalence against poliovirus types 1, 2, and 3 in children 6-12 and 36-59 months of age to guide future strategies to interrupt wild poliovirus transmission in high-risk areas. METHODS: Children attending 10 health facilities for minor illnesses who met criteria for study inclusion were eligible for enrollment. We recorded vaccination history, weight, and length and tested sera for neutralizing antibodies to poliovirus types 1, 2, and 3. RESULTS: Poliovirus type 1, 2, and 3 seroprevalences were 88% (95% confidence interval [CI], 84%-91%), 70% (95% CI, 66%-75%), and 75% (95% CI, 71%-79%), respectively, among 467 in the younger age group (n=467), compared with 100% (95% CI, 99%-100%), 97% (95% CI, 95%-98%), and 93% (91%-95%), respectively, among 447 children in the older age group (P<.001 for all serotypes). CONCLUSIONS: This seroprevalence study provided extremely useful information that was used by the program in India to guide immunization policies, such as optimizing the use of different OPV formulations in vaccination campaigns and strengthening routine immunization services. Similar surveys in populations at risk should be performed at regular intervals in countries where the risk of persistence or spread of indigenous or imported wild poliovirus is high.