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June 1989, Volume 39, Issue 6

Original Article


Mohammad Mubasbir Ahmad Khan  ( PMRC Central Research Centre and Public Health Division, National Institute of Health, Islamabad. )
Farhat Ara  ( PMRC Central Research Centre and Public Health Division, National Institute of Health, Islamabad. )
Mohammad Osman Yousuf  ( PMRC Central Research Centre and Public Health Division, National Institute of Health, Islamabad. )
Abdul Gbafoor  ( PMRC Central Research Centre and Public Health Division, National Institute of Health, Islamabad. )


Outbreak of acute gastroenteritis occurred during July to August, 1988 in districts of Mansehra, Swat and Muzaffarabad. Thirty cases, clinically diagnosed as cholera, were investigated. On examination, 22 (73.3%) cases were bacteriologically confirmed as cholera due to V. cholerae Eltor, ogawa. All strains were sensitive to chloramphenicol (JPMA 39:151, 1989).


The Eltor biotype discovered in 1905 caused four outbreaks in Celebes (Indonesia) in 1937-38, 1939-40, 1944 and 1957-58. In 1961-62 Eltor caused major epidemics spread throughout Southeast and South Asia, then westward1. Since then Eltor biotype has not only swept previously uninfected areas but has also displaced the classi­cal biotype from the Ganges river basin, where it remained endemic and epidemic until 19722. The global cholera problem at present is al­most exclusively caused by the Ogawa and Inaba serotypes of Eltor biotype of V. cholerae 01, though cases due to classical biotype still occur in Bangladesh3. The Eltor biotypes have greater “en­demic tendency” than classical vibrio because higher infection-to-case ratio and longer survival in the environment4. The endemicity of V. cholerae is not known but sporadic cases of gastroenteritis due to these organisms were seen in both children and adults in different parts of the country. An outbreak of V. cholerae Eltor ogawa was also reported in the District Mansehra in 19865. This study presents the results of investigations of the outbreaks of gastroenteritis during July to August, 1988 in dif­ferent areas of Pakistan.


During July to August, 1988, outbreaks of gastroenteritis were reported from districts of Ma­nsehra and Swat in North West Frontier Province (N.W.F.P.), Muzaffarabad in Azad Kashmir and Rawalpindi in Punjab. An increasing number of cases of gastroenteritis resembling cholera with the general complaints of watery diarrhoea and vomiting were reported in the month of July, 1988 in district Mansehra; 198 such cases were admitted in district headquarter hospital Mansehra, includ­ing 90 children and 108 adults. About 50 children had rice watery stools. Six children and two adults expired, while the rest were successfully treated. In the second week of August, 1988, increas­ing number of cases reported to Saidu Group of hospital, Swat, Bunir and Saidu Sharif subdivision of district Swat. In the third week of August, cases of gastroenteritis were reported from the villages Athmuqam, Dangli, Bagloota and Garrhi dopatta areas of Muzaffarabad district. In the same week gastroenteritis cases were also reported in and around Rawalpindi area. In all the above men- areas, cases were seen both in children and adults, Isolation procedures but generally gastroenteritis affected the adults. In total 30 patients were investigated of which 20 were males and 10 were females. All the patients presented with moderate to severe dehydration, watery diarrhoea of whitish or yellow colour of 1-3 days duration and vomiting. Severe dehydration and shock were seen in some cases. Few patients also camplained of abdominal cramps. Fever (37-38°C) was seen in some cases. Stool was characteristically rice watery and had varied frequency from 5-15/day and vomitus 2-5/day was seen in some patients. The faecal specimens from active cases of gastroenteritis and water samples from the diseased area were brought to the laboratory for the inves­tigation of aetiological agent of this outbreak.


Collection or Specimens
Faecal specimens of 30 patients were inves­tigated for causative agent of the outbreaks. The specimens collected in Cary-Blair and alkaline pe­ptone water (pH 8.6) were sent to our laboratory for microbiological analysis. Samples were processed immediately after arrival.
Twelve water samples were collected in steril­ized containers from different water sources of the infected areas for possible bacteriological examina­tion.
Laboratory Investigations
Faecal specimen was analysed for enteric pathogens according to methods described in "WHO Manual for Laboratory Investigation of Acute Enteric Infections4". The brief outline is given as follows:
1) Microscopic examination
Saline and iodine preparations examined for parasites.
ii) Bacteriological analysis
Faecal specimens examined for the pre­sence of Escherichia coli, Salmonella, Shigella, Vibrio cholerae, campylobacter and Yersinja enterocolitica. MacConkey agar plate was used for E.coli, Salmonella and Shigella species. Faecal sample was also inoculated on Thiosulphate citrate bile salt sucrose agar (TCBS) for vibrio cholerae and Butzlers agar plates for campylobacter. Yersinia selective agar base was used for Yersinia ente­rocolitica. Subcultures were made from selenite broth and alkaline peptone water on salmonella shigella agar (SSA), and TCBS respectively. Yersinia sele­ctive agar base plate was incubated at 32°C while Butzler\'s agar plate was kept at 43°C. All other plates were incubated at 37°C.
The suspected colonies were identified on the above mentioned media and characterized by using special differential media, e.g., triple sugar, iron agar or through a combination of standard biochemical tests. API 20E rapid system was also used where required. The following specialized determinations5 were made after preliminary ide­ntification.
i) Salmonella/Shigella
Serovarieties were determined by slide ag­glutination with specific antisera (Difco).
ii) Enterotoxigenic E.coli
Four lactose fermenting colonies (after biochemical identification) were exam­ined for enterotoxin production (LT and ST).
iii) Enteropathogenic E.coli
Sero-groups were first determined by polyvalent and monovalent OK antisera (Difco). The \'O\' serogroup was confirmed by tube agglutination test using heated suspension and specific monovalent an­tisera.
iv) Vibrio cholerae
Slide agglutination test with polyvalent 01, Inaba and Ogawa antisera (Difco) were done. Haemolysin test was also performed for biotyping of V. cholerae (ELTOR).
v) Campylobacter
Hippurate hydrolysis and other biochem­ical tests were performed.
vi) Virological examination
Virological investigations were carried out by Electron Microscopy6.
Antibiotic Sensitivity
Antibiotic susceptibility test of V. cholerae isolates’ was done by Kirby-Bauer method6 against chloramphenicol, tetracycline, Co-trimoxazole, Amoxil, Furoxone and Streptomycin.
Water samples were analysed according to WHO criteria, for the presence of coliforms and other pathogenic organisms7. Coliforms were detected by multiple-tube method in which me­asured volume of sample was inoculated into a series of tubes containing MacConkey broth with durham tube. The 10 ml volume of water was added to the same quantity of double strength medium (5 tubes). One set of five tubeseach containing 5 ml of single strength broth were inoculated with 1ml volume of water and the other set of five tubes were inoculated with 0.1 ml volume of water. After in­cubation at 37°C for 24-48 hours acid and gas posi­tive tubes were counted and most probable number (MPN) of coliforms were obtained. The samples were also examined for the presence of vibrio, sal­monella and shigella organisms.


The distribution of cases during outbreak of acute gastroenteritis in the 4 districts is shown in Table 1.

Districts Mansehra and Muzaffarábad were the most affected areas, followed by Swat and Rawalpindi areas. The fatality rate in the patients admitted in District headquarter hospital of district Mansebta was 4%. This table also presents the results of bacteriological investigations of 30 faecal samples from the hospitalized cases. The overall isolation rate of V. cholerae was 73.3%. All the strains isolated belonged to Eltor biotype ogawa. One sample of vomitus from Muzaffarabad was also positive for the same V. cholerae biotype. The water samples from the diseased area of Muzaffarabad did not yield any V.cholerae Eltor biotype (Table II).

However, a heavy growth of coliform (1609 +/100ml) in these samples indicates the contamination of water sources which can act as a source of diarrhoeal agents. The antibiotic susceptibility of all the 22 strains of Eltor is shown in Table III.

All the strains were found sensitive to chloramphenicol. 95% of strains were sensitive to tetracycline; followed by co-trimoxazole (77%), and Amoxil (73%). The resistance against furoxone and streptomycin was high.


The findings in the present studies suggest that the increasing incidence of Eltor ogawa cho­lera in the different areas of N.W.F.P. and Punjab is of significant public health importance. The clinical course of the disease in all cases present typical cholera like picture and the outbreak caused panic in local population. Similar outbreak caused by Eltor ogawa in District Mansehra in 19865 suggests possible endemic tendency of Eltor ogawa in this area. In 1988 outbreaks extended to the districts of Swat of N.W.F.P., Muzalfarabad of Azad Kashmir and Rawalpindi of Punjab.
The aetiological relationship of V.cholerae biotype Eltor ogawa is established by 73 percent isolation rate from the hospitalized cases of acute gastroenteritis. The age and sex distribution of the cases did not show any significant fmding. The out­break was proceeded by wide spread rainfall in these districts in the early part of the month of out­break. The ogawa serotype was the only aetiological agent (73.3%) found responsible for this out­break and this is similar to study reported earlier5. Eltor ogawa serotype was also the major causative agent (87.5%) in an outbreak reported from Manipur India. 7 These findings suggest the en­demic tendency of Eltor ogawa in these particular geographical locations. The source of infection and spread might be a human carrier, which is the main source of spread of Eltor cholera infection8. It is possible that carriers might have contaminated rivers, stre­ams and other water sources. Majority of the people in these areas do not get proper drinking water supply and hence depend on river, stream and pond water for drinking and other domestic purposes. The water samples of the diseased area of Muzaffarabad did not yield V.cholerae but they were heavily contaminated with coliform bacteria (1609+/100ml) which is an indicator of faecal pol­lution and could be a source of diarrhoeal agent.
All possible measures should be taken to supply safe drinking water in order to minimize the out­breaks of gastroenteritis. All the 22 strains of Eltor were found sensitive to chloramphenicol (100%), Tetracycline (95%), Co-trimoxazole (77%) and Amoxil (73%). Similar type of results were reported in another study from India7. The resis­tance against furoxone and streptomycin is found in this study.


1. Kamal, A.M. The seventh pandemic of cholera, in cholera, Edited by Barua D. Burrows W. Philadclphia, Saunders, 1974, p.1.
2. World Health Organization. Diarrhoeal Diseases Con­trol Programme. Cholera and other Vibrio associated diarrhoeas; report of a subgroup of the scientific work­ing group on epidemiology and etiology, Geneva, WHO 1979: 24-27 (WHO/DDC/EPE/ 80.3).
3. Zaman, K., Yunus, M. and Hossain, KM. Changing pat­tern of bio and serotypes of vibrio cholerae Olin rural Bangladesh (letter). JPMA., 1987; 37:57.
4. World Health Organization. Guide lines for cholera conS trol. Programme for control of diarrhoeal diseases. Geneva WHO., l9a6:4 (WHO/CDD/SER/80.4 REV 1).
5. Khan, M.A., Azmi, F.H., Israr, N., Rab, A.M., Iqbal, J., Ghafoor, A., Karamat, A.K., Burney, M.I. Gastroenteritis due to vibrio cholerae Eltor Ogawa. JPMA., 1988; 38:170.
6. Bauer, A.W., Kithy, W.M.M., Sherris, J.C. and Turck, PA; Antibiotic susceptibility testing by a standardized single disk method. Am. J.Clin. Pathol., 1966; 45:493.
7. Singh, Y.I., Singh,T.S., Dcvi, U. and Singh, N.B. Cholera outbreak in Manipur. J.Commun. Dis., 1986; 18:109.
8. Deb, B.C., De, S.P. and Pal, S.C. Study of an extensive outbreak of cholera in Cooch Behar during 1974. Indian J. Med. Res., 1979; 70:691.

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