International Journal of Infectious Diseases 14 (2010) e384–e389

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Epidemics of severe cholera caused by El Tor Vibrio cholerae O1 Ogawa possessing the ctxB gene of the classical biotype in Orissa, India B.B. Pal a,*, H.K. Khuntia a, S.K. Samal a, S.K. Kar a, B. Patnaik b a b

Microbiology Division, Regional Medical Research Centre (ICMR), Bhubaneswar 751023, Orissa, India Integrated Disease Surveillance Project, Directorate of Health Services, Government of Orissa, Orissa, India

A R T I C L E I N F O

A B S T R A C T

Article history: Received 29 August 2008 Received in revised form 28 April 2009 Accepted 15 June 2009

Background: We investigated the epidemic of cholera that occurred in Kashipur and Dasmantpur blocks of Orissa, reported during July–September 2007. Methods: Sixty-two rectal swabs and 28 water samples collected from diarrhea patients at different hospitals and villages were bacteriologically analyzed for the identification, antibiogram, and detection of toxic genes of Vibrio cholerae. Results: The cholera outbreaks were caused by V. cholerae O1 Ogawa biotype El Tor in both Kashipur and Dasmantpur blocks. All the V. cholerae isolates from the clinical and environmental samples were sensitive to tetracycline, gentamicin, azithromycin, and chloramphenicol, but were resistant to ampicillin, ciprofloxacin, norfloxacin, co-trimoxazole, nalidixic acid, neomycin, and furazolidone, except the water isolates, which were sensitive to ciprofloxacin and norfloxacin. The multiplex PCR assay revealed that all the clinical and environmental V. cholerae isolates were positive for the ctxA and tcpA genes, showing biotype El Tor. Interestingly, 88% of the clinical and environmental isolates of V. cholerae were El Tor biotype with mutation at the ctxB gene of the classical strain, as confirmed by mismatch amplification of mutation (MAMA)-PCR assay. Conclusions: This is the first report of the El Tor variant of V. cholerae O1 Ogawa having the ctxB gene of the classical strain with altered antibiogram causing epidemics of cholera in Orissa, India. ß 2009 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Corresponding Editor: J. Peter Donnelly, Nijmegen, the Netherlands Keywords: El Tor Vibrio cholerae O1 ctxB gene Classical Orissa

1. Introduction Vibrio cholerae causing severe diarrheal disorders has appeared in epidemic proportions in many developing countries and is endemic in Asia, Africa, and South America. The toxigenic V. cholerae serogroup O1 has frequently been reported from SubSaharan African countries. Serogroup O1 is classified into two biotypes, classical and El Tor. The seventh and most recent pandemic of cholera was caused by the El Tor biotype. The classical biotype reported in earlier years was believed to have become extinct in the recent past. A mutant of V. cholerae biotype El Tor was reported from Mozambique during January–March 2004.1 Genetic hybrids between El Tor and classical biotypes of O1 V. cholerae were reported from sporadic outbreaks in Bangladesh during 2002 and named Matlab variants.2 Sporadic outbreaks have been reported from many places, including Sangli District of Maharastra,3 Trivandrum, South India,4 and Kolkata.5 Outbreaks of diarrhea are one of the major public health problems in Orissa. The coastal districts of Orissa, situated in the eastern part of India, frequently report severe diarrheal disorders

* Corresponding author. Tel.: +91 674 2302801; fax: +91 674 2301351. E-mail address: [email protected] (B.B. Pal).

following floods and cyclones. Sporadic diarrheal outbreaks including cholera were reported after the super cyclone in 19996 and also during 2005,7 from which V. cholerae O1 Ogawa and Inaba serotypes, biotype El Tor strains were isolated. Outbreaks of cholera due to V. cholerae O1 biotype El Tor during 1993 were reported from Koraput and Nabarangpur districts.8 During the months of July–September 2007 an epidemic of severe cholera affecting large ethnic groups was reported from Kashipur Block of Rayagada District, spreading to adjacent blocks of bordering districts, including Laxmipur and Dasmantapur of Koraput District, Thuamul Rampur of Kalahandi District, and areas of Gajapati District in Orissa. We report herein the epidemiological investigation and the causative organism of this recent cholera epidemic.

2. Methods 2.1. Sample collection Rectal swabs from patients with diarrhea were collected in Cary–Blair transport medium at various public health centers (PHCs) and community health centers (CHCs) of Kashipur and Dasmantapur blocks and also from the households of affected

1201-9712/$36.00 – see front matter ß 2009 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijid.2009.06.020

B.B. Pal et al. / International Journal of Infectious Diseases 14 (2010) e384–e389

villages. These rectal swabs were collected from patients prior to the initiation of any antibiotics. The samples were transported to the Regional Medical Research Center laboratory within 24 h of collection for bacteriological analysis. At the same time, environmental water samples were collected from the streams, rivers, chua (small pond in paddy field), nala (small stream), tube wells, and household water supplies from various affected villages.

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identification of cholera toxin of classical and El Tor biotypes. For this, a conserved forward primer (FW-Com) and two allele-specific primers, Rv-cla and Rv-elt, were designed that can amplify ctxB of classical and El Tor biotypes, respectively.12 Using the above technique the environmental and clinical isolates of all V. cholerae O1 strains were subjected to simplex PCR assay to detect the V. cholerae O1 El Tor variant that harbors classical ctxB and V. cholerae O1 that harbors El Tor ctxB separately.

2.2. Processing of samples 3. Results Rectal swabs were inoculated onto MacConkey agar, thiosulfate citrate bile salt sucrose (TCBS) agar, and Hektoen enteric agar (HEA) and enrichment was done in selenite F broth and alkaline peptone water (APW).9 Similarly, water samples were processed through membrane filtration and sedimentation, and enriched in double-strength APW medium and then sub-cultured on TCBS plates to look for the growth of V. cholerae strains. V. cholerae was identified by standard biochemical test. Serotyping was carried out for confirmation of V. cholerae using antisera obtained from Becton and Dickinson (BD, USA). 2.3. Antimicrobial susceptibility testing The sensitivity and the resistance patterns of V. cholerae O1 strains were tested with antibiotic-impregnated commercial disks (Hi-Media, Mumbai, India) using ampicillin (10 mg), chloramphenicol (30 mg), co-trimoxazole (25 mg), ciprofloxacin (5 mg), furazolidone (100 mg), gentamicin (10 mg), neomycin (30 mg), nalidixic acid (30 mg), norfloxacin (10 mg), streptomycin (10 mg), tetracycline (30 mg), azithromycin (15 mg), and polymyxin B (50 mg). V. cholerae O1 strains were sub-cultured in tryptic soy broth (BD, USA) and plated on Mueller–Hinton agar (BD, USA). Plates were incubated for 24 h at 37 8C. Characterization of strains as susceptible or resistant was determined based on the size of the inhibition zones around each antibiotic disk in accordance with the manufacturer’s instructions, following the Kirby–Bauer technique (1966).10 2.4. PCR assay A multiplex PCR-based assay was employed to determine the presence of the A-subunit of cholera toxin gene (ctxA) and to biotype the V. cholerae strains by targeting tcpA (encoding the major structural subunit of the toxin-coregulated pilus), which is specific for El Tor and classical strains by the method described by Keasler and Hall.11 2.5. Mismatch amplification mutation assay (MAMA)-PCR to differentiate the cholera toxin B subunit of classical and El Tor biotypes of V. cholerae O1 The MAMA-PCR was designed to detect the nucleotide sequence difference at position 203 of the ctxB gene for the

The clinical signs and symptoms in the diarrhea patients were sudden onset of belching abdominal pain within 5–6 h, with rice watery stool, vomiting, muscular cramping, and rapid progress of severe dehydration. The severity of the cholera was due to the El Tor variants of the ctxB gene of the classical strain of V. cholerae, as evidenced by MAMA-PCR assay. Analysis by month and year of the severe diarrheal attacks and deaths in Kashipur Block during last 4 years indicated that the diarrheal attacks increased significantly in the months of July–September 2007 in comparison to previous years (2004–2006). The data obtained from the Health Department, Government of Orissa revealed that the outbreak affected six blocks (Kashipur, Kolnara, Dasmantpur, Laxmipur, Thuamul Rampur, and Mohana) of four adjacent western districts of Orissa, namely Rayagada, Koraput, Gajapati, and Kalahandi. Diarrhea cases and deaths by block and district for July–October 2007 in the four districts of Orissa are shown in Table 1 and Figure 1. Out of the six blocks of the four affected districts, 358 villages were affected, with a population at risk of 123 546. One hundred and sixty-two deaths occurred, with an attack rate (AR) of 6.64% and a case-fatality rate (CFR) of 1.97%. The outbreak began with the index case (35-year-old male) on July 13, 2007 in the village of Andirakanch, Maikanch Gram Panchayat, Kashipur Block and subsided by September 30, 2007. Figure 2 shows the distribution of affected villages in Kashipur Block by month (July–September 2007). The villages of Tikirapadar under Chandragiri, Maikanch, Godibali, and Hadiguda showed the highest attack rates, which exceeded 30%; the lowest incidences of diarrhea cases were reported in Talajhiri and some villages of Tikiri Gram Panchayat (GP) and Sunger GP with attack rates varying from 4% to 10%. The incidence of acute diarrhea cases from July 13 to October 10, 2007 in the six affected blocks is shown in Figure 3. A total of 62 rectal swabs were collected from hospitalized diarrhea patients of both Kashipur and Dasmantpur blocks (52 from hospitals and 10 from the affected villages) during the month of August 2007. Simultaneously, 28 environmental water samples from sources commonly used by people were collected from the affected villages of the two blocks (22 from Kashipur and six from Dasmantpur block: nala = 7, chua = 7, house water = 3, bore well = 2, stream = 4, dug well = 2, river = 2, water reservoir = 1). It was found that 97.4% of rectal swabs from Kashipur Block and 78.6% from Dasmantpur Block were positive for V. cholerae O1

Table 1 Diarrhea cases and deaths by district and block; July 13–October 10, 2007a Period

District

Blocks

Villages affected

Population at risk

No. of cases

No. of deaths

Attack rate

CFR

July to Oct 10, 2007

Rayagada

Aug to Oct 10, 2007

Koraput

Kashipur Kolnara Dasmantpur Laxmipur Thuamul Rampur Mohana

92 24 127 34 20 61 358

35 963 7093 35 574 14 808 4492 25 616 123 546

4015 103 2409 1239 253 187 8206

52 11 43 18 31 7 162

11.16 1.45 6.77 8.37 5.63 0.73 6.64

1.29 10.68 1.78 1.45 12.25 3.74 1.97

Sept to Oct 10, 2007 Total

Kalahandi Gajapati

CFR, case-fatality rate. a Source: State Health Surveillance Cell.

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Figure 1. Orissa blocks affected by diarrhea, July–September 2007.

Ogawa (Table 2). Similarly, eight out of the 28 (28.6%) water samples (two from nala, three from chua, one from river, and two from spring water) were positive for V. cholerae O1 Ogawa biotype El Tor. All the strains of V. cholerae isolated from clinical samples were resistant to ciprofloxacin, norfloxacin, ampicillin, streptomycin, neomycin, nalidixic acid, furazolidone and co-trimoxazole, whereas the water isolates were sensitive to ciprofloxacin, norfloxacin, gentamicin, azithromycin, tetracycline and chloramphenicol. The clinical V. cholerae isolates were sensitive to gentamicin, azithromycin, chloramphenicol, and tetracycline. The multiplex PCR assay showed that all V. cholerae isolates were positive for ctxA and tcpA genes, showing biotype El Tor. Interestingly 88% of the V. cholerae strains isolated from the diarrhea patients and environmental samples were sensitive to polymyxin B, showing phenotypic classical biotype. These strains were Voges–Proskauer (VP)-positive and positive for El Tor hemolysis by tube agglutination method exhibiting phenotypic trait of El Tor. The MAMA-PCR assays for the differentiation of the cholera toxin B subunit of classical and El Tor biotypes of V. cholerae O1 indicated that most of the strains were a V. cholerae O1 Ogawa biotype El Tor variant that harbors the classical ctxB gene (Figure 4). 4. Discussion Contamination of water sources is the main cause of transmission of Cholera, a water-borne disease. During the rainy season, people usually go to their paddy fields for farming and drink water from the river, chua, nala, streams, etc. in the field, becoming infected in this way. The water samples collected from springs, chua, nala and rivers from the diarrhea-affected villages were found to be contaminated with V. cholerae O1 Ogawa. We conducted an investigation of the diarrhea outbreak in the village of Parbatia, Kashipur Block during the year 2002, which was due to V. cholerae O1 Ogawa biotype El Tor (unpublished). Niyogi et al.8

reported an outbreak of cholera due to V. cholerae O1 biotype El Tor in Koraput and Nabarangpur districts of Orissa during May–June 1993. The outbreak was mostly confined to the schedule caste and schedule tribe population who are socially backward, mostly illiterate, and poor. The majority of the diarrhea patients were over 20 years of age (78.1%). Analysis of the incidence of severe diarrhea cases during the years 2004–2007 in Kashipur Block indicated that the highest number of cases was recorded during July–August 2007 in comparison to the previous three years (2004–2006). Ninetytwo out of the 499 villages of Kashipur Block were affected by the diarrheal disorder. Due to population migration and water source contamination, the infection spread to Thuamul Rampur of Kalahandi District, Laxmipur Block of Koraput District, and Mohana Block of Gajapati District, which are close to Kashipur Block. This indicated that the localized diarrhea outbreak of Kashipur Block took an epidemic pattern. The early reporting of the causative organism, which was isolated from stool and water samples, chlorination of different water sources, and intervention with appropriate control measures kept the cholera epidemic in check in this region. A high percentage of resistance to ciprofloxacin, norfloxacin, nalidixic acid, and furazolidone of the V. cholerae O1 Ogawa was observed in the present study, in contrast to the strain isolated during the 1999 and 2005 cholera outbreaks from the state, which were sensitive to ciprofloxacin and norfloxacin.6,7 Fluoroquinolone-resistant V. cholerae strains were reported from South India in 2002,13 a high prevalence of furazolidone resistance has been reported in Bangladesh,14 and a progressive increase in ciprofloxacin and norfloxacin resistance among V. cholerae O1 strains isolated between 1995 and 2000 from Kolkata has been reported.15 The emergence of fluoroquinolone resistance in V. cholerae needs attention as it complicates the therapeutic use of these drugs. All the environmental and clinical isolates of V. cholerae were positive for ctxA and tcpA genes, showing biotype El Tor, and they were hybrids of a V. cholerae O1 Ogawa biotype El Tor variant that

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Figure 2. Diarrhea-affected villages of Kashipur Block, Rayagada District by month, July–September 2007.

harbors the classical ctxB gene as determined by simplex MAMAPCR assay. However, the clinical and environmental isolates of V. cholerae exhibited different antibiograms; this might be due to different mutations at their respective antibiotic virulence genes. Retrospective analysis of V. cholerae strains by MAMA-PCR assay indicated that the hybrid strain of El Tor variants of ctxB gene of the classical strain emerged in Koraput District during 2000, appearing in Kashipur Block of Rayagada District in 2002, and subsequently in coastal districts in very small numbers during 2005, 2006, and 2007. Interestingly, no cholera outbreak caused by this hybrid strain of V. cholerae O1 Ogawa was reported during these periods. Of note is that this hybrid strain of V. cholerae O1 Ogawa emerged

in epidemic form during 2007 in western parts of Orissa. Recently, three variants of El Tor biotypes of V. cholerae O1 have been described, including (1) the Matlab variants, which cannot be biotyped, having a combination of classical and El Tor features,16 (2) the Mozambique variants that have a typical El Tor genome but a tandem repeat of classical prophage located in the small chromosome,1 and (3) altered El Tor strains, which have an El Tor CTX prophage but produce cholera toxin of the classical type.17 Our clinical and environmental isolates of V. cholerae O1 from the present cholera epidemic may be related to the same strains of El Tor variants that produce cholera toxin of the classical type, however this needs further analysis by random amplification of

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Figure 3. Epicurve showing incidence of diarrhea cases in six blocks of four districts of Orissa, July 13–October 10, 2007.

Table 2 Bacteriological analysis of rectal swabs collected from diarrhea patients from different outbreak areas

Total samples Culture-positive Escherichia coli Vibrio cholerae O1 Ogawa Inaba O139 Salmonella Shigella Aeromonas Culture-negative

polymorphic DNA-PCR (RAPD-PCR), ribotyping, and pulsed field gel electrophoresis (PFGE), etc.

Kashipur

Dasmantpur

5. Conclusion

40 39 (97.5%) 0 38 (97.4%) 37 (94.9%) 1 (2.6%) 0 0 1 (2.6%) 0 1 (2.5%)

22 14 (63.6%) 2 (14.3%) 11 (78.6%) 11 (78.6%) 0 0 0 0 1 (7.1%) 8 (36.4%)

This is the first report of a high percentage of resistant V. cholerae O1 Ogawa biotype El Tor with ctxB gene of classical strain having epidemic potential being reported from the western part of two districts of Orissa, India. It is likely that future outbreaks/ epidemics of diarrheal disorders in Orissa may occur due to this El Tor variant of V. cholerae with classical ctxB gene, which warrants close monitoring in other parts of Orissa and India. Acknowledgements The authors are grateful to Dr G.B. Nair, Director and Dr T. Ramamurthy, Deputy Director of the National Institute of Cholera and Enteric Diseases (NICED), Kolkata for providing the MAMAPCR assay technique for establishing the El Tor variants of V. cholerae O1, before publication, and for their valuable comments and suggestions. We are also thankful to the Director, Directorate of Health Services, Government of Orissa, CDMO of Koraput and Rayagada districts, ADMO (PH), Rayagada, Dr H. Das, (MO) Kashipur CHC, and the MO of Dasmantpur CHC for their kind co-operation, help and providing necessary assistance during the investigation. The help rendered by Dr B. Dwibedi (RO) and Dr A.S. Kerketta (SRO) from Regional Medical Research Centre, Bhubaneswar is acknowledged. We are thankful to Mithun Karmakar, GIS mission consultant, NRHM, Orissa for preparing the map. Conflict of interest: No conflict of interest to declare. References

Figure 4. Simplex MAMA-PCR assay for the detection of representative hybrid classical and El Tor Vibrio cholerae strains isolated from the Kashipur and Dasmantpur area during August–September 2007. Lane 1, ladder; lane 2, hybrid El Tor reference strain; lane 3, classical strain; lanes 4, 6, 7, 9, 10, 12–18, clinical and water isolates of V. cholerae O1 (O) with ctxB mutation with classical primer; lanes 5 and 8, clinical and water isolates of V. cholerae O1 (O) negative for ctxB mutation; lane 11, negative control; lanes 19 and 20, negative for El Tor ctxB mutation with El Tor primer.

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