NIH Public Access: Evaluation of Enrichment Method For Detection of Vibrio

Evaluation of Enrichment Method for Detection of Vibrio
cholerae O1 using a Rapid Dipstick Test in Bangladesh

Christine Marie George, PhD,
Department of International Health Program in Global Disease Epidemiology and Control Johns
Hopkins Bloomberg School of Public Health 615 N. Wolfe Street, Room E5535 Baltimore,
Maryland 21205-2103, Telephone: (410) 955 -2485
Mahamud-ur Rashid, MS,
International Centre for Diarrhoeal Disease Research, Bangladesh
David A. Sack, MD,
Hopkins Bloomberg School of Public Health
R. Bradley Sack, MD, ScD,
Hopkins Bloomberg School of Public Health
K. M. Saif-Ur-Rahman, MBBS,
Andrew S Azman, MS,
Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health
Shirajum Monira, PhD,
Sazzadul Islam Bhuyian,
K. M. Zillur Rahman, MS,
M. Toslim Mahmud, MS,
Munshi Mustafiz, MS, and
Munirul Alam, PhD, MS
Abstract
BackgroundCulturing is generally considered to be the gold standard for detecting Vibrio
cholerae in stool, though it is not always feasible in resource-limited settings. The Crystal VC
dipstick test allows for rapid stool testing for the diagnosis of cholera in the field. However,
NIH Public Access
Author Manuscript
Trop Med Int Health. Author manuscript; available in PMC 2014 June 21.
Published in final edited form as:
Trop Med Int Health. 2014 March ; 19(3): 301307. doi:10.1111/tmi.12252.
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previous studies have found low specificities (49%79%) associated with direct testing of stool for
cholera using this kit when compared to culturing.
MethodsIn the present study conducted in Dhaka, Bangladesh in 2013, we compare direct
testing using the Crystal VC dipstick test and testing after enrichment for 6-hours in Alkaline
Peptone Water (APW) to bacterial culture as the gold standard. Samples positive by dipstick but
negative by culture were also tested using PCR.
ResultsStool was collected from 125 patients. The overall specificities of the direct testing and
testing after 6-hour enrichment in APW compared to bacterial culture were 91.8% and 98.4%
(p=0.125) respectively, and the sensitivities were 65.6% and 75.0% (p=0.07), respectively.
ConclusionThe increase in the sensitivity of the Crystal VC kit with the use of the 6 hour
enrichment step in APW compared to direct testing was marginally significant. The Crystal VC
dipstick was found to have a much higher specificity than previously reported (9198%).
Therefore this method provides a promising screening tool for cholera outbreak surveillance in
resource limited settings where elimination of false positive results is critical.
Introduction
The World Health Organization estimates that there are between 35 million cholera cases
and more than 100,000 cholera deaths per year.
1
In recent years there have been major
cholera outbreaks around the world including Haiti
2
, Cameroon
3
, Guinea-Bissau
4
, and the
Democratic Republic of the Congo.
5
Cholera case management when provided properly is
very effective.
6
Almost all cholera patients can be effectively treated through administration
of oral rehydration solution (ORS) and intravenous fluid.
7,8
Effective control measures and
case management for cholera rely on early detection of outbreaks during cholera epidemics.
Through surveillance of acute watery diarrhea cases entering health facilities cholera can be
detected early and provisions can be made for life saving ORS, and intravenous rehydration
fluid in those who are severely dehydrated. Furthermore, cholera outbreaks can be
controlled with water sanitation and hygiene interventions and the distribution of reactive
oral cholera vaccine.
9
Culturing of stool specimens using appropriate media is considered to be the gold standard
for detecting Vibrio cholerae (VC) in stool.
1012
However, this method requires a laboratory
and generally takes 23 days.
13
Further, in resource limited settings these methods are often
not feasible. A commercially available rapid dipstick test, Crystal VC
, allows for rapid

stool testing for the diagnosis of cholera in the field. However, while previous studies have
found high sensitivities ranging from 9297%, they found low specificities (49%79%) with
direct testing of stool using this kit compared to culture and PCR.
1419
The high number of
false positives found in previous studies suggests that this tool, when carried out with fresh
stool, is not reliable in accurately identifying true cholera cases. This has limited its
usefulness as a surveillance method since one should avoid declaring a case to be cholera
unless one is extremely sure that the case is truly caused by V. cholerae O1 or O139.
Alkaline peptone water (APW: 1% peptone, 1% NaCl, pH 8.4) is a commonly used
enrichment medium for VC.
10,11,20
In the present study we evaluate if enrichment for 6-
hours in APW can be used to increase the sensitivity and specificity of the Crystal VC rapid
George et al. Page 2
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dipstick test in comparison to the direct testing method when the bacterial culture method is
used as the gold standard.
Methods
Ethics
Ethical approval for the present study was obtained from the Johns Hopkins Bloomberg
School of Public Health Institutional Review Board and by the Research Review Committee
and the Ethical Review Committee at the International Centre for Diarrhoeal Disease
Research, Bangladesh (iccdrb). Informed consent was obtained for all patients enrolled in
the present study, for patients under 18 years of age parental consent was obtained.
Study Population
This study took place at the iccdr,b hospital in Dhaka, Bangladesh between May and July
2013. Patients presenting at the iccdr,b hospital with moderate to severe clinical dehydration
and acute watery diarrhea were recruited for the present study. Patients were typically
enrolled within 3 hours of being admitted. Moderate to severe dehydration was defined
using World Health Organization guidelines. Acute watery diarrhea was defined as (self-
reported) 3 or more loose stools in a 24 hour period in the last 3 days. All of the patients
enrolled in the study received intravenous (IV) fluids and ORS for rehydration followed by
oral antibiotics as either Azithromycin or Ciprofloxacin. None of the enrolled patients had
received oral cholera vaccine. Stool samples were collected in 100 ml stool cups for analysis
from all patients enrolled in the study. Our sample size included all patients found to meet
our study eligibility criteria between May to July 2013.
Dipstick Procedure
The Crystal VC dipstick test kit (16IC101-10, Span Diagnostics, Surat, India) evaluated in
the present study for the detection of V.cholerae in stool costs 19 USD and includes 10 tests
(1.9 USD per test). These kits were procured in February 2013. The dipstick test has
monoclonal antibodies specific to both V.cholerae (VC) O1 and O139 lipopolysaccharides
(LPS) and uses vertical flow immunochromatography. The LPS detection of the kit is 10
ng/ml for VC O1 and 50 ng/l for VC 0139.
25
A recent study reported that the minimum
detectable limit of the kit was 10
6
CFU of VC O1/ml and 10
7
CFU of VC O139/ml.
14
This
kit can be stored in temperatures ranging between 4 to 30 degrees Celsius, and under humid
conditions.
A watery section of the stool was collected from the stool cup using the pipet supplied with
the dipstick kit then 23 drops (200 l) of stool were placed in the kit sample processing
bottle for dilution. The processing bottle was then shaken. Four drops of the liquid from the
processing bottle was then added to the kit test tube, and then the dipstick was inserted. The
final result of the dipstick test was read after 15 minutes. Possible results were as follows:
positive reactive for VC O1, positive reactive for VC O139, positive reactive for VC O1 and
O139, negative non-reactive for VC O1 and O139, and invalid (no bands present on
dipstick).
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For the dipstick samples analyzed after enrichment in APW, the following procedure was
used: 400 l of the watery portion of the stool sample from each patient was transferred to a
vial containing 3 ml of APW broth and kept at 37C. Stool samples were transferred to
APW broth within 2 hours of collection. After 6-hours of enrichment in APW, 200 l of this
enrichment broth was placed into the dipstick processing bottle, and analyzed using the
dipstick testing procedure described previously.
Only the study supervisor had previous experience using the Crystal VC dipstick test. A half
day training was conducted on how to use the kit for all study personnel. The direct dipstick
testing was conducted on the hospital ward, and the testing after the 6-hour enrichment in
APW water was conducted in the laboratory.
Stool Culture
The same APW samples used for the 6-hour dip stick test were used for culture. After 6-
hours of enrichment in APW, 510 l of enriched broth was streaked using an inoculating
loop onto Thiosulphate Citrate Bile Sucrose Agar (TCBS) and Taurocholate Tellurite
Gelatin Agar (TTGA) then incubated at 37 C for 1824 hours. Presumptive colonies were
sub-cultured on Gelatin Agar and again incubated at 37 C for 1824 hours.
21,22
Serogrouping
Vibrio cholerae colonies from gelatin agar plates were tested to determine their serogroups
using slide agglutination with polyvalent antiserum, followed by monoclonal and VC
serogroup O1 and O139-specific antisera as previously published.
23,24
Polymerase Chain Reaction (PCR) Analysis
Selected samples that were positive by dipstick but negative by culture were analyzed by
PCR to determine if the dipstick test was able to identify VC O1 and VC O139 positive
samples not detectable by the culture method. The following procedure was used: 400 l of
the watery portion of the stool sample from each patient was enriched in 3ml of APW at 37
C for 1824 hours. Then 1ml of enriched sample was used for preparation of template DNA
using standard procedure. The DNA was then analyzed by a multiplex PCR for concurrent
detection of wbe and wbf sequences specific for O1 and the O139 serogroups of VC,
respectively, and for ctxA specific sequences. After amplification, 9 l of each reaction
mixture was subjected to electrophoresis on a 2% agarose gel using a horizontal
electrophoresis apparatus (Horizon 11.14; Life Technologies, Gibco BRL). The gel
containing amplified DNA was stained with ethidium bromide and visualized using a UV
transilluminator. Images of the transilluminator were digitized using a one-dimensional Gel
documentation system (Bio-Rad).
13
Statistical Methods
The sensitivity, specificity, positive predictive value, and negative predictive value were
estimated for both direct testing with the dipstick and use of the dipstick test after 6-hours of
enrichment in APW. The sensitivity is the proportion of those with cholera that have a
positive dipstick test, and the specificity is the proportion of those without cholera who have
a negative dipstick test. A false positive result is a positive finding by dipstick test that is
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negative by culture. A false negative result is a negative finding by dipstick test that is
positive by culture. Positive predictive value (PPV) is the proportion of those with a positive
dipstick test who have cholera, and the negative predictive value (NPV) is the proportion of
those with a negative dipstick test who do not have cholera. The PPV and NPV are functions
of the population prevalence of cholera in addition to the sensitivity and specificity of the
test. The 95% confidence intervals for sensitivity, specificity, PPV, and NPV were
calculated using the exact method.
25
We estimated p-values from McNemars test for
equality of the sensitivity and specificity of direct testing and 6 hour enrichment testing.
Results
A total of 125 patients presenting with moderate to severe clinical dehydration and acute
watery diarrhea were recruited from icddr,b hospital in Dhaka, Bangladesh between May
and July 2013. The median age of patients recruited was 32 years (range: 370 years), and
50% of the patients were female. Fifty one percent of the screened patients were found to be
culture positive for VC O1. The cholera prevalence among patients admitted to icddr,b
hospital in Dhaka in the previous year (May 2012May 2013) was 7 per 100 diarrheal cases
for all age groups combined (Personal Communication: Dr. ASG Faruque, icddrb).
The overall sensitivity of the direct testing and testing after 6-hour enrichment in APW
compared to bacterial culture for the presence of VC O1 was 65.6% (95% Confidence
Interval (CI) 52.7%77.1%) and 75.0% (95% CI 62.6%85.0%) respectively, the specificity
was 91.8% (95% CI 81.9%97.3%) and 98.4% (95% CI 91.2%100%) respectively (Table
1). Tables 2 presents the cross tabulations. The p-values between the direct testing and the 6
hour enrichment for the sensitivity and specificity using the McNemars test were p = 0.07
and p= 0.125, respectively.
Four stool samples that were dipstick VC O1 positive by when tested directly, but VC O1
dipstick negative after the 6 hour enrichment step and negative by culture were analyzed by
PCR to determine if the dipstick test was able to identify VC O1 positive samples not
detectable by the culture method. All four of these samples were found to be negative by
PCR for VC O1 and VC O139.
Seven stool samples were found to be positive for VC O139 after directing testing using the
dipstick, two of these samples were also dipstick positive for VC O1 with direct testing
(Table 3). None of these stool samples were culture or PCR positive for VC O139.
However, two of these VC O139 positive and VC O1 negative samples by direct dipstick
testing were found to be culture and PCR positive for VC O1. No samples were found to be
dipstick positive for VC O139 after the 6-hour APW enrichment step.
Discussion
This study represents the first evaluation comparing direct testing of stool using the Crystal
VC dipstick test to dipstick testing after a 6-hour enrichment step in Alkaline Peptone Water
(APW) using bacterial culture as the gold standard. We observed much higher specificities
then previously reported for the Crystal VC dipstick test, 98% after 6-hour enrichment
compared to 49%79%
1419
found in previous studies. The increase in the sensitivity of the
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Crystal VC kit with the use of the 6 hour enrichment step in APW compared to direct testing
was marginally significant (p=0.07). Furthermore, only one sample after the 6-hour
enrichment in APW gave a VC O1 positive finding by dipstick and VC O1 negative finding
by culture compared to 5 samples with direct dipstick testing. However, this increase in
specificity associated with the enrichment method was not statistically significant (98% vs.
91%).
Accurate diagnostic tools for cholera are urgently needed for cholera surveillance in both
epidemic and endemic settings. However techniques such as bacterial culture are usually not
feasible in low resource settings. Earlier versions of a Crystal VC dipstick prototype
developed by Institute Pasteur found sensitivities ranging from 92100% and specificities
ranging from 84100%.
22,26
However, recent studies of this dipstick test have found lower
specificities ranging from 49%80%.
1416,18,19,27
In Ley et al. 50.8% of positive dipstick
tests were false positives.
15
For cholera surveillance a high specificity is important to identify true cholera cases quickly,
and to prevent unnecessary mobilization of resources.
17
Early cholera detection allows for
more time for control programs to be put in place that distribute vaccinations and promote
hand washing and water treatment. In addition, an early warning allows time to stock
previsions of life saving oral rehydration salts, antibiotics, and intravenous rehydration fluid
for those who are severely dehydrated.
There is only one other published study to date, to our knowledge, that has evaluated the
performance of the Crystal VC dipstick test with direct testing compared to testing after
enrichment in APW. This study conducted in Mozambique compared direct testing of stool
to rectal swab samples enriched in APW for four hours before testing. Much higher
specificities were observed with the enriched rectal swab samples compared to the directly
tested stool (97% vs. 77%).
28
However this study used a prototype version of the Crystal
VC dipstick when it was manufactured by Institute Pasteur.
In the present study we observed lower sensitivities than previously reported for the Crystal
VC kit, 66% with direct testing and 75% with 6-hour enrichment in APW compared to 92
97%
1416
in previous evaluations. The reason for the high number of dipstick negative and
culture positive samples for VC O1 is unclear, particularly since the study only included
acute watery diarrhea cases presenting with severe clinical dehydration who likely had a
high bacterial load of V.cholerae.
We found four VC O1 dipstick positive results with direct testing that were negative for VC
O1 by culture and PCR. Previous studies have discussed the possibility that dipstick false
positive stool samples could lack live organisms, but have lipopolysaccharide antigens in
sufficient quantities to react with the dipstick test, for example in cholera cases that took
antibiotics.
14,26
However, Ley et al. found no significant difference between in cholera
dipstick sensitivities between patients receiving and not receiving antibiotics.
15
In the present study we found 2 stool samples which were VC O139 positive and VC O1
negative by direct dipstick but were positive for VC O1 by culture and PCR. This finding is
puzzling and warrants further investigation. Findings from previous studies suggest that
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bacteria in the family of Vibrio mimicus or Aeromonas trota could share antigens with VC
O139.
25,26
Therefore it is possible that these bacteria are causing O139 false positive
readings.
It is important to note that no samples were VC O139 dipstick positive after the 6-hour
APW enrichment step. This finding suggests that this kit may also be effective at reducing
VC O139 false positive dipstick results.
The use of clinically defined case definitions of cholera without systematic microbiological
confirmation can lead to misclassification of disease, including false positives.
29
Using low-
cost easy to use high specificity diagnostic tools like the one described in this study can
improve estimates of cholera incidence, particularly in low-resource settings where cholera
data is often scant. The improved epidemiologic resolution provided by a test like Crystal
VC could lead to more realistic computational models of cholera transmission
17,30,31
,
ultimately leading to improved estimates of the potential impact of cholera prevention and
control measures, and a better understanding of the cholera transmission dynamics.
Our present study had several limitations. One major limitation of this study was the small
sample size of 125 suspected cholera cases. This sample size was selected based on the
number of patients that met the study eligibility criteria between May to July 2013. We
conducted a power calculation to determine the sample size needed based on our present
effect size of 0.09 for sensitivity at a 95% confidence level with a power of 80%. We found
that the required sample size would be 971 patients. Therefore we were underpowered to
assess an association for sensitivity at the effect size found in the present study. Future
studies should evaluate the APW enrichment method using a larger sample size that is
sufficiently powered to detect a significant association at the effect size found in the present
study. A second limitation is that all of our cases had acute watery diarrhea, moderate to
severe clinical dehydration, and received intravenous rehydration. Therefore these findings
may not be generalizable to patients presenting with less severe symptoms. Wang et al. in
Mozambique found higher sensitivities with patients receiving IV rehydration compared to
those not receiving IV therapy.
28
However, a more recent evaluations in Zanzibar
15
and in
India
14
found no such association. Future studies should determine if there are differences
in the performance of the dipstick test with 6-hour enrichment in APW by case severity and
bacterial load. A third limitation of the present study is the use of whole stool instead of
rectal swab samples for our fecal specimen collection. Future studies should evaluate if the
Crystal VC can be used effectively on rectal swab samples. Finally, all of the dipstick testing
was conducted at icddrb hospital. Therefore we could not evaluate the performance of the
dipstick test after 6-hour enrichment in APW water in a field setting.
Conclusion
The Crystal VC dipstick was found to have a much higher specificity than previously
reported for this kit. Further, the 6 hour enrichment step in APW for the dipstick test led to a
marginally significant increase in sensitivity compared to direct testing, suggesting that the
enrichment method may be beneficial in improving the sensitivity of the dipstick test. Based
on these findings the Crystal VC dipstick provides a promising screening tool for cholera
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outbreak surveillance in resource limited settings where elimination of false positive results
is critical. The improved data from systematic use of this kit can be combined with models
of cholera transmission to help make decisions related to where and how to best allocate
interventions like cholera vaccine. However, future research needs to be done to evaluate the
APW enrichment method using a larger sample size, and to determine the reason for the
lower than previously reported sensitivities in the present study.
Acknowledgments
The project was funded by NIAD 5R01 AI039129-14 and 3R01 AI039129-13S1, and by funds from the Johns
Hopkins Center for Global Health. We would also like to thank all of study participants, research assistants, and
project staff who were involved in this project. In addition, we would like to thank the Delivering Oral Cholera
Vaccine Effectively (DOVE) project for their support in implementing this study.
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Table 1
Performance of the Rapid Dipstick (Crystal VC) Test for Diagnosis of Vibrio cholerae O1 in 125 patients
Compared to Bacterial Culture.
Sensitivity%
*
(95% CI) Specificity%
*
(95% CI)
Positive Predictive Value
%
*
(95% CI)
Negative Predictive Value
%
*
(95% CI)
Direct Dipstick
Stool Testing
65.6% (52.7%, 77.1%) 91.8% (81.9%, 97.3%) 89.4% (76.9%, 96.5%) 71.8% (60.5%, 81.4%)
Dipstick Stool
Testing after 6-
hour Enrichment
in APW
75.0% (62.6%, 85.0%) 98.4% (91.2%, 100%) 98.0% (89.2%, 100.0%) 79.0% (68.1%, 87.5%)
*
exact binomial confidence intervals
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Table 2
Direct and Enrichment Dipstick Testing Compared to Culture
Culture Total
+
Direct Dipstick + 42 5 47
22 56 78
Total 64 61 125
Enrichment Dipstick + 48 1 49
16 60 76
Total 64 61 125
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Evaluation of Enrichment Method for Detection of Vibrio

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