Evolving epidemiology of poliovirus serotype 2 following withdrawal of the serotype 2 oral poliovirus vaccine.

Although there have been no cases of serotype 2 wild poliovirus for more than 20 years, transmission of serotype 2 vaccine-derived poliovirus (VDPV2) and associated paralytic cases in several continents represent a threat to eradication. The withdrawal of the serotype 2 component of oral poliovirus vaccine (OPV2) was implemented in April 2016 to stop VDPV2 emergence and secure eradication of all serotype 2 poliovirus. Globally, children born after this date have limited immunity to prevent transmission. Using a statistical model, we estimated the emergence date and source of VDPV2s detected between May 2016 and November 2019. Outbreak response campaigns with monovalent OPV2 are the only available method to induce immunity to prevent transmission. Yet our analysis shows that using monovalent OPV2 is generating more paralytic VDPV2 outbreaks with the potential for establishing endemic transmission. A novel OPV2, for which two candidates are currently in clinical trials, is urgently required, together with a contingency strategy if this vaccine does not materialize or perform as anticipated.

Strengthening the evidence base for health programming in humanitarian crises.

Given the growing scale and complexity of responses to humanitarian crises, it is important to develop a stronger evidence base for health interventions in such contexts. Humanitarian crises present unique challenges to rigorous and effective research, but there are substantial opportunities for scientific advance. Studies need to focus where the translation of evidence from noncrisis scenarios is not viable and on ethical ways of determining what happens in the absence of an intervention. Robust methodologies suited to crisis settings have to be developed and used to assess interventions with potential for delivery at scale. Strengthening research capacity in the low- to middle-income countries that are vulnerable to crises is also crucial.

Molecular characterization of high-level-cholera-toxin-producing El Tor variant Vibrio cholerae strains in the Zanzibar Archipelago of Tanzania.

Analysis of 1,180 diarrheal stool samples in Zanzibar detected 247 Vibrio cholerae O1, Ogawa strains in 2009. Phenotypic traits and PCR-based detection of rstR, rtxC, and tcpA alleles showed that they belonged to the El Tor biotype. Genetic analysis of ctxB of these strains revealed that they were classical type, and production of classical cholera toxin B (CTB) was confirmed by Western blotting. These strains produced more CT than the prototype El Tor and formed a separate cluster by pulsed-field gel electrophoresis (PFGE) analysis.

Culture-independent real-time PCR reveals extensive polymicrobial infections in hospitalized diarrhoea cases in Kolkata, India.

Culture-independent identification of diarrhoeal aetiological agents was performed using DNA harvested from diarrhoeal stool specimens with SYBR-Green-based real-time PCR targeting Vibrio cholerae, Vibrio parahaemolyticus, Campylobacter spp., Shigella spp. and three different pathotypes of diarrhoeagenic Escherichia coli. Conventional culture-dependent methods detected bacterial enteropathogens in 68 of 122 diarrhoeal stool specimens. Of 68 specimens, 59 (86.8%) had a single pathogen and the remaining nine (13.2%) had polymicrobial infections with multiple pathogens. Re-analysis of the 68 specimens by culture-independent real-time PCR methods showed that 25 (36.8%) specimens contained single pathogen and 43 (63.2%) specimens contained mixed infections with multiple pathogens. The prevalence of such high levels of polymicrobial infections would not have been detected without using real-time PCR. Culture-dependent analysis assigned 54 of the 122 selected archived specimens as 'no known aetiology'. However, re-analysis of these samples by real-time PCR showed the presence of single or multiple pathogens among 34 (63%) of these specimens. Estimation of relative pathogen load by real-time PCR in the stool specimens indicated that the inability of conventional culture-dependent methods to detect the pathogens was related to lower colony-forming units of the pathogen, as reflected by lower C(t) values. Detection of high levels of polymicrobial infection by real-time PCR indicates that in the settings like Kolkata and its surroundings, where cholera and other enteric diseases are endemic, the concept of one pathogen one disease might need to be re-evaluated.

Evaluation of a rapid dipstick test for identifying cholera cases during the outbreak.

BACKGROUND & OBJECTIVES: Intermittent cholera outbreaks are major problem in many of the states of India. It is essential to identify cholera at the earliest for timely mobilization of public health responses and to abort the outbreaks. The present study was a part of a diarrhoeal outbreak investigation in Secunderabad, India, during May 2009 where the usefulness of Crystal VC rapid dipstick kit was assessed for detecting the aetiologic agent of the outbreak. METHODS: Stool specimens were collected from 15 hospitalized patients with acute watery diarrhoea and analyzed for detection of cholera vibrios using Crystal VC rapid dipstick kit and the usefulness of the kit was determined by comparative analysis of the same set of specimens using both microbiological and real-time PCR (RT-PCR) based assays. RESULTS: Detection of Vibrio cholerae O1 from 10 of 15 specimens was recorded using dipstick assay. Microbiological methods detected V. cholerae O1 positivity among 11 specimens. However, RT-PCR based assay showed all 15 specimens positive for the presence of V. cholerae O1. In addition, the same assay showed that the pathogen load in the dipstick as well as RT-PCR positive specimens ranged from 10 6 colony forming units (cfu)/ml or more. INTERPRETATION & CONCLUSIONS: Crystal VC kit had the potential to identify cholera cases in 10 min in field conditions without having good laboratory support. Therefore, dipstick kit may be considered as cholera detecting tool in diarrhoeal outbreak investigations. Specimens from clinically typical cholera cases, if negative by dipstick, should be reanalyzed by culture based methods.

Vibrio cholerae typing phage N4: genome sequence and its relatedness to T7 viral supergroup.

OBJECTIVES: In countries where cholera is endemic, Vibrio cholerae O1 bacteriophages have been detected in sewage water. These have been used to serve not only as strain markers, but also for the typing of V. cholerae strains. Vibriophage N4 (ATCC 51352-B1) occupies a unique position in the new phage-typing scheme and can infect a larger number of V. cholerae O1 biotype El Tor strains. Here we characterized the complete genome sequence of this typing vibriophage. METHODS: The complete DNA sequence of the N4 genome was determined by using a shotgun sequencing approach. RESULTS: Complete genome sequence explored that phage N4 is comprised of one circular, double-stranded chromosome of 38,497 bp with an overall GC content of 42.8%. A total of 47 open reading frames were identified and functions could be assigned to 30 of them. Further, a close relationship with another vibriophage, VP4, and the enterobacteriophage T7 could be established. DNA-DNA hybridization among V. cholerae O1 and O139 phages revealed homology among O1 vibriophages at their genomic level. CONCLUSIONS: This study indicates two evolutionary distinctive branches of the possible phylogenetic origin of O1 and O139 vibriophages and provides an unveiled collection of information on viral gene products of typing vibriophages.

Emergence and progression of Vibrio cholerae O1 El Tor variants and progenitor strains of Mozambique variants in Kolkata, India.

Analysis of 75 Vibrio cholerae O1 strains isolated from hospitalized patients in Kolkata from 1989 to 1994 revealed the existence of true El Tor along with El Tor variants that possessed the classical allele of ctxB (ctxB(cl)) in strains having an El Tor backbone. Based on the existence of different combinations of ctxB and rstR alleles and their localization sites in the genome, these strains were classified into multiple genetic groups. Of 75 clinical strains, 11 were identified as non-toxigenic. These 8 strains were also devoid of pTLC, which is uncommon among the O1 strains. However, Mozambique variants isolated in 2004 were typically negative for pTLC, but these strains possessed tandemly arranged CTX prophages with ctxB(cl) in the small chromosome. Genetic manipulation studies with laboratory-generated kanamycin-tagged pCTX-Kan (derived from tandemly arranged small chromosome-localized ctxB(cl) bearing CTX prophages of 1992 VC53, a progenitor strain of the Mozambique variant) demonstrated that integration specificity of the pCTX-Kan was somewhat towards small chromosome. Such integration could be the prime step towards generation of the Mozambique variant. Based on the existence of multiple alleles of CTXϕ and their infections with non-toxigenic strains, we propose that the El Tor variant strains could have emerged following these genetic events. This study demonstrated existence of different 'intermediate strains' in a time frame that overlapped with a period of V. cholerae O139 emergence. Identification of these intermediate strains gave impetus to believe stepwise generation of the El Tor variants, and all these events profoundly influenced V. cholerae epidemiology in the following years.

Randomized placebo controlled human volunteer trial of a live oral cholera vaccine VA1.3 for safety and immune response.

A live oral cholera vaccine developed from a non-toxigenic Vibrio cholerae O1 El Tor strain VA1.3 was tested in a double-blind randomized placebo controlled study for safety and immunogenicity in 304 men aged between 16 and 50 years from Kolkata, India. A dose of 5 x 10(9)CFU (n=186) or a placebo (n=116) containing the diluent buffer was administered. The vaccine did not elicit adverse events except in two vaccine recipients with mild diarrhoea and vomiting. None excreted the vaccine strain. Vibriocidal antibody response developed in 105/186 (57%) and 5/116 (4%) in vaccine and placebo recipients, respectively. In a subgroup, anti-CT antibody rose (> or =2-folds) in 23/30 (77%) and 6/19 (32%) in vaccine and placebo recipients, respectively. These studies demonstrate that VA1.3 at a dose of 5 x 10(9) is safe and immunogenic in adults from a cholera endemic region.

Incidence, virulence factors, and clonality among clinical strains of non-O1, non-O139 Vibrio cholerae isolates from hospitalized diarrheal patients in Kolkata, India.

The incidence of Vibrio cholerae non-O1, non-O139 strains from hospitalized patients with acute diarrhea constituted 27.4% (n = 54) of the total 197 V. cholerae strains isolated from patients in Kolkata, India, in 2003. Of 197 strains, 135 were identified as O1 serotype Ogawa and 2 were identified as O139. In the same time period, six O1 background rough strains that possessed all known virulence factors were identified. Serotype analysis of the non-O1, non-O139 strains placed 42 strains into 19 serogroups, while 12 remained O nontypeable (ONT); the existing serotyping scheme involved antisera to 206 serogroups. Detection of a good number of ONT strains suggested that additional serogroups have arisen that need to be added to the current serotyping scheme. The non-O1, non-O139 strains were nontoxigenic except for an O36 strain (SC124), which regulated expression of cholera toxin as O1 classical strains did. Additionally, strain SC124 carried alleles of tcpA and toxT that were different from those of the O1 counterpart, and these were also found in five clonally related strains belonging to different serogroups. Strains carrying tcpA exhibited higher colonization in an animal model compared to those lacking tcpA. PCR-based analyses revealed remarkable variations in the distribution of other virulence factors, including hlyA, rtxA, Vibrio seventh pandemic island I (VSP-I), VSP-II, and type III secretion system (TTSS). Most strains contained hlyA (87%) and rtxA (81.5%) and secreted cytotoxic factors when grown in vitro. Approximately one-third of the strains (31.5%) contained the TTSS gene cluster, and most of these strains were more motile and hemolytic against rabbit erythrocytes. Partial nucleotide sequence analysis of the TTSS-containing strains revealed silent nucleotide mutations within vcsN2 (type III secretion cytoplasmic ATPase), indicating functional conservation of the TTSS apparatus.

Biotyping of Vibrio cholerae O1: time to redefine the scheme.

Considering the recent emergence of "hybrid biotype" and "El Tor variant", we propose to redefine the biotyping scheme for Vibrio cholerae O1 serogroup. The existing biotyping scheme has limitations and causes confusion as many of the hybrid biotype and El Tor variant strains have phenotypic and genetic changes. A revised biotyping scheme will play a significant role to understand the ecology, epidemiology and nature of infection of V. cholerae O1 strains in future.

Intestinal parasitism and Vibrio cholerae infection among diarrhoeal patients in Kolkata, India.

In this study, we have analysed the epidemiological significance of the concurrent infections caused by Vibrio cholerae and intestinal parasites among different age groups of hospitalized diarrhoeal patients in Kolkata. A total of 3556 stool samples collected during 1996-2004 were screened for vibrios and parasites. The seasonality of V. cholerae and parasitic infections were studied in detail. The detection rates for Ascaris lumbricoides and Giardia lamblia infection were more than for the hookworm, Trichuris trichiura and Entamoeba histolytica. V. cholerae O1 was identified as the predominant serogroup among diarrhoeal patients. The highest rates for V. cholerae infection were in the 2-5 years age group and combined infection of V. cholerae and parasites was recorded among children aged between 2 and 10 years.

Spread of cholera with newer clones of Vibrio cholerae O1 El Tor, serotype inaba, in India.

During 2004 and 2005, cholera was recorded in 15 states of India, with 7 outbreaks. The newly emerged Vibrio cholerae O1 Inaba had a different antibiogram and ribotype, different pulsotypes, and different mutations in the wbeT gene. Due to the absence of serogroup O139, the Inaba serotype may have acquired the potential to affect the population at large.

Concomitant infection of enterotoxigenic Escherichia coli in an outbreak of cholera caused by Vibrio cholerae O1 and O139 in Ahmedabad, India.

In Ahmedabad, a major city in the state of Gujarat, India, an outbreak of acute secretory diarrhea caused by Vibrio cholerae O1 Ogawa El Tor, V. cholerae O139, and multiple serotypes of enterotoxigenic Escherichia coli (ETEC) occurred in January 2000. All of the representative V. cholerae O1 and O139 isolates examined harbored the ctxA gene (encoding the A subunit of cholera toxin) and the El Tor variant of the tcpA gene (encoding toxin-coregulated pilus). ETEC isolates of different serotypes were positive for the elt gene, encoding heat-labile enterotoxin. To further understand the molecular characteristics of the pathogens, representative isolates were examined by ribotyping and pulsed-field gel electrophoresis (PFGE). Ribotyping showed that the isolates of V. cholerae O1 Ogawa exhibited a pattern identical to that of the prevailing clone of O1 in areas where cholera is endemic in India, and all of the O139 isolates were identical to the BII clone of V. cholerae O139. PFGE of the representative O1 Ogawa isolates exhibited an identical pattern, comparable to the H pattern of the new clone of O1 reported in Calcutta, India. PFGE analysis of the V. cholerae O139 isolates showed identical patterns, but these differed from the PFGE patterns of O139 isolates reported during 1992 to 1997 in Calcutta. ETEC isolates showed genetic heterogeneity among isolates belonging to the same serotype, although the identical PFGE pattern was also observed among ETEC isolates of different serotypes. Antibiograms of the isolates were unusual, because all of the O139 isolates were resistant to nalidixic acid. Likewise, all of the E. coli isolates showed resistance to ciprofloxacin, norfloxacin, and nalidixic acid. This is a unique outbreak, and we believe that it is the first in which V. cholerae and ETEC were concomitantly involved.

Complex humanitarian emergencies: a major global health challenge.

Complex humanitarian emergencies have been a major political, security and public health feature of the post-Cold War world. These man-made disasters account for more morbidity and mortality than all natural and technological disasters combined. In order to deliver effective aid during complex humanitarian emergencies, international relief agencies must have a solid understanding of the political and social climates in which they are operating. In addition, they should base their health interventions on objective epidemiological data, especially standardized rates of morbidity and mortality. Most deaths during complex humanitarian emergencies are due to preventable causes, especially increased rates of infectious diseases malnutrition and violent trauma. The most appropriate health interventions are therefore based on the models of public health and primary health care, emphasizing disease prevention and health promotion. The field of humanitarian assistance has become increasingly professionalized in recent years, with its own professional standards, literature, research agenda and training opportunities. It is an unfortunate reflection on the current state of international affairs that the number of complex humanitarian emergencies and the enormous levels of suffering associated with them are unlikely to decline in the foreseeable future.

Rehabilitating public health infrastructure in the post-conflict setting: epidemic prevention and preparedness in Kosovo.

The war in Kosovo in 1999 resulted in the displacement of up to 1.5 million persons from their homes. On the subsequent return of the refugees and internally displaced persons, one of the major challenges facing the local population and the international community, was the rehabilitation of Kosovo's public health infrastructure, which had sustained enormous damage as a result of the fighting. Of particular importance was the need to develop a system of epidemic prevention and preparedness. But no single agency had the resources or capacity to implement such a program. Therefore, a unique six-point model was developed as a collaboration between the Kosovo Institute of Public Health, the World Health Organization, and an international, non-governmental organization. Important components of the program included a major Kosovo-wide baseline health survey, the development of a province-wide public health surveillance system, rehabilitation of microbiology laboratories, and the development of a local capacity for epidemic response. While all program objectives were met, important lessons were learned concerning the planning, design, and implementation of such a project. This program represents a model that potentially could be replicated in other post-conflict or development settings.

Modification of the multiplex PCR for unambiguous differentiation of the El Tor & classical biotypes of Vibrio cholerae O1.

BACKGROUND & OBJECTIVES: Biotyping of Vibrio cholerae O1 using multiplex PCR (ctxA-tcpA) exploits the nucleotide sequence differences of the major subunit protein of the toxin co-regulated pilus (TCP) gene (tcpA) to differentiate between the classical and El Tor biotypes. However, the presence of classical biotype specific tcpA amplicon with the El Tor strains often complicates the interpretation. The effect of PCR variables on the amplification of biotype specific tcpA in the multiplex PCR has been investigated. METHODS: Reference strains of toxigenic V. cholerae O1 belonging to classical and El Tor biotypes were selected to optimize the PCR variables for the unambiguous biotype determination by multiplex PCR. RESULTS: In the multiplex PCR assay, a reduction in the reaction volume from 100 microliters to 25 microliters and the annealing temperature of 64 degrees C, the El Tor strain produced ctxA amplicon (302 bp) along with tcpA amplicons of 618 bp and 472 bp which are specific for classical and El Tor tcpA respectively. The simplex PCR with biotype specific tcpA primer pairs showed the amplification of either 472 bp or 618 bp tcpA amplicon with El Tor template. With the classical biotype strain, the specific primer pair yielded tcpA amplicon of the expected size. Lowering of PCR annealing temperature from 64 to 60 degrees C resulted in the elimination of the amplification of the nonspecific tcpA amplicon with El Tor strain. INTERPRETATION & CONCLUSION: A comparison of the theoretical melting temperature (Tm) values of the reacting primers, and their alignment to the biotype specific tcpA revealed the basis of unambiguous biotyping of V. cholerae O1 at a PCR annealing temperature of 60 degrees C.

Cluster-analysis & patterns of dissemination of multidrug resistance among clinical strains of Vibrio cholerae in Calcutta, India.

BACKGROUND & OBJECTIVES: Antimicrobial resistance among Vibrio cholerae has been monitored for several years in Calcutta. To investigate the changing trends in multidrug resistance (MDR) among different serogroups of V. cholerae and to perform software assisted cluster analysis the current study was undertaken. METHODS: Strains isolated from patients with cholera and "cholera-like" diarrhoea admitted in the Infectious Diseases Hospital, Calcutta were analysed. Eight hundred and forty V. cholerae strains isolated from 1992 through 1997 were tested for susceptibility to 11 antibiotics. Cluster analysis was done using SPSS software. RESULTS: Most of the strains exhibited MDR with fluctuating trends as the resistance profile diverged each year. A total of 119 different resistance profiles exhibited by V. cholerae O1, O139 and non-O1, non-O139 serogroups were analysed by cluster combination method. During 1993 and 1994, 53 per cent of V. cholerae O139 and 82 per cent of V. cholerae O1 serogroups, respectively, exhibited maximal number of new resistance patterns. The frequency of new resistance patterns among V. cholerae non-O1, non-O139 was constantly high (33-47%) during 1995 to 1997. INTERPRETATION & CONCLUSIONS: With a few exceptions, preponderance of the resistance profiles was generally not confined to any serogroup. The cluster analysis depicted dissemination of some of the resistance patterns commonly found among V. cholerae non-O1, non-O139 belonging to different serogroups to the O139 serogroup in the succeeding years. In this study we have shown that the V. cholerae strains are resistant to several antibiotics with constant change in the MDR profiles. It is imperative to define the susceptibility pattern of the strains to determine the effective drug of choice for the treatment of cholera.

Rapid method for species-specific identification of Vibrio cholerae using primers targeted to the gene of outer membrane protein OmpW.

The distribution of genes for an outer membrane protein (OmpW) and a regulatory protein (ToxR) in Vibrio cholerae and other organisms was studied using respective primers and probes. PCR amplification results showed that all (100%) of the 254 V. cholerae strains tested were positive for ompW and 229 ( approximately 98%) of 233 were positive for toxR. None of the 40 strains belonging to other Vibrio species produced amplicons with either ompW- or toxR-specific primers, while 80 bacterial strains from other genera tested were also found to be negative by the assay. These studies were extended with representative number of strains using ompW- and toxR-specific probes in DNA dot blot assay. While the V. cholerae strains reacted with ompW probe, only one (V. mimicus) out of 60 other bacterial strains tested showed weak recognition. In contrast, several strains belonging to other Vibrio species (e.g., V. mimicus, V. splendidus, V. alginolyticus, V. fluvialis, V. proteolyticus, V. aestuarianus, V. salmonicida, V. furnissii, and V. parahaemolyticus) showed weak to strong reactivity to the toxR probe. Restriction fragment length polymorphism analysis and nucleotide sequence data revealed that the ompW sequence is highly conserved among V. cholerae strains belonging to different biotypes and/or serogroups. All of these results suggest that the ompW gene can be targeted for the species-specific identification of V. cholerae strains. The scope of this study was further extended through the development of a one-step multiplex PCR assay for the simultaneous amplification of ompW and ctxA genes which should be of considerable value in the screening of both toxigenic and nontoxigenic V. cholerae strains of clinical as well as environmental origin.