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Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street E5537, Baltimore, MD 21205, USA
Manual for Detecting Vibrio cholerae O1 and O139 from
Fecal Samples and from Environmental Water Using a
Dipstick Assay1
Amanda Debes, Subhra Chakraborty, Mohammad Ali, David A. Sack
From the DOVE Project
Based in the Department of International Health
Johns Hopkins Bloomberg School of Public Health
March 28, 2014
Correspondence: David A. Sack, M.D.
Professor, Department of International Health
Johns Hopkins Bloomberg School of Public Health
615 N. Wolfe Street E5537
Baltimore, Maryland 21205
[email protected]
1
Note: This is a “working paper” which describes the use of a dipstick test to detect Vibrio cholerae O1
or O139 from fecal samples and from environmental samples. Being a working paper” we expect that
improvements and modifications will be made in the future; however, we also believe that the methods
described here will be useful for programs attempting to control cholera. The DOVE Project and the
authors welcome your suggestions for improving the methods or for improving the explanation of how to
carry out these assays. The manual describes the use of Crystal VC because most field reports have used
this product, but the DOVE Project will update the manual as more experience is gained with other rapid
tests.
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Contents
1. Introduction .................................................................................................................... 3
2. The objectives of surveillance ......................................................................................... 3
3. The dipstick test kit ......................................................................................................... 4
4. Testing fecal specimens .................................................................................................. 6
5. Testing environmental water specimens........................................................................ 9
Materials and supplies .................................................................................................. 10
Procedure ...................................................................................................................... 11
6. Conclusion ..................................................................................................................... 12
7. References .................................................................................................................... 13
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1. Introduction
Surveillance plays an important role in the effective control of cholera, by detecting cases early
and by following the spread of the infection in time and space. The decision to release oral
cholera vaccines (OCVs) from the World Health Organization (WHO) stockpile also depends on
the capacity of the member states to produce timely and quality epidemiological and
laboratory surveillance data concerning the risk of cholera and the number of cholera cases and
deaths from the area to be vaccinated. Systematic collection, analysis, and dissemination of
epidemiological information through the surveillance system will facilitate epidemiological
forecasts and preparation of adequate control measures. However, establishing a high quality
disease surveillance system is often thought to be expensive and may be considered impossible
to accomplish by many developing countries. This is especially true following a natural disaster
when there are many other priorities following the emergency. We describe here a low cost
method which incorporates cholera dipsticks to detect Vibrio cholerae O1 from patient
specimens and water samples from the affected area.
2. The objectives of surveillance
The primary objective of cholera surveillance is early detection of cholera and prompt response
to cases and outbreaks of the disease. Specific objectives include:
•
to report cholera cases and deaths immediately
•
to detect hotspots for cholera
•
to detect high risk groups for whom preventative programs, to include WASH and OCV,
may be especially appropriate
•
to monitor the course of an outbreak
•
when appropriate, to monitor the effectiveness of cholera prevention programs, to
include an OCV vaccination campaign
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Microbiological surveillance needs to occur within the framework of an epidemiological
surveillance. Epidemiological surveillance can be carried out using different methods, and these
are described in a separate manual.
3. The dipstick test kit
The Crystal VC test kit (16IC101-10, Span Diagnostics, Surat, India) described in this manual is
the kit about which there is most documented experience in the field. 2 This test uses
monoclonal antibodies specific for the lipopolysaccharides (LPS) of V. cholerae (VC)
serogroups O1 and O139 in a vertical flow immunochromatography dipstick. The LPS
detection of the kit is 10 ng/ml for VC O1 and 50 ng/ml for VC O139 [1]. A recent study
reported that the minimum detectable limit of the kit was 106 Colony Forming Units (CFU) of
VC O1/ml and 107 CFU of VC O139/ml [2]; our laboratory found that about 107 CFU/ml were
needed for both serogroups to yield a positive test. The test costs 1.9 USD per test when
purchased in bulk.
Crystal VC is available in two formats. One has a line for only V. cholerae O1 and one has lines
for both O1 and O139. Each dipstick has a control line to ensure that the test is able to detect
the antigen. The kit can be stored in temperatures ranging between 4 and 30˚C and in humid
conditions. The kit is not FDA approved, but it is marketed in India and has been used in
several field studies. According to published reports (3-4), the dipstick test has a sensitivity of
about 80–90% and a specificity of about 60–70% when used directly with patients’ fecal
specimens. The relatively low specificity suggests that an improvement in the test procedure is
needed to be sure that a positive test represents a true positive. Especially in areas without
known cases of cholera, declaring a cholera outbreak, when in fact there is no cholera, would
be very unfortunate.
2
Other manufacturers produce similar rapid tests for V. cholerae, but there is less field
experience with these tests. As experience grows, this manual will be updated.
4
Reasons for false positive results are not known, but it would seem that some fecal samples
contain materials (other than V. cholerae) that cross-react with the antibody and show up as a
positive line. In some case, technicians without sufficient training or experience may not
adhere to the test instructions exactly as they are outlined in the product insert. This may
include attributing faint lines as being positive or evaluating the test after the prescribed 15
minutes. In some instances, there may in fact have been V. cholerae antigen in the specimen,
but the organism could not be cultured because the patient had already taken antibiotics.
There are methods, e.g. Polymerase Chain Reaction (PCR), that can detect a V. cholerae signal,
but these are not practical for most situations where cholera is being considered and a rapid
result is needed. Thus, the gold standard remains detection of V. cholerae O1 or O139 using
culture.
A solution for ensuring that a positive test represents a true positive result (is reacting to V.
cholerae rather than a cross-reacting material), is to first place the fecal specimen in alkaline
peptone water (APW) and let this incubate for 6 hours, allowing the V. cholerae (if present) to
grow and the LPS signal to be amplified. V. cholerae grow well at a wide range of temperatures,
so in most tropical countries the APW does not have to be in an incubator; putting the APW
tube at room temperature is acceptable if an incubator is not available. After 6 hours, the APW
fluid is tested to detect V. cholerae using the instructions on the package, as if the APW were a
fecal sample.
When the fecal sample is incubated in the APW, the cross-reacting material (if present) is
diluted so much that it no longer causes a false positive line to show up on the dipstick. While
the cross-reacting material is diluted, the V. cholerae bacteria will grow and the LPS antigen will
be amplified. Thus, the test is less likely to yield a false positive and should provide a more
accurate result. The APW enrichment method is a logical procedure and is based on sound
microbiological concepts, but it is still undergoing field testing to document its sensitivity and
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specificity. It does depend on the ability of the bacteria to grow in the APW; thus, a patient who
has already received antibiotics may be more likely to yield a false negative. However,
specimens from all patients are much less likely to yield false positive results.
If there are questions about the accuracy of the test, some specimens can be sent to a central
laboratory for culture using transport medium (e.g. Cary-Blair medium). The specimens sent to
the central lab can be taken either directly from the fecal specimen or from the APW that
yielded the positive dipstick test. Confirming the positive dipstick test is especially important
when confirming cases in an area that is not known to have cholera.
4. Testing fecal specimens
Materials and supplies
o Fecal specimen or rectal swab
o Preprinted specimen ID labels
o Sterile cotton-tipped wooden swabs
o Tube rack
o 50 ml conical tube
o 25 ml alkaline peptone water (APW)
o Crystal VC dipstick kit
o Cary-Blair transport medium tube
o Disposable latex gloves
o Phosphate buffer solution (PBS)
o Biohazard bags/container
o Chlorine/bleach (if used for decontamination)
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Either stool specimens or rectal swabs of patients who present to the hospitals/health facilities
for treatment of acute watery diarrhea can be tested. Stool specimens are generally collected in
a cup , the bucket under a patient’s bed or from the diapers of small children. Stool specimens
must be taken from stool that has not been treated with chlorine and should not be collected in
a recipient that has held strong chlorine solutions. A rectal swab can be collected using a cotton
swab; generally it is best to wet the swab with saline or water before inserting into the rectum.
When collecting the rectal swab, ensure that there is fecal material on the swab. For patients
with severe watery diarrhea, a stool specimen is best because it may be difficult to detect fecal
material on a swab when the stool is like water.
If there is a fecal specimen, the cotton swab should be inserted into the cup containing the
fecal specimen and rotate the swab to ensure that the cotton tip is coated in the fecal matter.
Insert the cotton swab inoculated
with feces (from previous step or
from rectal swab) into a tube of APW
and break the wooden stick so that
the swab is left in the APW tube.
Place the APW tube in a safe place
(an incubator at 35–37˚C if available)
for 6 hours (range 5–8 hours).
After incubation, place 2–4 drops of
the APW into the kit test tube and
place a Crystal VC dipstick into the
test tube. Leave the dipstick strip in
Figure 1 Negative sample on the left, positive on the right
the test tube. Observe the dipstick to see if lines develop on the control line as well as the test
line. A positive line will generally be visible within 5 to 10 minutes, but do not record lines that
appear after 15 minutes.
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To validate the dipstick test, some of the positive samples should be sent to a microbiology
laboratory test for confirmation by culture by following the procedure shown in Figure 2. This
chart shows that when a positive result is seen, the original fecal sample, as well as a sample
from the APW, may be sent to the laboratory for culture by placing a small amount of the
specimen in Cary-Blair transport medium. These specimens to be sent to the laboratory should
be collected promptly, but once in transport medium the specimen will be valid for up to a
week.
The number of specimens to send
to the microbiology lab depends
on the setting. During a confirmed
outbreak, only a few specimens
need be sent. These specimens
sent for microbiology will be
important to confirm both the
diagnosis as well as antibiotic
sensitivity. If an unexpected
Figure 2. Procedure for detecting V cholerae O1 from a fecal
specimen using dipstick method
outbreak of diarrhea is occurring,
or if sporadic cases are being
detected, a higher proportion of positive cases (say 10–20%) should be confirmed to ensure
that these positive specimens do not represent false positives.
Please note that the specimens and the APW are biohazardous. They need to be discarded
safely, according to local procedures for hospital contaminated waste. Universal precautions in
the laboratory need to be observed, including protective clothing and proper decontamination
of materials. Precautions must be taken for transfer of materials to ensure samples are
properly maintained and safely transferred; this includes the triple packaging system. This
includes the use of a primary waterproof, leakproof receptacle containing the specimen. This
receptacle is surrounded by absorbent material in case of leakage, then contained within a
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second waterproof and leakproof receptacle. Several specimens wrapped in primary
receptacle’s can be transported within the same secondary receptacle. Finally the third, outer
receptacle is the outer shipping package that can protect the inner contents from damage while
being transferred.
5. Testing environmental water specimens
Cholera is a “waterborne disease,” meaning that the bacterium causing cholera, V. cholerae, is
transmitted through contaminated water. Although it is transmitted in water, it can also be
transmitted in contaminated food either from water or from contaminated hands.
The vibrio can survive in water for a very long time, perhaps indefinitely, and can then infect
other people. More often, water is contaminated from a patient with cholera who has
defecated into a sewage system that allows for the feces to contaminate the water supply.
Surveillance for V. cholerae in water is suggested as part of cholera surveillance for two
reasons:
•
Firstly, if one identifies a contaminated water source, plans can be implemented to
decontaminate the water prior to consuming or to repairing the water supply that may
be broken.
•
Secondly, surveillance of water bodies may provide an early warning of cholera in the
area prior to detection of cases being reported. Often, an outbreak may be preceded by
a few sporadic cases that are not known to be cholera and thus are not reported. They
may be excreting into the environment and these bacteria could be detected with an
ongoing surveillance program.
This manual describes how water surveillance can be carried out using the dipstick method. The
overall procedure is shown in Figure 3. Surveillance should be carried out in a systematic
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manner so that a water sample is collected from likely contaminated water sources on a regular
basis. For example, selected sites may be sampled every 15 or 30 days. The selected sites
should be ones that have a high probability of contamination if cholera should occur. These
may be ponds, canals, drainage ditches, or even faucets where people obtain their water.
Materials and supplies
o Closable bottle(s) to hold 1.5-3 l of water. They should be clean, but do not need to
be sterile
o ~3L per water sample from environmental water sources
o Preprinted environmental specimen ID labels
o Plastic bottle for filtration (such as a 1.5 liter water bottle
o Pocket knife (to remove base from water bottles for filtration)
o Medical gauze for filtration (sterile gauze is best, but clean gauze can also be used)
o Sterile cotton-tipped wooden swabs
o Tube rack
o 50 ml conical tube
o 20 ml 2X alkaline peptone water (APW)
o Crystal VC dipstick kit
o Cary-Blair transport medium tube
o Disposable latex gloves
o Phosphate buffer solution (PBS)
o Biohazard bags/container
o Chlorine/bleach (if used for decontamination)
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Procedure
Collect 3 liters of water from the site and close lid, place a specimen ID label on the bottle used
for collection, and send to the lab. 3 Fold the contents of one (sterile) medical gauze packet into
a square and twist it into the neck/spout of a second water bottle whose base has been cut off;
the neck of the water bottle works as a funnel and the water is poured into the filtered through
the gauze. . If packaged medical gauze is not an option, gauze can be cut into 4x4 squares and
folded into a square similarly to medical gauze. The gauze must fit tightly into the spout of the
water bottle to ensure that the pressure from the water does not dislodge the gauze filter
during filtration. Slowly pour the water through the cotton gauze filter while ensuring that the
filter is not displaced from the funnel. Once the entire water sample has passed through the
filter, remove the gauze from the water bottle neck/spout and place in a pre-labeled (with
specimen ID) 50 ml conical tube containing 20 ml of 2X APW4. Place the labeled sample tube in
a safe place and allow the tube to incubate for 24 hours (range 22–26 hours). Incubation should
be in an incubator at 35–37₀C if available, but if not available, incubation can be at room
temperature. After incubation, using the dropper in the Crystal CV kit, aliquot 2–4 drops of the
APW enriched specimen sample into kit test tube and place a Crystal VC dipstick into the test
tube. Observe the dipstick to see if lines develop on the control line as well as the test line. A
positive line will generally be visible within 5 to 10 minutes, but do not record lines that appear
after 15 minutes. (See Figure 1 for examples of positive and negative specimens.)
If the specimen is positive, this result may be confirmed by sending a specimen to the
microbiology laboratory. This is done by inoculating a pre-labeled Cary-Blair transport medium
tube and sending this to the laboratory. Dip a cotton-tipped wooden stick into the APW
3
Alternatively, the filtration and the incubation can be started in the field if the field worker is trained.
4
2X APW is double strength alkaline peptone water. We assume that the gauze will contain about 20 ml
of water, so when this is placed into 20 ml of 2X APW, the resulting solution will have the correct
concentration of APW.
11
Figure 3. Procedure for detecting V. cholerae O1 from environmental water
enriched specimen and place the swab into a Cary-Blair transport tube. Break off the tip of the
cotton-tipped wooden dipstick, close the tube tightly, and prepare documents for transfer
according to the triple packaging system described above, and transfer to the microbiology
laboratory for further processing.
Remember that after incubation, the APW is biohazardous and should be discarded safely.
6. Conclusion
The low cost surveillance method using the dipstick has the potential to improve control of
cholera by providing an early warning system, detecting cholera hotspots, identifying high risk
groups for whom Water, Sanitation and Hygiene (WASH) interventions and OCV may be
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especially appropriate, monitoring the course of an outbreak, and monitoring the effectiveness
of the interventions which were undertaken.
7. References
1. Clopper C, Pearson ES. The use of confidence or fiducial limits illustrated in the case of the
binomial. Biometrika 1934;26:404-13.
2. Mukherjee P, Ghosh S, Ramamurthy T, et al. Evaluation of a rapid immunochromatographic
dipstick kit for diagnosis of cholera emphasizes its outbreak utility. Jpn J Infect Dis 2010;63:2348.
3. Ley B, Khatib AM, Thriemer K, von Seidlein L, Deen J, Mukhopadyay A, Chang NY, Hashim R,
Schmied W, Busch CJL, Reyburn R, Wierzba TF, Clemens JD, Wilfing H, Enwere G, Aguado T,
Jiddawi MS, Sack D, Ali SM. Evaluation of a rapid dipstick (Crystal VC) for the diagnosis of cholera
in Zanzibar and a comparison with previous studies. PLoS ONE 2012; 7(5):e3693.
4. Mukherjee P, Ghosh S, Ramamurthy T, Bhattacharya MK, Nandy RK, Takeda Y, Nair
GB, Mukhopadhyay AK. Evaluation of a rapid immunochromatographic dipstick kit for diagnosis
of cholera emphasizes its outbreak utility. Jpn J Infect Dis. 2010;63(4):234-8.
5. Chakraborty S, Alam M, Scobie HM, Sack DA. Adaptation of a simple dipstick test for detection
of Vibrio cholerae O1 and O139 in environmental water. Frontiers in microbiology. 2013; 4: 320.
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