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 Table of Contents  
Year : 2018  |  Volume : 6  |  Issue : 1  |  Page : 24-27

Evaluation of a rapid dipstick test (Crystal Vc®) for the diagnosis of cholera in Maiduguri, Northeastern Nigeria

Department of Medicine, College of Medical Sciences, University of Maiduguri, Borno State, Nigeria

Date of Web Publication11-Jun-2018

Correspondence Address:
Dr. Ballah Akawu Denue
Department of Medicine, College of Medical Sciences, University of Maiduguri, Bama Road PMB 1069, Borno State
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/amhs.amhs_20_18

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Background: Cholera is a preventable diarrheal disease associated with rapidly progressing dehydration. Early detection of the causative agent, Vibrio cholerae (VC) among symptomatic patients is a key step in cholera outbreak management to minimize disease spread and mortality. Materials and Methods: We prospectively evaluated the performance of Crystal VC®, a commercially available test kit for rapid detection of VC serotypes 01 and 0139 directly from stool samples. Patients included in this study were those admitted to the cholera treatment unit from August 14, 2017, to September 20, 2017, during the cholera outbreak in Maiduguri, northeastern Nigeria. Conventional bacterial stool culture is considered the gold standard and was used as the comparator. Results: A total of 156 stool specimens were collected and tested. Compared with stool culture results, the Crystal VC® test had sensitivity (SE) of 95.1% and specificity (SP) of 59.3%. The positive and negative likelihood ratios were 2.33 and 0.08, respectively. The positive predictive value (PPV) was 81.5% while the negative predictive value (NPV) was 86.5%. There was no gender variation with respect to SE, SP, PPV, and NPV. Conclusion: The cholera rapid dispstick test (RDT), Crystal VC®, is a useful tool for diagnosis of diarrheal disease due to VC serotypes 01 and 0139 and may provide an initial alert in an outbreak situation. It may also be used for case surveillance in cholera epidemic prone situations resulting from displacement to camps or overcrowded shelters due to human conflict or natural disasters. Although it is less sensitive than conventional stool culture, it is a convenient and simple test to perform with faster turnaround time.

Keywords: Cholera, diarrhea, dipstick, Maiduguri, rapid test, vibrio

How to cite this article:
Denue BA. Evaluation of a rapid dipstick test (Crystal Vc®) for the diagnosis of cholera in Maiduguri, Northeastern Nigeria. Arch Med Health Sci 2018;6:24-7

How to cite this URL:
Denue BA. Evaluation of a rapid dipstick test (Crystal Vc®) for the diagnosis of cholera in Maiduguri, Northeastern Nigeria. Arch Med Health Sci [serial online] 2018 [cited 2023 Jan 28];6:24-7. Available from: https://www.amhsjournal.org/text.asp?2018/6/1/24/234094

  Introduction Top

Cholera remains a global public health threat especially in underdeveloped nations where more than 90% of cases occur.[1] In regions where it is endemic, there is an increase in both incidence and number of geographical areas reflecting a failure of socioeconomic infrastructure and difficulties in implementation of control measures.[2] Despite attempts at interventions, most African countries struggle to retain expertise, access to laboratory equipment and consumables and maintain full capabilities to diagnose this disease. This is compounded by frequent major natural disasters and internal strife.[2],[3]

Isolation of Vibrio cholerae (VC) through stool culture is regarded as the standard method of diagnosis.[4] Further, isolates obtained for culture may be subjected to antimicrobial susceptibility testing or characterization of the strain through genetic or molecular methods.[4],[5]

However, prompt management of cholera requires early detection to avoid high morbidity and mortality associated with the delayed institution of appropriate measures.[3],[4],[5]

Cholera is endemic in poor communities due to lack of basic amenities or when there is damage to sanitary and sewage facilities due to damage from human conflict and natural disasters. In underdeveloped and developing regions such as sub-Saharan Africa and southeast Asia, conventional culture and molecular diagnostic testing such as polymerase chain reaction (PCR) are not readily available due to the dearth of facilities and expertise.[5],[6] In such settings, the use of a low cost, efficient, and simple to perform rapid diagnostic tests (RDT) such as Crystal VC ® is necessary for the prompt diagnosis of cholera.[7]

Researchers at the Institut Pasteur, Paris, developed a rapid dipstick test for the detection of VC from stool specimens. Through a licensure agreement with Span Diagnostics (India), the test is commercially available and branded as crystal VC rapid dipstick (VC). The test is simple and can be performed by an untrained laboratory scientist using the manufacturer's manual. The test is based on immunochromatography and colorimetric reporting and detects VC O1 and O139 antigens binding to antibodies fixed on a nitrocellulose strip.[8]

Although several cholera RDTs including Crystal VC have been used for early detection of cholera outbreaks, to the best of our knowledge, the performance of crystal VC rapid dipstick (VC) test has not been validated in northeastern Nigeria. Against this background, we evaluated the sensitivity, specificity (SP), positive predictive value (PPV), and negative predictive value (NPV) of crystal VC rapid dipstick (VC) test using culture as the gold standard from stool samples of patients during the ongoing cholera outbreak in Maiduguri, Borno State.

  Methods Top

Participants were recruited from patients that presented for care from August 14, 2017, to September 20, 2017, with diarrheal disease at the cholera treatment center located at Muna Ward, in Maiduguri Metropolis during the ongoing cholera outbreak in Borno State. Consent for participation was obtained from the patient or caregiver if the patient was <10 years of age. A stool sample was collected from each patient in a plastic specimen container. Patient demographics, as well as clinical signs and symptoms, were recorded.

Rapid test procedure

The rapid diagnostic test was conducted on each stool sample using the Crystal VC ® test (Span Diagnostics Ltd., India) as per the manufacturer's instructions included in the test kit. The laboratory scientists were previously trained on the use of RDTs for early detection of epidemic-prone diseases by the WHO as part of the response to health crises caused by “Boko Haram” insurgency in Northeastern Nigeria. About 200 μl of diarrheal stool was transferred from the specimen container to a sterile clean glass test tube labeled with a patient identification code. Semisolid samples were diluted with saline if necessary. A VC test dipstick was placed in the test tube such that approximately one centimeter of the strip was immersed in feces. After 10 min of incubation, the dipstick was removed, and test results were read. The appearance of a colored line in the specified region was observed, which served as an index to interpret the presence or absence of cholera in the test specimen. Tests were interpreted as positive or negative for VC. Two indeterminate results were observed and were repeated and reviewed by two independent scientists.

Transport and culture

Samples that tested positive by RDT were transported in Cary-Blair transport medium, within 2 h of collection from the patient, to the Microbiology Department at the University of Maiduguri Teaching Hospital, located approximately 5 km from the CTU. On arrival to the Microbiology department, samples were received by designated laboratory personnel. Specimens were immediately plated onto thiosulfate citrate bile salt sucrose medium (Eiken Chemical, Tokyo, Japan) and incubated overnight at 37°C. Yellow (sucrose +) colonies suggestive of VC culture-positive samples were subsequently transported to the Central Public Health Laboratory, Yaba, Lagos State for serogroup testing. All the samples belonged to serogroup 01, serotype Ogawa.

Data analysis

Sensitivity (true positive [TP] rate) was defined as the probability that patients with laboratory-confirmed cholera had a positive RDT. SP (true negative [TN] rate) was the probability that patients with no laboratory-confirmed cholera had a negative RDT. The PPV was the probability that patients with a positive RDT had VC isolated from stool culture. The NPV was the probability that patients with a negative RDT had no VC isolated from a stool culture. The false positive or FP rate (calculated as FP/[FP + TN] or 1-SP) was the proportion of stool samples with no VC isolated on culture but showed a positive RDT result. The false negative (FN) rate (calculated as FN/[TP + FN] or 1-sensitivity) was the proportion of stool samples with VC isolated on culture but showed a negative RDT result.

The comparison of unpaired samples was done using Chi-squared test; comparison of paired samples was done using McNemar's test. Confidence intervals (CIs) were calculated using the exact method. Calculations were done using Stata, version 10 (StataCorp, College Station, TX, USA). Performance indicators were calculated using Excel 2010 (Microsoft, WA, USA).

  Results Top

Among the 156 patients included in this study, 87 (55.8%) were males with a male:female ratio of 1.3:1. The mean (± standard deviation) age of participants was 18.12 ± 19.40 years. Females were significantly (P = 0.001) older than males with a mean (±standard deviation) age of 19.99 ± 18.36 years compared to 16.64 ± 20.16 years, respectively (P = 0.003) [Table 1]. Most patients in the study were between 2 and 20 years, as shown in [Figure 1]. A total of 112 patients were culture positive for VC, with 54 negative culture results. Of the 102 culture positive cases, 97 were positive and 5 were negative by dipstick test. Rapid dipstick assay indicated 119 positive and 37 negative results. Out of the 119 that were positive by rapid cholera dipstick test, 22 were negative (false positive). Conversely, of the 37 negative results by rapid dipstick assay, 5 were culture positive for VC (FN) as depicted in [Table 2].
Table 1: Age and sex distribution of the participants

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Figure 1: Distribution of the patients based on defined age group

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Table 2: Detection of  Vibrio cholerae Scientific Name Search  (VC) in 156 stoolsample by dipstick test versus stool culture

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Overall, the sensitivity (SN), SP, PPV, and NPV were 95.1% (95% CI, 88.9%–98.4%), 59.3% (95% CI, 45.0%–72.4%), 81.5% (95% CI, 76.1%–85.9%), and 86.5% (95% CI, 72.6%–93.9%), respectively [Table 3]. The positive likelihood ratio indicating the ratio between true and false positive was 2.33, while the negative likelihood ratio indicating the ratio between FN and true negative by rapid diagnostic assay was 0.08. Stratification by gender or severity of disease (data not shown) did not identify any significant variation in the performance of the rapid diagnostic test based on these factors [Table 3].
Table 3: Detection of Vibrio cholerae 1 in 156 stool sample by dipstick test versus stool culture based on sex

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  Discussion Top

Effective management of cholera outbreaks requires immediate identification of VC to minimize disease spread and avoid the devastating consequences of such epidemics.[3],[9] The use of culture or PCR-based diagnostic approaches in many endemic countries is restricted by insufficient laboratory capacity. The challenges posed by the dearth of laboratory facilities and expertise hamper outbreak detection and subsequent control measures in cholera-endemic regions.[10] Rapid diagnostic tests (RDT) have been used to detect initial cases of cholera during outbreaks in settings where culture or PCR is not readily available. In this study, we evaluated the performance of Crystal VC ® RDT, one of several available RDT, using stool culture as the gold standard among patients during the ongoing cholera outbreak in Maiduguri, Borno State, northeastern Nigeria. Borno State is the epicenter of Boko Haram insurgency with an estimated 1.4 million internally displaced persons, many of whom are living in camps and squatter settlements.[11] The Crystal VC ® RDT is a dipstick test based on the detection of the lipopolysaccharide of VC O1 and O139 by monoclonal antibodies and uses a one step, vertical-flow immunochromatography principle and colloidal gold particles-conjugated antibodies for detection of bound antigens.[12]

This study sought to validate the performance of the branded Crystal VC ® RDT compared to stool culture as a gold standard. Our evaluation showed a sensitivity of 95.1%, SP of 59.3%, PPV of 81.5%, and NPV of 86.5%.

Several previous studies sought to validate both the prototype test developed by Institut Pasteur [12],[13],[14],[15] and the branded Crystal VC ® RDT, India,[8],[16],[17] with culture as a gold standard. Most studies suggest the ability of the prototype test to detect VC in stool samples or rectal swabs is better than the branded Crystal VC ® RDT, India. The prototype tests are more complicated to perform, and thus may not be suitable for use in the field or resource-limited settings compared to the Crystal VC ® RDT. Conversely, the Crystal VC ® and other RDTs require minimal technical skill, and test results are available within 10 min. Furthermore, test kits may be stored at room temperature in a humidity-proof plastic bag and can easily be transported to the field. For instance, validation studies of the Institut Pasteur prototype test by Nato et al.[12] in Madagascar reported a sensitivity of 98.5% and SP of 96%, with PPV of 95.6% and NPV 99%. Bhuiyan et al.,[13] in Bangladesh obtained a sensitivity of 96%, SP 92%, PPV 93%, and NPV 95%. Similar study by Wang carried out in Mozambique reported a sensitivity 93%, SP 77%, and of PPV 74%.[15]

Studies conducted using Crystal VC ® RDT include a study by Harris et al.[8] carried out in Guinea-Bissau that reported a sensitivity of 97%, SP of 76%, PPV 87%–89%, and NPV 92%–93%. Mukherjee et al.[16] study from India revealed a sensitivity of 92%, SP of 73%, PPV 64%, and NPV of 95%. Ley et al.,[17] from Zanzibar, Tanzania reported a sensitivity of 93%, SP of 49%, PPV 47%, and NPV 94%.

Our study is in agreement with previous similar studies that evaluated the performance of Crystal VC ® RDT with a reported sensitivity >90%.[15],[16],[17] The SP of 59.3% obtained in this study is lower than Harris et al.[8] and Mukherjee et al.[16] but higher than 49% obtained by Ley et al.[17] The WHO global task force on cholera control recommended sensitivity of at least 90% and SP of at least 85% as a minimum performance standard for cholera RDTs to limit the proportion of false positives to an acceptable level.[18] In this evaluation, the sensitivity was within the acceptable limit, but we obtained a lower SP of 59.3%. It should be noted that the SP result may be underestimated as it is possible for culture result to underestimate the actual number of TP cases. The PPV that predicts the presence of the disease when the test is positive of 81.5% indicates that RDT has limitation for individual diagnostic confirmation but useful in outbreak detection.

Evaluation of both the prototype developed by Institute Pasteur and the branded Crystal VC ® VC, India, has been shown to be inferior to culture in detecting VC. An ideal cholera RDT should be highly sensitive so as not to miss the diagnosis of cases and be sufficiently specific when used in detecting outbreak.[5],[8],[18] However, culture sensitivity can be affected by different parameters, such as the initial bacterial load, experience of the technician, prior administration of antimicrobial treatment, sample storage conditions, and delay between sample collection and inoculation. In this study, highly experienced laboratory scientists performed the culture within two hours of receipt of the samples.


All our samples belonged to VC serogroup 01, therefore the performance on serogroup 0139 was not evaluated in this study.

  Conclusion Top

The cholera RDT Crystal VC ® is a useful tool for diagnosis of diarrheal disease due to VC serotypes 01 and 0139 or to identify initial cases in an outbreak situation. It can also be used for case surveillance in cholera epidemic prone settings resulting from displacement of individuals to camps or overcrowded shelters due to human conflict or natural disasters. Although it is less sensitive than conventional culture, it is a convenient, low cost, and simple test to perform with faster turnaround time.


Crystal VC ® cholera RDT is an easy test to perform that requires minimal skill, expertise, or stringent requirement for storage. It is recommended for use as an adjunct for prompt diagnosis of diarrheal disease caused by VC in areas of limited laboratory infrastructure and outbreak situations.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

World Health Organization. Cholera. Wkly Epidemiol Rec 2016;91:433-40.  Back to cited text no. 1
Ali M, Nelson AR, Lopez AL, Sack DA. Updated global burden of cholera in endemic countries. PLoS Negl Trop Dis 2015;9:e0003832.  Back to cited text no. 2
Shikanga OT, Mutonga D, Abade M, Amwayi S, Ope M, Limo H, et al. High mortality in a cholera outbreak in Western Kenya after post-election violence in 2008. Am J Trop Med Hyg 2009;81:1085-90.  Back to cited text no. 3
Keddy KH, Sooka A, Parsons MB, Njanpop-Lafourcade BM, Fitchet K, Smith AM, et al. Diagnosis of Vibrio cholerae O1 infection in Africa. J Infect Dis 2013;208 Suppl 1:S23-31.  Back to cited text no. 4
Dick MH, Guillerm M, Moussy F, Chaignat CL. Review of two decades of cholera diagnostics – How far have we really come? PLoS Negl Trop Dis 2012;6:e1845.  Back to cited text no. 5
Alam M, Hasan NA, Sultana M, Nair GB, Sadique A, Faruque AS, et al. Diagnostic limitations to accurate diagnosis of cholera. J Clin Microbiol 2010;48:3918-22.  Back to cited text no. 6
Boncy J, Rossignol E, Dahourou G, Hast M, Buteau J, Stanislas M, et al. Performance and utility of a rapid diagnostic test for cholera: Notes from Haiti. Diagn Microbiol Infect Dis 2013;76:521-3.  Back to cited text no. 7
Harris JR, Cavallaro EC, de Nóbrega AA, Dos S Barrado JC, Bopp C, Parsons MB, et al. Field evaluation of crystal VC rapid dipstick test for cholera during a cholera outbreak in Guinea-Bissau. Trop Med Int Health 2009;14:1117-21.  Back to cited text no. 8
Pena ES, Bwire G, Dzotsi E, Bonnet MC, Hessel L. New momentum in prevention, control and elimination of cholera in Africa: Priority actions identified by affected countries. Wkly Epidemiol Rec Health Sect Secr Leag Nations 2016;91:305-14.  Back to cited text no. 9
World Health Organization. Cholera 2014. Wkly Epidemiol Rec 2015;90:517-28.  Back to cited text no. 10
Displacement Tracking Matrix. DTM Nigeria, Round XIII Report; December 2016. Available from: http://www.nigeria.iom.int/dtm. [Last accessed on 2017 Oct 20].  Back to cited text no. 11
Nato F, Boutonnier A, Rajerison M, Grosjean P, Dartevelle S, Guénolé A, et al. One-step immunochromatographic dipstick tests for rapid detection of Vibrio cholerae O1 and O139 in stool samples. Clin Diagn Lab Immunol 2003;10:476-8.  Back to cited text no. 12
Bhuiyan NA, Qadri F, Faruque AS, Malek MA, Salam MA, Nato F, et al. Use of dipsticks for rapid diagnosis of cholera caused by Vibrio cholerae O1 and O139 from rectal swabs. J Clin Microbiol 2003;41:3939-41.  Back to cited text no. 13
Kalluri P, Naheed A, Rahman S, Ansaruzzaman M, Faruque AS, Bird M, et al. Evaluation of three rapid diagnostic tests for cholera: Does the skill level of the technician matter? Trop Med Int Health 2006;11:49-55.  Back to cited text no. 14
Wang XY, Ansaruzzaman M, Vaz R, Mondlane C, Lucas ME, von Seidlein L, et al. Field evaluation of a rapid immunochromatographic dipstick test for the diagnosis of cholera in a high-risk population. BMC Infect Dis 2006;6:17.  Back to cited text no. 15
Mukherjee P, Ghosh S, Ramamurthy T, Bhattacharya MK, Nandy RK, Takeda Y, et al. Evaluation of a rapid immunochromatographic dipstick kit for diagnosis of cholera emphasizes its outbreak utility. Jpn J Infect Dis 2010;63:234-8.  Back to cited text no. 16
Ley B, Khatib AM, Thriemer K, von Seidlein L, Deen J, Mukhopadyay A, et al. Evaluation of a rapid dipstick (Crystal VC) for the diagnosis of cholera in Zanzibar and a comparison with previous studies. PLoS One 2012;7:e36930.  Back to cited text no. 17
The Global Task Force on Cholera Control. World Health Organization. Available from: http://www.who.int/cholera/task_force/en/. [Last accessed on 2016 Sep 08].  Back to cited text no. 18


  [Figure 1]

  [Table 1], [Table 2], [Table 3]

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