Original Article

Prevalence of Cholera in Pediatric Patients with Acute

Dehydrating Diarrhea
Vijesh S. Kuttiat, Rakesh Lodha, Bimal Das and Utkarsh Kohli

Department of Pediatrics, All-India Institute of Medical Sciences, New Delhi, India

ABSTRACT
Objective. To estimate the prevalence of culture-confirmed cholera in patients with acute dehydrating diarrhea, at a tertiary
care center in north India, during a 6-month period from March to August, 2006.

Methods. We studied 145 children, who presented to the pediatric emergency services of a tertiary care teaching hospital
in north India with acute dehydrating diarrhea. Each patient had his/her stool sample collected for Vibrio cholerae culture and
hanging drop preparation for darting motility. The stool specimen for hanging drop analysis was immediately transported to
the emergency laboratory, where a trained technician prepared the slides and examined them for darting motility characteristic
of Vibrio cholerae.

Results. V. cholerae was isolated in 36 (24.8%) patients. Forty-nine (33.7%) patients had a positive hanging drop examination.
Hanging drop examination had a sensitivity and specificity of 85.8% and 81.7%, respectively. Severe dehydration (OR 4.3;
P<0.01) and hanging drop positivity (OR 12.42; P<0.001) were associated with higher odds of cholera after adjustment for
other risk factors.

Conclusion. Cholera is an important cause of acute watery diarrhea in pediatric patients in urban north India and should be
ruled out in all children presenting with acute dehydrating diarrhea, particularly those with severe dehydration. Hanging drop
test is useful for diagnosis in the emergency setting. [Indian J Pediatr 2010; 77 (1) : 67-71] E-mail: utkarshkohli @hotmail.
com

Key words : Cholera; Pediatric; Acute dehydrating diarrhea

Diarrhea is a major cause of morbidity and mortality in is one of the few causes of childhood diarrhea where
the developing world. Cholera is an important contributor antibiotics are indicated and are known to improve
to the burden of diarrheal diseases, and its incidence outcome.6
varies across the developing world ranging from 0.5/
The present study was carried out to estimate the
1000/year in Indonesia to 4/1000/year in Mozambique.1
prevalence of culture-confirmed cholera in patients with
Incidence of cholera in India has been estimated to be 1.6/
acute dehydrating diarrhea at a tertiary care centre in
1000 population/year1 or 40/1000 cases of acute diarrhea.2
north India, during a 6-month period from March to
Cholera is a significant contributor to morbidity in the
August, 2006. We also evaluated were stool hanging drop
pediatric population in India and other developing
preparation slides from these patients for a secondary
countries. 1-3 Recently, concern has been raised about
analysis to determine the sensitivity and specificity of this
widespread under-reporting of cholera cases in the
rapid and inexpensive diagnostic test to accurately
developing world, particularly in south Asia, due to social
diagnose cholera.
and economic considerations. 4, 5 More importantly,
routine screening for cholera is not incorporated in our
national diarrheal diseases control program, leading to MATERIAL AND METHODS
under-diagnosis of this important condition.4 Implications
of under-diagnosis of cholera are potentially serious as it
We enrolled 145 patients, who presented to the pediatric
emergency services of a tertiary care teaching hospital in
Correspondence and Reprint requests : Dr Utkarsh Kohli, MD. north India with acute dehydrating diarrhea between
Research Fellow, Division of Clinical Pharmacology, Department of March and August, 2006. Children presenting during the
Medicine, Vanderbilt University Medical Center, Nashville, duty hours of the participating physician team were
Tennessee-37203, USA.
considered for enrollment. All children presenting with
[Received March 22, 2009; Accepted September 23, 2009]

Indian Journal of Pediatrics, Volume 77January, 2010 67

 Vijesh S. Kuttiat et al
diarrheal symptoms were independently assessed by two
physicians for signs of dehydration as per World Health
Organization (WHO) criteria7 and the patients were only
included if the findings of both the physicians were in
agreement.
Each patient with acute dehydrating diarrhea had his/
her stool sample collected for Vibrio cholerae culture and
hanging drop preparation for darting motility. The stool
samples were mixed with Venkataraman-Ramakrishnan
(VR) transport medium and transported to the
bacteriology laboratory within 8 hours of collection as per
WHO recommendations.8 Vibrio cholerae remains viable in
this medium for upto 6 weeks after stool collection and
the medium can be stored at room temperature after
collection.8 The stool specimen for hanging drop analysis
was immediately transported to the emergency
laboratory, where a trained technician prepared the slides
and examined them for darting motility characteristic of
Vibrio cholerae. All enrolled subjects were managed as per
standard WHO case management guidelines.7
The stool specimens were cultured directly on
thiosulphate citrate bile salt sucrose (TCBS) agar and
incubated at 37o Celcius for 16-20 hours. Pre-enrichment of
stool samples in alkaline peptone broth was also carried
out with subsequent subculture on thiosulphate citrate
bile salt sucrose (TCBS) agar. Sucrose fermenting yellow
colored colonies were selected and further identified as V.
cholerae by biochemical tests. Final confirmation was done
using a slide agglutination test with specific anti-sera
against V. cholerae (Denka Seiken, Tokyo, Japan).
Antibiotic sensitivity testing was carried out on Mueller
Hinton agar (BecktonDickinson, USA) by Kirby-Bauer
disc diffusion method using Clinical and Laboratory
Standards Institute (CLSI) guidelines.9 Antibiotics used in
the sensitivity testing were ampicillin (10 g), tetracycline
(30 g), septran (trimethoprim- sulfamethoxazole) (30 g),
nalidixic acid (30 g) and chloramphenicol (30 g).
Eschericia coli ATCC 25922 strain was used as quality
control strain. Antibiotic sensitivity profile of each isolate
was recorded as resistant, intermediate and sensitive
based on the zone diameter according to the guidelines of
CLSI.9
Statistical analysis: Data is expressed as median and
interquartile range from all normally distributed data are
expressed as mean S.D. Unadjusted and adjusted odds
ratios for having cholera, as defined by a positive stool
culture for Vibrio cholerae, were calculated using logistic
regression models. Covariates included severity of
dehydration, stool hanging drop positivity, stool
consistency, and stool frequency. Continuous variables
like stool frequency and stool duration were recoded as
categorical variables. Stool frequency per hour in 8 hours
prior to presentation to the pediatric emergency was
recoded as a categorical variable and patients were
divided into 3 categories: those with < 1 loose stool/hr, 1-
68
2 loose stools/hr and >2 loose stools/hr. Analyses were
performed with the statistical software STATA10
(StataCorp, College Station, TX).
RESULTS
One hundred and forty-five patients (median age: 2 year;
interquartile range: 1/6-12 year; 80 males) were enrolled
in the present study. The median duration of diarrhea for
at the time of presentation was 24 (2-120) hr, and the
median stool frequency (stools per hr) in the 8 hours prior
to presentation was 0.73 (0.12-3) loose stools/hr. Thirty-six
(24.8%) patients had culture positivity and 49 (33.7%)
patients had a positive hanging drop examination.
Hanging drop examination had a sensitivity and
specificity of 85.8% and 81.7%, respectively, for detecting
a patient with positive Vibrio cholerae culture (Table 1).
All the subjects had watery stools; 10.5% had typical
rice watery stools as assessed by the enrolling physicians.
TABLE 1.. Demographic Characteristics
Parameter (n=145) Median (IQR)
Age (yrs) 2 (1/6-12)
Sex Male/Female 80/65
Duration (hrs) 24 (2-120)
Frequency (stools/hr) 0.73 (0.12-3)
Consistency Watery/Rice Watery* 129/15
Vomiting Yes/No* 125/19
Fever Yes/No** 36/107
Dehydration Some/Severe 76/69
Hanging Drop Positive/Negative 49/96
Stool Culture Positive/Negative 36/109
* Information could not be recorded for 1 child
** Information could not be recorded for 2 children
Fig. 1. Month wise break-up of dehydrated and Vibrio cholerae
culture positive patients.
Proportion of both culture positive and hanging drop
positive patients was significantly higher in patients with
rice watery than watery stools (46.7% vs 21.7% [P=0.033]
for stool culture and 60% vs 30.2% [P= 0.021] for hanging
drop examination, respectively). Approximately half
(47.5%) of the patients had severe dehydration at the time
Indian Journal of Pediatrics, Volume 77January, 2010
 Prevalence of Cholera in Pediatric Patients with Acute Dehydrating Diarrhea

TABLE 2. Risk Factors for Culture-Confirmed Cholera with Their Respective Odds Ratios.

Risk factor Unadjusted analysis Adjusted analysis

Odds ratio P Odds ratio P

Rice water diarrhea 3.15 0.04 1.55 0.58

Severe dehydration 4.97 <0.001 4.3 0.008
Hanging drop positive 18.43 <0.001 12.42 <0.001
Fever 0.40 0.08 0.77 0.71
Sex 0.55 0.12 0.89 0.83
Frequency/hr 1-2$ 1.07 0.89 0.91 0.89
Frequency/hr >2$ 6.46 0.04 4.3 0.25
Duration 24-48 hrs* 0.63 0.38 0.58 0.43
Duration >48 hrs* 0.24 0.03 0.36 0.25

*Baseline group: Duration less than 24 hr

$
Baseline group: Frequency/hr < 1

of enrollment. Proportion of both culture positive and
hanging drop positive patients was significantly higher in
patients with severe dehydration than some dehydration
(39.1% vs 11.8% [P=0.0001] for stool culture and 43.4% vs
23.6% [P=0.007] for hanging drop, respectively). A third
of the patients (33.7%) had a positive stool hanging drop
preparation. Stool culture positivity was significantly
higher in patients with positive hanging drop preparation
(P<0.0001), and hanging drop positivity was the most
important indicator of culture confirmed cholera
(Adjusted odds ratio=12.4; P<0.001).
The unadjusted odds ratio (OR) for having cholera was
6.5 times higher in subjects with >2 loose stools/hr than
those with <1 loose stool/hr and 1-2 loose stools/hr
(P=0.04). Stool frequency did not have a significant effect
on odds for having cholera after correction for other
important risk factors like dehydration (P=0.25).
Those who presented with diarrheal duration of more
than 48 hrs had significantly lower odds in favor of
cholera than those who presented within 48 hrs of onset of
diarrhea (OR=0.24; P=0.03). This effect was lost after
adjustment for other risk factors like hanging drop
positivity, severity of dehydration and stool frequency
(P=0.25) (Table 2). Also analyzed was the sensitivity
pattern for the isolates of Vibrio cholerae (Table 3). The
majority of the isolates were resistant to trimethoprim-
sulfamethoxazole, nalidixic acid, and ampicillin.
DISCUSSION
A very high prevalence (24.8%) of culture positive cholera,
TABLE 3. Antibiotic Sensitivity Pattern of Vibrio cholerae Isolates.
Antibiotic Sensitive (%) Partially Resistant (%)
Sensitive (%)
Ampicillin 6 (17.1) 1 (2.9) 28
Tetracycline 19 (54.3) - 16
Septran 4 (11.4) - 31
Nalidixic acid 1 (3) - 33
Chloramphenicol 23 (67.7) 1 (3) 10
as defined by culture positivity, was found in pediatric
patients with acute dehydrating diarrhea during the
period from March to August, 2006. While current
national guidelines do not recommend routine screening
for cholera in patients with acute dehydrating diarrhea,
the present study data presents a strong case in favor of
routine screening of children with acute dehydrating
diarrhea for cholera presenting to the hospital.
Hanging drop examination had a sensitivity of 85.8%
and a specificity of 81.7% in the patients. It is inexpensive
and can be rapidly performed in an emergency setting,
and hence, can play an important role in rapid
identification of cholera patients for appropriate therapy.
Stool cultures reveal the pattern of Vibrio cholerae strains
prevalent in the community and their antibiotic
susceptibility and thus help in guiding appropriate
antimicrobial therapy. The majority of the Vibrio cholerae
isolates were resistant to commonly prescribed antibiotics
like trimethoprim-sulfamethoxazole, nalidixic acid and
ampicillin. Interestingly, almost half of the cultured Vibrio
cholerae were sensitive to chloramphenicol and
tetracycline (Table 3). These results indicate a change in
antibiotic susceptibility pattern of circulating Vibrio
cholerae strains over the last decade with increased
resistance to commonly prescribed antibiotics like
nalidixic acid.10
While the hanging drop examination was able to detect
approximately 50% of culture positive cholera in earlier
studies10, 11 it had high sensitivity and specificity in the
present study patients. This could be attributed to
immediate transportation of stool samples to the
emergency laboratory and proper training of the
technicians. Prevalence of cholera in pediatric patients
seeking emergency care for acute watery diarrhea has
been estimated to be 10% in an earlier hospital-based
study from the same city.11 The proportion of dehydrated
(80)
(45.7) patients in this study was not reported. 11 Since patients
(88.6) with acute dehydrating diarrhea, were studied the
(97) prevalence of Vibrio cholerae is higher in the present study.
(29.4) The present results further support the notion that cholera

Indian Journal of Pediatrics, Volume 77January, 2010 69

 Vijesh S. Kuttiat et al
is an important cause of morbidity in pediatric patients
with acute diarrhea in urban Delhi, especially those who
have concomitant dehydration.
More importantly, data from the present study and
other studies suggest a significant increase in cholera
prevalence over last 15 years. Stool culture positivity for
cholera was found to be 0.35% in acute watery diarrhea
patients from the same city in 1995. The proportion of
dehydrated patients was not reported in this study.11
Due to lack of financial resources and trained
manpower, laboratory facilities for the accurate diagnosis
of cholera are not widely available in the developing
world. Therefore, efforts have been made to identify
clinical parameters that are predictive of cholera, in
patients presenting with acute watery diarrhea, in order
to start them on appropriate fluid and antimicrobial
therapy. Earlier studies have identified risk factors such as
age more than 24 mth, history of hospitalization for
diarrhea, short duration of illness, rice water stools, high
purge rate (>12 in 24 hr), severe dehydration, and
vomiting (>4/hr), as clinical predictors of a higher cholera
risk in patients with acute watery diarrhea. 10, 12 While
clinical parameters like rice water diarrhea, severe
dehydration, stool frequency >2/hr and duration of
illness < 48 hr were predictive of higher risk of cholera in
the present study patients, only severe dehydration was
associated with a higher cholera risk after adjustment for
other risk factors (including hanging drop positivity).
Currently available guidelines do not recommend
microscopy of stool in children with acute diarrhea.13, 14
There has been emphasis on the clinical presentation for
diagnosis. However, the present study suggests that
clinical features have limited use. In contrast, a simple
laboratory test, stool hanging drop examination is helpful
in predicting cholera in children presenting with acute
dehydrating diarrhea, particularly those with severe
dehydration.
Cholera is known to cause severe diarrhea that can be
rapidly dehydrating, and these sick patients are more
likely to be referred to tertiary care centers like ours for
management. Therefore, these results should not be
extrapolated to other settings including the outpatient
clinics. Also, we did not ascertain the socio-economic
status of the patients. Cholera is more prevalent in
individuals of lower socio-economic status as these
groups have inadequate access to clean drinking water
and proper sanitation. 15, 16 Patients from lower socio-
economic strata are more likely to seek care from a facility
like the present one where medical care is provided at a
nominal cost. Thus, the prevalence of data obtained from
the cohort may not reflect the community prevalence of
cholera.
The present study was carried out in March to August
which is the peak cholera season in Delhi. In the present
70
study, the proportion of Vibrio Cholerae culture positive
patients ranged from a low of approximately 20% in May,
June and August to a high of 38.2% in July. These findings
are highly significant as there was no ongoing cholera
epidemic in Delhi during the study period. However,
since the study was carried out in the peak cholera season
it is possible that true prevalence of cholera for the year
2006 could be lower.3, 17 Also, patients who had received
antibiotic therapy prior to presentation to the study site
and fulfilled other inclusion criteria were included in the
study. Antibiotic therapy prior to obtaining stool culture
may reduce the yield stool culture for Vibrio cholerae and
bias the prevalence estimates towards a lower value.
Therefore, it is possible that some patients with cholera
were not picked up on stool culture and the true
prevalence of culture-confirmed cholera in the present
study population was higher.
CONCLUSION
Cholera is an important cause of acute watery diarrhea in
children in urban north India and should be ruled out in
all children presenting with acute dehydrating diarrhea,
particularly those with severe dehydration. The hanging
drop test is useful for diagnosis in emergency setting.
Contributions: RL, BD, VSK and UK were involved in planning the
study. Data collection and data entry was done by VSK and UK. UK
analyzed the data and drafted the manuscript. BD carrid out the
microbiological studies.
Conflict of Interest: None of the authors have a interest relevant to
the work presented.
Role of Funding Source: No external funding.
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