A novel and simple mixture as point-of-use water treatment agent to produce safe drinking water.

BACKGROUND: People in rural Bangladesh have a poor understanding of the link between use of contaminated surface water and disease. An inexpensive point-of-use water treatment agent was developed to purify surface water. METHODS: Surface water was collected from various sources in Bangladesh from February 2007 to January 2008. Microbiological and physicochemical parameters of raw and treated surface water were analysed. Water was treated with a mixture of alum potash, bleaching powder and lime, or with each agent individually. RESULTS: Raw water was contaminated with bacteria, the counts for total coliforms, faecal coliforms and faecal streptococci being 26,431, 14,548 and 240 colony-forming units (cfu) 100 ml(-1), respectively. These counts fell to 0 cfu 100 ml(-1) after treatment with the mixture. The count of artificially introduced Vibrio cholerae was also reduced to 0 cfu 100 ml(-1) after treatment. Treatment of raw water altered the pH from 6.90 to 6.87, turbidity from 21.61 to 3.55 nephelometric turbidity units (NTU), residual chlorine from 0 to 0.09 mg litre(-1), conductivity from 124.03 to 229.96 µS cm(-1), and total dissolved solids from 59.40 to 199.25 mg litre(-1). All these results of treatment were within the range recommended by the WHO as acceptable for drinking water. CONCLUSION: The mixture of alum potash, bleaching powder and lime described can be safely used to disinfect contaminated surface water to make it suitable for drinking and other household purposes in Bangladesh.

Genetic characterization of Vibrio cholerae O1 strains isolated in Zambia during 1996-2004 possessing the unique VSP-II region of El Tor variant.

New variants of Vibrio cholerae O1 have appeared in different time-frames in various endemic regions, especially in Asia and Africa. Sixty-nine strains of V. cholerae O1 isolated in Zambia between 1996 and 2004 were investigated by various genotypic techniques to determine the lineage of virulence signatures and clonality. All strains were positive for Vibrio seventh pandemic Islands (VSP)-I and VSP-II and repeat toxin (RTX) gene clusters attesting their El Tor lineage. Interestingly, strains isolated in recent times (2003-2004) were identified as an altered variant (El Tor biotype that harbours El Tor type rstR but produce classical ctxB) that replaced completely the progenitor El Tor strains prevalent in 1996-1997. Recent altered variant strains differed from prototype El Tor strains isolated earlier in that these strains lacked two ORFs, VC0493 and VC0498, in the VSP-II region. PFGE analysis revealed two major clonal lineages in the strains; cluster A represented the strains isolated before 2003 and cluster B the altered strains isolated in 2003-2004. Cluster A was closely related to prototype El Tor reference strain isolated in Bangladesh in 1971. Cluster B was found to be matched with Bangladeshi altered strains but was different from the hybrid strains isolated from Mozambique and Bangladesh. This report provides important information on the genesis of altered strains of V. cholerae O1 isolated in Zambia and emphasizes the need for further studies to follow the trends of evolutionary changes.

Multilocus variable-number tandem repeat analysis of Vibrio cholerae O1 El Tor strains harbouring classical toxin B.

Atypical Vibrio cholerae O1 strains - hybrid strains (strains that cannot be classified either as El Tor or classical biotype) and altered strains (El Tor biotype strains that produce classical cholera toxin) - are currently prevalent in Asia and Africa. A total of 74 hybrid and altered strains that harboured classical cholera toxin were investigated by multilocus variable-number tandem repeat analysis (MLVA). The results showed that the hybrid/altered strains could be categorized into three groups and that they were distant from the El Tor strain responsible for the seventh cholera pandemic. Hybrid/altered strains with a tandem repeat of the classical CTX prophage on the small chromosome were divided into two MLVA groups (group I: Mozambique/Bangladesh group; group III: Vietnam group), and altered strains with the RS1-CTX prophage containing the El Tor type rstR and classical ctxB on the large chromosome were placed in two MLVA groups (group II: India/Bangladesh group; group III: India/Vietnam group).

Classical RS1 and environmental RS1 elements in Vibrio cholerae O1 El Tor strains harbouring a tandem repeat of CTX prophage: revisiting Mozambique in 2005.

Currently, Vibrio cholerae O1 serogroup biotype El Tor strains producing classical type cholera toxin (altered strains or El Tor variants) are prevalent in Asia and in Mozambique. Mozambican strains collected in 2004 contained a tandem repeat of CTX prophage on the small chromosome and each CTX prophage harboured the classical rstR and classical ctxB. We found that the majority of the strains collected in 2005 in Mozambique contained extra elements on the large chromosome in addition to the tandem repeat of CTX prophage on the small chromosome. New type RS1 elements RS1(cla) and RS1(env), and a CTX(env) with rstR(env) and the classical ctxB were identified on the large chromosome of the Mozambican isolates collected in 2005.

Cholera outbreaks caused by an altered Vibrio cholerae O1 El Tor biotype strain producing classical cholera toxin B in Vietnam in 2007 to 2008.

Vibrio cholerae O1 isolates collected during cholera outbreaks occurring from late 2007 to early 2008 in northern Vietnam were revealed to represent an altered strain containing the RS1 element followed by a CTX prophage harboring El Tor type rstR and classical ctxB on the large chromosome.

Classification of hybrid and altered Vibrio cholerae strains by CTX prophage and RS1 element structure.

Analysis of the CTX prophage and RS1 element in hybrid and altered Vibrio cholera O1 strains showed two classifiable groups. Group I strains contain a tandem repeat of classical CTX prophage on the small chromosome. Strains in this group either contain no element(s) or an additional CTX prophage or RS1 element(s) on the large chromosome. Group II strains harbor RS1 and CTX prophage, which has an E1 Tor type rstR and classical ctxB on the large chromosome.

Characteristics of a pandemic clone of O3 : K6 and O4 : K68 Vibrio parahaemolyticus isolated in Beira, Mozambique.

The genetic characteristics of Vibrio parahaemolyticus strains isolated in 2004 and 2005 in Mozambique were assessed in this study to determine whether the pandemic clone of V. parahaemolyticus O3 : K6 and O4 : K68 serotypes has spread to Mozambique. Fifty-eight V. parahaemolyticus strains isolated from hospitalized diarrhoea patients in Beira, Mozambique, were serotyped for O : K antigens and genotyped for toxR, tdh and trh genes. A group-specific PCR, a PCR that detects the presence of ORF8 of the filamentous phage f237, arbitrarily primed PCR, PFGE and multilocus sequence typing were performed to determine the pandemic status of the strains and their ancestry. All strains of serovars O3 : K6 (n=38) and O4 : K68 (n=4) were identified as a pandemic clonal group by these analyses. These strains are closely related to the pandemic reference strains of O3 : K6 and O4 : K68, which emerged in Asia in 1996 and were later found globally. The pandemic serotypes O3 : K6 and O4 : K68 including reference strains grouped into a single cluster indicating emergence from a common ancestor. The O3 : K58 (n=8), O4 : K13 (n=6), O3 : KUT (n=1) and O8 : K41 (n=1) strains showed unique characteristics different from the pandemic clone.

Is HIV infection associated with an increased risk for cholera? Findings from a case-control study in Mozambique.

OBJECTIVE: As residents of sub-Saharan Africa are at high risk for HIV and cholera, it is biologically plausible that immune suppression caused by HIV infection predisposes to cholera. Our aim was to assess the potential association between both diseases. METHODS: We conducted a case-control study in Beira, Mozambique, a high-risk area for HIV and cholera. Between 1 January 2005 and 30 June 2006, experienced counsellors invited 132 suspected cholera cases and 528 age- and sex-matched controls to an HIV counselling and testing centre. RESULTS: Forty (30%) of the invited cases and 127 (24%) of the invited controls came for HIV testing. No significant differences in demographic and socio-economic baseline characteristics were detected between participants and non-participants. Twenty five of 167 (15%) individuals who underwent testing were found HIV-positive. The probability of a positive HIV-test was highest in participants between 40 and 49 years; 6 of 14 (43%) tested HIV-positive. Nine of 40 (23%) cholera cases were found to be HIV-infected compared with 16 of 127 (13%) controls (adjusted odds ratio 2.6; 95% CI 0.9-7.5; P = 0.08). DISCUSSION: The findings suggest that in a cholera-endemic area, HIV infection is associated with an increased risk for cholera. More research in HIV endemic settings is needed to confirm the findings and to explore the effect of HIV-related immunosuppression on the transmission of cholera.

Characterization of enterotoxigenic Escherichia coli from diarrhoeal patients in Bangladesh using phenotyping and genetic profiling.

A total of 99 isolates out of 370 colonization factor (CF)-positive, well-characterized enterotoxigenic Escherichia coli (ETEC) strains belonging to 13 different CF types isolated from diarrhoeal patients admitted to the hospital of the International Centre for Diarrhoeal Disease Research, Bangladesh, were tested. The isolates were selected at random based on expression of the major CFs prevailing in Dhaka, Bangladesh, from 1996 to 1998. These isolates were characterized by O-antigenic serotyping, randomly amplified polymorphic DNA (RAPD) analysis and biochemical fingerprinting using the PhenePlate (PhP) system. The 99 ETEC isolates belonged to 10 O serogroups, the predominant ones being O6 (n=28), O115 (n=20) and O128 (n=20). Most isolates of serogroup O6 (CS1+CS3, 11/14; CS2+CS3, 5/8) belonged to the same PhP/RAPD type (H/f), whereas other isolates of serogroup O6 (n=12) belonged to different PhP/RAPD types (Si/f and F/c). Eleven serogroup O128 (CFA/I) isolates belonged to the same PhP/RAPD type (E/b), whereas the other O128 isolates formed different PhP/RAPD types. Fifteen (75%) serogroup O115 isolates (together with fourteen isolates from serogroups O25, O114, O142 and O159) demonstrated two closely related common groups by PhP typing (A and A1) and belonged to the same PhP/RAPD type (A/a). Three major clonal groups were identified among the ETEC strains in this study, largely based on O-antigenic type, CF expression pattern and toxin profile.

Multiply antibiotic-resistant Vibrio cholerae O1 biotype El Tor strains emerge during cholera outbreaks in Zambia.

Antibiotic resistance data, made available from laboratory records during eight cholera outbreaks between 1990 and 2004 showed Vibrio cholerae serogroup O1 to have a low level of resistance (2-3%) to tetracycline during 1990-1991. Resistance increased for tetracycline (95%), chloramphenicol (78%), doxycycline (70%) and trimethoprim-sulphamethoxazole (97%) in subsequent outbreaks. A significant drop in resistance to tetracycline and chloramphenicol followed the adoption of a national policy to replace tetracycline with erythromycin for treating cholera. Sixty-nine strains from cholera outbreaks in Zambia between 1996 and 2004, were examined for antibiotic resistance and basic molecular traits. A 140 MDa conjugative, multidrug-resistant plasmid was found to encode tetracycline resistance in strains from 1996/1997 whereas strains from 2003/2004 were resistant to furazolidone, but susceptible to tetracycline, and lacked this plasmid. PCR revealed 25 of 27 strains from 1996/1997 harboured the intl1 class 1 integron but lacked SXT, a conjugative transposon element. Similar screening of 42 strains from 2003/2004 revealed all carried SXT but not the intl1 class 1 integron. All 69 strains, except two, one lacking ctxA and the other rstR and thus presumably truncated in the CTX prophage region, were positive for important epidemic markers namely rfbO1, ctxA, rstR2, and tcpA of El Tor biotype. Effective cholera management is dependent on updated reports on culture and sensitivity to inform the choice of antibiotic. Since the emergence of antibiotic resistance may significantly influence strategies for controlling cholera, continuous monitoring of epidemic strains is crucial.

Genetic characteristics of Matlab variants of Vibrio cholerae O1 that are hybrids between classical and El Tor biotypes.

The Matlab variants of Vibrio cholerae O1, defined as hybrids between the classical and El Tor biotypes, were first isolated from hospitalized patients with acute secretory diarrhoea in Matlab, a rural area of Bangladesh. These variants could not be categorized as classical or El Tor biotypes by phenotypic and genotypic tests, and had representative traits of both the biotypes. A number of virulence-associated genes and/or gene clusters were screened by PCR and DNA sequencing. El Tor-specific gene clusters, Vibrio seventh-pandemic islands (VSP)-I and -II and repeat toxin (RTX) were present in the genome of these variants, indicating their El Tor lineage, whereas the nucleotide-sequence-derived CtxB amino acid sequence of these strains grouped them under the classical biotype. Matlab variants possessed all the necessary genes to initiate pandemics. The genetic relatedness of Matlab variants to the V. cholerae strains recently isolated in Mozambique is another important observation of this study, which underscores the epidemiological significance of Matlab variants.

Field evaluation of a rapid immunochromatographic dipstick test for the diagnosis of cholera in a high-risk population.

BACKGROUND: Early detection of cholera outbreaks is crucial for the implementation of the most appropriate control strategies. METHODS: The performance of an immunochromatographic dipstick test (Institute Pasteur, Paris, France) specific for Vibrio cholerae O1 was evaluated in a prospective study in Beira, Mozambique, during the 2004 cholera season (January-May). Fecal specimens were collected from 391 patients with acute watery nonbloody diarrhea and tested by dipstick and conventional culture. RESULTS: The overall sensitivity and specificity of the rapid test compared to culture were 95% (95% confidence interval [CI]: 91%-99%) and 89% (95% CI: 86%-93%), respectively. After stratification by type of sample (rectal swab/bulk stool) and severity of diarrhea, the sensitivity ranged between 85% and 98% and specificity between 77% and 97%. CONCLUSION: This one-step dipstick test performed well in the diagnosis of V. cholerae O1 in a setting with seasonal outbreaks where rapid tests are most urgently needed.

Multilocus sequence typing (MLST) analysis of Vibrio cholerae O1 El Tor isolates from Mozambique that harbour the classical CTX prophage.

Vibrio cholerae O1 isolates belonging to the Ogawa serotype, El Tor biotype, harbouring the classical CTX prophage were first isolated in Mozambique in 2004. Multilocus sequence typing (MLST) analysis using nine genetic loci showed that the Mozambique isolates have the same sequence type (ST) as O1 El Tor N16961, a representative of the current seventh cholera pandemic. Analysis of the CTX prophage in the Mozambique isolates indicated that there is one type of rstR in these isolates: the classical CTX prophage. It was also found that the ctxB-rstR-rstA-rstB-phs-cep fragment was PCR-amplified from these isolates, which indicates the presence of a tandem repeat of the classical CTX prophage in the genome of the Mozambique isolates. The possible origin of these isolates and the presence of the tandem repeat of the classical prophage in them implicate the presence of the classical CTX phage.

Pandemic serovars (O3:K6 and O4:K68) of Vibrio parahaemolyticus associated with diarrhea in Mozambique: spread of the pandemic into the African continent.

Forty-two episodes of Vibrio parahaemolyticus infections were detected in Beira, Mozambique, from January to May 2004. The majority of the isolates (81%) belonged to the pandemic serovars (O3:K6 and O4:K68) of V. parahaemolyticus. The pandemic serovars were positive by group-specific PCR (GS-PCR) and a PCR specific for open reading frame ORF8 (ORF8-PCR), which are molecular markers of the pandemic clone, and were positive for tdh but negative for trh. The remaining 19% of the strains also possessed the tdh gene but were GS-PCR and ORF8-PCR negative and did not belong to the pandemic serovars. Patients with V. parahaemolyticus infection were older (mean age, 27 years) than patients infected by other diarrheal agents (mean age, 21 years). Ten percent of diarrhea patients from whom no V. parahaemolyticus was cultured were severely dehydrated, but none of the V. parahaemolyticus cases were severely dehydrated. This is the first report of the isolation of pandemic strains of V. parahaemolyticus in sub-Saharan Africa and clearly indicates that the pandemic of V. parahaemolyticus has spread into the African continent.

Isolation and characterization of provisional serovar Shigella boydii E16553 from diarrhoeal patients in Bangladesh.

In previous studies with strains of the Shigella dysenteriae provisional serovars E22383 and E23507 from diarrhoeal stools from patients in Bangladesh, two strains of Shigella species were identified as Shigella boydii provisional serovar E16553 by a reference laboratory. Further tests with an antiserum to an international type strain of the provisional serovar E16553 identified an additional 15 isolates. None of the isolates reacted with antisera to the established Shigella serovars or any other provisional serovars reported so far and all showed biochemical reactions typical of S. boydii. All of the isolates harboured the 140 MDa invasion plasmid, had the ipaH gene and produced keratoconjunctivitis in the guinea pig eye. All isolates were susceptible to ampicillin, sulfamethoxazole-trimethoprim, nalidixic acid, ciprofloxacin and mecillinam but eight strains were resistant to tetracycline. A single PFGE type (type A) was shown for all 17 clinical isolates, indicating a common source of origin. The pulsotype of the Bangladeshi isolates was closely related to that of a Japanese strain but was different from that of the type strain. On the basis of these biochemical, serological and virulence markers, and diverse geographical origin, it is recommended that the provisional status of serovar E16553 be changed and that it be included in the international serotyping classification scheme as S. boydii 19.

Effectiveness of mass oral cholera vaccination in Beira, Mozambique.

BACKGROUND: New-generation, orally administered cholera vaccines offer the promise of improved control of cholera in sub-Saharan Africa. However, the high prevalence of human immunodeficiency virus (HIV) infection in many cholera-affected African populations has raised doubts about the level of protection possible with vaccination. We evaluated a mass immunization program with recombinant cholera-toxin B subunit, killed whole-cell (rBS-WC) oral cholera vaccine in Beira, Mozambique, a city where the seroprevalence of HIV is 20 to 30 percent. METHODS: From December 2003 to January 2004, we undertook mass immunization of nonpregnant persons at least two years of age, using a two-dose regimen of rBS-WC vaccine in Esturro, Beira (population 21,818). We then assessed vaccine protection in a case-control study during an outbreak of El Tor Ogawa cholera in Beira between January and May 2004. To estimate the level of vaccine protection, antecedent rates of vaccination were compared between persons with culture-confirmed cholera severe enough to have prompted them to seek treatment and age- and sex-matched neighborhood controls without treated diarrhea. RESULTS: We assessed the effectiveness of the vaccine in 43 persons with cholera and 172 controls. Receipt of one or more doses of rBS-WC vaccine was associated with 78 percent protection (95 percent confidence interval, 39 to 92 percent; P=0.004). The vaccine was equally effective in children younger than five years of age and in older persons. A concurrently conducted case-control study designed to detect bias compared persons with treated, noncholeraic diarrhea and controls without diarrhea in the same population and found no protection associated with receipt of the rBS-WC vaccine. CONCLUSIONS: The rBS-WC vaccine was highly effective against clinically significant cholera in an urban sub-Saharan African population with a high prevalence of HIV infection.

Diverse CTX phages among toxigenic Vibrio cholerae O1 and O139 strains isolated between 1994 and 2002 in an area where cholera is endemic in Bangladesh.

PCR surveillance of the rstR genes of CTX phages in Vibrio cholerae O1 and O139 showed no relationship between the incidence of disease and changes in the rstR but showed variations in their presence in O1 and O139 strains and the occurrence of multiple types in a few strains.

Phenotypic and genotypic characterization of provisional serotype Shigella flexneri 1c and clonal relationships with 1a and 1b strains isolated in Bangladesh.

The serotypes of 144 strains of Shigella flexneri serotype 1 (serotypes 1a, 1b, and 1c) isolated from patients attending the Dhaka treatment center of the International Centre for Diarrhoeal Disease Research, Bangladesh, between 1997 and 2001 were serologically confirmed by using commercially available antisera and a panel of monoclonal antibodies specific for S. flexneri group and type factor antigen (MASF). Among serotype 1 isolates, the prevalence of provisional serotype S. flexneri 1c increased from 0 to 56% from 1978 to 2001 in Bangladesh. Detailed biochemical studies revealed that none of the strains of serotype 1 produced indole, while all the strains fermented mannose, mannitol, and trehalose. Twenty percent of the serotype 1c and all the serotype 1a strains fermented maltose and 53% of the serotype 1c strains and 60% of the serotype 1a strains fermented arabinose, whereas all serotype 1b strains were negative for fermentation of these sugars. Only 18% of serotype 1b strains were resistant to nalidixic acid, and most of the serotype 1c and 1b strains were resistant to ampicillin, tetracycline, and trimethoprim-sulfamethoxazole. All the strains of serotypes 1a and 1b and about 88% of the serotype 1c strains were found to be invasive by the Sereny test, had a 140-MDa plasmid, and had Congo red absorption ability. Plasmid profile analysis showed that 26% of the strains of serotype 1 contained identical patterns. Most of the serotype 1c strains (72%) had the 1.6-MDa plasmid, which was not found in either serotype 1a or 1b strains. A self-transmissible middle-range plasmid (35 to 80 MDa) was found in some strains carrying the multiple-antibiotic-resistance gene. Pulsed-field gel electrophoresis analysis yielded three types (types A, B, and C) with numerous subtypes among the serotype 1c strains, whereas serotypes 1b and 1a yielded only one type for each serotype, and those types were related to the types for serotype 1c strains. Ribotyping analysis yielded three patterns for serotype 1c strains and one pattern each for serotype 1a and 1b strains which were similar to the patterns for the serotype 1c strains. Overall analysis of the results concluded that subserotype 1c is closely related to serotypes 1a and 1b. Furthermore, the high rate of prevalence of serotype 1c necessitates the commercial production of antibody against this subserotype to allow the determination of the actual burden of shigellosis caused by provisional serotype 1c.

Prevalence of the pandemic genotype of Vibrio parahaemolyticus in Dhaka, Bangladesh, and significance of its distribution across different serotypes.

Sixty-six strains of Vibrio parahaemolyticus belonging to 14 serotypes were isolated from hospitalized patients in Dhaka, Bangladesh, from January 1998 to December 2000. Among these, 48 strains belonging to four serotypes had the pandemic genotype and possessed the tdh gene. A marker (open reading frame ORF8) for a filamentous phage previously thought to correspond to the pandemic genotype was found to have a poor correlation with the pandemic genotype.