Manual for Detecting Vibrio cholerae O1 and O139 from Fecal Samples and from Environmental Water Using a Dipstick Assay 1

Transcription

1 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 Assay 1 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 dsack@jhsph.edu 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. 1

2 Contents 1. Introduction The objectives of surveillance The dipstick test kit Testing fecal specimens Testing environmental water specimens... 9 Materials and supplies Procedure Conclusion References

3 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 3

4 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 10 6 Colony Forming Units (CFU) of VC O1/ml and 10 7 CFU of VC O139/ml [2]; our laboratory found that about 10 7 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

5 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 5

6 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) 6

7 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 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 Figure 1 Negative sample on the left, positive on the right test tube. Leave the dipstick strip in 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. 7

8 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. Figure 2. Procedure for detecting V cholerae O1 from a fecal specimen using dipstick method 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 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 8

9 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 9

10 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 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) 10

11 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 APW 4. Place the labeled sample tube in a safe place and allow the tube to incubate for 24 hours (range hours). Incubation should be in an incubator at ₀ C 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

12 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 12

13 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: 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: 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):e 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): 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: