Abstract
257 stool samples from patients with a clinical diagnosis of cholera and gastroenteritis were received in the microbiology department of Grant Medical College, Mumbai. The samples were further subjected to standard microbiological methods. 30 (11.67%) of them yielded growth of Vibrio cholerae. The strain showed maximum sensitivity to Gentamycin followed by Chloramphenicol, Ciprofloxacin, Tetracycline and Ampicillin. All these isolates belong to the biotype El Tor. The major serotype was Ogawa.

All strains were found to belong to 5 different phage types suggesting that the isolates were from different environmental sites.

Introduction
Vibrio cholerae is the aetiologic agent of Asiatic cholera in humans, a potentially severe diarrhoeal disease that has been responsible for causing seven pandemics.1-3 Diarrhoeal illness ranks first among infectious diseases in terms of incidence and mortality in developing countries. Cholera has a fear complex associated with it due to its high mortality, so it is often under reported.

Material and Methods
257 stool samples were received in the department of microbiology with clinically suspected gastroenteritis over a one-year period from hospitalized patients. All the samples were subjected to hanging drop preparation (HDP) and the preliminary report of HDP was communicated immediately. Simultaneously, samples were inoculated into the alkaline peptone water media for 6 hours at 37^o C.^2,4

Further inoculation was done on Blood agar, Mac Conkeys agar and TCBS agar media and inoculated aerobically for 24 hours at 37^o C.^2,5 Colonies grown were further subjected to a battery of biochemical tests and identification was done by conventional methods.^1,2,4,5 The antibiotic sensitivity was done against Chloramphenicol (30 mcg), Gentamycin (30 mcg), Tetracycline (30 mcg), Cefotaxime (30 mcg), Ciprofloxacin (5 mcg), Ampicillin (10 mcg) Netilmycin (30 mcg), Amikacin (30 mcg) and Ceftizoxime (30 mcg) by modified Kirby-Bauer disc diffusion method.^4,5 Antibiograms were performed as epidemiological markers and to provide a guide for antibiotic changes, if required. Isolates confirmed to be Vibrio cholerae were sent for phage typing to National Institute of Cholera and Enteric Diseases (NICED).

Observations and Results
Over the period of study, Vibrio cholerae was isolated from 30 stool samples i.e.11.67% of the samples received. 13 of these (43.33%), presented with acute gastroenteritis, only 17 (56.66%) presented with the classical symptoms of cholera i.e. rice water stool and severe dehydration.

In the current year, there was no significant difference in the incidence of cholera or gastroenteritis in adults and in children. In the 30 confirmed cases the distribution between adults and children was 54% and 46% respectively (Table 2). However a clear male preponderance was seen in both age groups. The isolation rate of Vibrio cholerae from suspected gastroenteritis was almost double that seen in children (Table 2).

Hanging drop preparation had a specificity of 88.32% and sensitivity of 76.66% in diagnosing cholera (Table 1) taking culture as a gold standard. Fig. 1 shows an increase in incidence of confirmed cases of cholera during the monsoon with a peak in July. All the Cholera isolates showed a higher sensitivity to Gentamycin (80%) followed by Chloramphenicol (73.33%) and Ciprofloxacin (60%), Tetracycline and Ampicillin 56.7% each (Fig. 2).

Table 1 : Shows the results of HDP positive vis a vis culture positive No of cases No of isolates HDP + / Culture + HDP-ve / Culture + 257 ) 30(11.67% 23( 76.66% ) 7(23.33%)	Table 2 : Age and sexwise distribution of Cholera patients Age group No of cases No of isolates Males No (%) Females No (%) Adults >=14 yrs 111 18 (83.33%) 15 (16.21%) 3 (16.66%) Children < 14 yrs 146 12 (8.21%) 8 (66.66%) 4 (33.33%) Total 257 30 (11.67%) 23 (76.66%) 7 (23.33%)
Fig. 1 : Seasonal variation of cholera sases.	Fig. 2 : Antibiotic sensitivity pattern of Vibrio cholera isolates.

In the present study, 14 i.e. (46.66%) strains were multiple drug resistant. All 30 isolates belong to El Tor Ogawa type. Basu and Mukherjee typing scheme showed that all the isolates are of T4 phage type. However “New scheme’’ revealed that 9 were of T-27 phage type, 8 were of T-26, 5 were of T-12 and one was T-24. Thus the “New scheme’’ is more specific compared with Basu and Mukherjee typing scheme, and gives an indication of the different phage types present in the Mumbai city.

Discussion
Parameters like rice water stools and presence of severe dehydration were significant parameters associated with positive stool culture. In the present study, 56.66% patients were having rice water stools and severe dehydration. Amin V etal⁶ reported 0.7% cases with rice water stool, vomiting and severe dehydration. In a study carried out by Kamble et al7 64.70% cases with severe rice water loose motions and 66.66% cases had severe dehydration. The hanging drop preparation method could assist in the rapid diagnosis of Cholera, but has its limitations, as results would depend on the expertise of the person interpreting the results. In our study, it showed a sensitivity of 76.6%, specificity of 88.3% and positive predictive value of 8.9%, negative predictive value 97%. These results correlate with Amin et al6 with sensitivity of 51.5%, specificity of 100% and positive predictive value of 100% in their study.

In the present study only 8.21% culture positivity, was seen in children aged less than 14 years with clinical suspicion of cholera. However, Amin etal⁶ reported 64% positivity below 5 years of age group. Singh etal⁸ observed that the hospitalization rates of cholera were the highest in children aged less than 5 years and decline significantly with increasing age. In a later study by them⁹ they reported 19% culture positivity in infants and 1.4% positivity in children less than 5 years of age group. No significant increase in isolation in children vis a´ vis in adults was observed in our study.

A clear male preponderance was seen in the study. In children out of 12 positive cases 8 i.e.66.66% were males and in adults out of 18, 15 i.e. 83.33% were males. Similar findings were reported by Singh et al.⁹ Rathna K et al10 found no significant difference in the incidence among males (48.6%) and females (51.4%). However Singh et al8 observed males predominantly in the 20-year age group only.

Occurrence of Cholera cases was observed throughout the year with a peak in July. Out of 30 confirmed cases, 13 i.e. 43.3% alone were in July. Other studies also show an increase in incidence in the monsoon.7,11 Singh et al’s⁸ study shows most of the cases during May to September (Summer and Monsoon months) while January to March (Winter) was completely free of Cholera.

Our antibiotic susceptibility pattern was similar to Sundaram et al and Kaur et al.^12-14 i.e. 46.66% strains were multiple drug resistant. Similar results were reported in other studies.^8,11,14

In this study, all 30 isolates belong to El Tor Ogawa type, which correlates with a number of studies.^10,11,12,14 Basu and Mukherjee typing scheme showed all 30 isolates of T4 phage type. Sundaram S P et al showed predominant phage type T2(34%) and T4(61%) respectively from Tamil nadu in their study.¹² An earlier study in Mumbai found that the strains belonged to phage types T2 and T4; T2 showed a higher prevalence (76.5%).15 Kaur et al observed T2 phage type in the early stages of their study.¹³ In the other studies predominant phage types were T2 and T4 respectively.^11,14 However “New scheme” showed that the isolates in the study were from 4 different phage types with a predomination of T27 i.e. 30% followed by T26 i.e. 26.66%. In Sundaram et al study, the ‘’New scheme’’ revealed 22 phage types, with T27 i.e. 68.2% followed by T26 i.e. 12.3% predominantly¹². In Kaur et al study, the ‘’New scheme’’ showed T27 phage type predominantly in later stages.¹³ The spectrum of phage types found over a one-year period in Mumbai suggests that they originated from different sites and some of them may have been imported from other cities.

Conclusion
To conclude, this retrospective analysis shows that 43% of the patients with Vibrio cholerae infection do not present with classical symptoms of Cholera. Hanging drop preparation method still has a limited role to play in the diagnosis and 4 circulating phage types seem to be prevalent in Mumbai during the study period.

References

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