Comparison of cholera rapid diagnostic test and culture results during a cholera outbreak response, Moyo District, West Nile, Uganda, June-July 2025

Summary

Background: During outbreak response, cholera rapid diagnostic tests (RDTs) are used for rapid screening, while stool culture serves as the reference method for laboratory confirmation. However, the field performance of cholera RDTs may vary by setting, specimen quality, timing of collection, circulating strains, and implementation conditions. In Uganda, limited outbreak-specific evidence is available on how well cholera RDT results agree with stool culture results under routine response conditions. We compared the performance of cholera rapid diagnostic testing against culture and sensitivity results obtained from routine outbreak response specimen to determine the level of test accuracy of the cholera rapid diagnostic testing kit, Moyo District, Uganda, June-July 2025.

Methods: Stool specimens were collected from suspected cholera cases defined as was any person aged ≥5 years presenting with dehydration or a death from acute watery diarrhea in Moyo District during June-July 2026, and any person aged 2 years or more with acute watery diarrhea, Moyo District during June-July 2025. Samples were tested using the Crystal VC O1/O139 cholera RDT at field laboratories and subsequently transported in Cary-Blair transport medium under cold-chain conditions (2–8°C) to the national microbiology reference laboratory for culture confirmation. Results from specimens with both RDT and culture outcomes were matched and analyzed using a 2×2 contingency table. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and overall agreement were calculated using culture as the gold standard method.

Results: A total of 29 stool specimens underwent both RDT and culture testing. Twenty-two (75.9%) specimens tested positive by RDT, while 19 (65.5%) were confirmed positive by culture. Nineteen specimens were positive by both methods, three were RDT-positive but culture-negative, and no culture-positive specimens were missed by the RDT. The sensitivity of the RDT was 100% (19/19), specificity was 70% (7/10), PPV was 86% (19/22), NPV was 100% (7/7), and overall agreement between RDT and culture was 90% (26/29).

Conclusion: Cholera RDTs demonstrated optimal sensitivity and high agreement with culture testing under outbreak response conditions in a border district of Uganda. The findings support the use of RDTs for rapid screening and early detection of suspected cholera cases at peripheral health facilities. However, the occurrence of false-positive results highlights the continued importance of culture confirmation for outbreak verification, surveillance, and public health decision-making.

Introduction

Cholera is an acute diarrheal disease caused by toxigenic strains of Vibrio cholerae and remains a significant cause of morbidity and mortality globally, particularly in low-resource settings with poor access to clean water and sanitation [1]. Although cholera is preventable and treatable, outbreaks continue to occur frequently in many African countries, including Uganda [1,2].

Timely laboratory confirmation supports case management, surveillance, and implementation of public health interventions such as water sanitation measures and vaccination campaigns [3,4].

Stool culture remains the gold standard for confirmation of cholera [5]. However, culture capacity is often centralized at national reference laboratories, leading to delays in diagnosis and reporting during outbreaks occurring in remote districts. Cholera rapid diagnostic tests (RDTs) offer an important alternative for early screening and detection because they can be deployed at peripheral health facilities with minimal infrastructure requirements [6].

Despite RDT operational advantages, there is limited local information describing how the two testing methods compared under actual outbreak response conditions. Understanding the level of agreement between RDT and culture results is important for interpreting test results, supporting laboratory quality assurance, and informing the continued use of RDTs during outbreak response activities [7].

On 7 June 2025, the Ministry of Health confirmed a cholera outbreak in Moyo district in West Nile, Uganda. We compared the performance of cholera rapid diagnostic testing against culture and sensitivity results obtained from routine outbreak response specimen to determine the level of test accuracy of the cholera rapid diagnostic testing kit.

Methods

Study setting: The study was conducted in Moyo District, located in the West Nile region of northern Uganda along the Uganda–South Sudan border, where cross-border population movement increases vulnerability to cholera transmission. During the June–July 2025 cholera outbreak, suspected cases were identified at health facility level using the national case definition, and stool specimens were tested using cholera rapid diagnostic tests for rapid screening and immediate response action. In line with Uganda’s cholera testing approach, specimens were also referred through the national specimen referral system to designated laboratories for stool culture and antimicrobial susceptibility testing, providing an opportunity to compare RDT results with culture findings under routine outbreak response conditions [8].

Sample collection, packaging, transportation, and testing

Stool specimens were collected from suspected cholera cases in Moyo District during June–July 2026 by trained healthcare workers following standard specimen collection and biosafety procedures. One fresh stool specimen was collected from each suspected case in a sterile, leak-proof container; a portion was tested onsite using the Crystal VC O1/O139 cholera rapid diagnostic test according to the manufacturer’s instructions, while a portion from the same specimen was placed in Cary-Blair transport medium for culture confirmation and antimicrobial susceptibility testing at the National Microbiology Reference Laboratory. Specimens were assigned unique identification numbers, accompanied by completed case investigation forms, packaged using the triple packaging system, maintained at 2–8°C, and transported through the national specimen referral system. At the reference laboratory, specimens were cultured for Vibrio cholerae using standard procedures, with species identification and antimicrobial susceptibility testing performed using the VITEK® 2 Compact system; results were recorded and shared with the outbreak response team.

Data management and analysis

Data from specimens that had both RDT and culture results available were extracted from routine laboratory records. Results from the two testing methods were matched using specimen identification numbers and reviewed for completeness. A two-by-two comparison table was developed to determine agreement between the RDT and culture methods. The numbers of true positives, true negatives, false positives, and false negatives were calculated. Sensitivity, specificity, positive predictive value, negative predictive value, and overall agreement were subsequently determined to describe the level of comparability between the two methods.

Ethical considerations

This activity was conducted as a non-research determination to support laboratory quality assurance and program improvement during outbreak response activities. The assessment utilized generated outbreak response data and did not involve additional specimen collection or patient interventions beyond standard outbreak response procedures. Rapid diagnostic test and culture results were matched using anonymized patient identifiers.

Results

Cholera diagnostic cascade during the response

A total of 30 suspected cholera cases were sampled during the outbreak period. One sample didn’t meet the laboratory acceptance criteria (lacked a request form) and was therefore not tested. Rapid diagnostic tests were performed on each of the 29 samples collected from suspected cases, of which 22 tested positive (Figure 1). Nineteen samples were confirmed positive for Vibrio cholerae by culture (Figure 1).

Comparability of Cholera rapid diagnostic tests against culture testing

Table 1:  Comparability between cholera rapid diagnostic tests and culture results

A total of 29 samples were analysed using both rapid diagnostic tests (RDT) and culture methods. Of these, 22 samples tested positive by RDT, while 19 were confirmed positive by culture, the gold standard. Among the RDT-positive results, 19 corresponded with culture-confirmed cases, indicating that the RDT correctly identified all true cholera cases (Table 1). However, 3 samples tested positive by RDT but were negative by culture, representing false positives.

Table 2: 2X2 contingency table showing comparability between rapid diagnostic tests performed and culture results (gold standard)

A total of 29 stool samples were tested using both the cholera rapid diagnostic test (RDT) and stool culture, with culture used as the gold standard. Of the 29 samples, 22 tested positive by RDT and 19 tested positive by culture. Nineteen samples were positive on both RDT and culture (true positives), three samples were RDT-positive but culture-negative (false positives), no samples were RDT-negative but culture-positive (false negatives), and seven samples were negative by both methods (true negatives) (Table 2).

The sensitivity of the RDT was 100% (19/19), and the specificity was 70% (7/10). The positive predictive value (PPV) was 86.4% (19/22), while the negative predictive value (NPV) was 100% (7/7). The overall accuracy of the RDT compared with culture was 90% (26/29).

Discussion

This evaluation assessed the performance of the cholera rapid diagnostic test (RDT) against stool culture as the gold standard in this outbreak response.   The assessment found a high level of agreement between cholera RDT and culture results. All culture-confirmed cholera cases included in the assessment were detected by the RDT, indicating that the test was able to identify specimens that were confirmed positive by the gold standard method. Three specimens produced positive RDT results that were not confirmed by culture.  This finding suggests that while cholera RDTs are useful for rapid screening and early outbreak detection, culture confirmation remains important for accurate diagnosis and surveillance reporting. Similar findings have been reported in evaluation of culture and cholera RDTs, where high sensitivity was observed and varying specificity [12,13]

Rapid diagnostic tests can play an important role in early case detection at district level, while culture testing remains essential for confirmation and surveillance [8,9].

Study limitations: Only 29 specimens were available for comparison, limiting the number of observations included in the assessment. Culture was used as the reference method for comparison; however, culture results may be affected by factors such as specimen quality, transport conditions, prior antimicrobial exposure, and laboratory processing procedures.

Conclusion: The assessment provides operational evidence that cholera RDTs can effectively support rapid screening during outbreak response activities, while culture testing remains important for laboratory confirmation and surveillance.

Acknowledgements: We thank the District Health Team, District Task Force for coordinating the response and providing access to records and communities. We acknowledge the National Microbiology Reference Laboratoryand Yumbe RRH for laboratory support, and the US Centres for Disease Control and Prevention for technical and financial support through the Uganda Public Health Fellowship Program. We thank CDC Uganda.

Conflict of interest: The authors declare no competing interests.

Authorship contributions: PP drafted the initial version of the manuscript. SG, MN AND MO revised the article for substantial intellectual content. PP, PZ, MN and JW participated in the outbreak investigation.  SG, GN, SN also supervised the field activity.

Copyright and licensing: All materials in the Uganda Public Health Bulletin are in the public domain and may be used and reprinted without permission; citation as to source, however, is appreciated. Any article can be reprinted or published. If cited as a reprint, it should be referenced in the original form.

References

1. Cholera. Cholera Outbreak Response Field Manual [Internet]. Available from: https://choleraoutbreak.org/book-page/cholera
2. Almagro-Moreno S, Taylor RK. Cholera: environmental reservoirs and impact on disease transmission. Microbiol Spectr. 2013;1(2): OH–0003.
3. Azman AS, Rudolph KE, Cummings DAT, Lessler J. The incubation period of cholera: a systematic review. J Infect. 2013;66(5):432–8.
4. Paye NG, Miller RHB, Zhou C, Weamie SJY, Davies TA. Systematic study and meta-analysis of risk factors associated with cholera outbreaks in Africa: a review. Adv Infect Dis. 2021;11(2):240–60.
5. Cholera [Internet]. World Health Organization. Available from: https://www.who.int/news-room/fact-sheets/detail/cholera
6. Cholera – Global situation [Internet]. World Health Organization. [cited 2023 Oct 4]. Available from: https://www.who.int/emergencies/disease-outbreak-news/item/2022-DON426
7. World Health Organization (WHO). Reports exponential rise in cholera cases in Africa | UN News [Internet]. Available from: https://news.un.org/en/story/2023/02/1133337
8. Uganda Ministry of Health. Prevention and Control of Cholera Operational Guidelines for the National and District Health Workers & Planners, 2017
9. Baumgartner ET, Williams KN, Rai E, Rosser EN, Marasini RP, Dahal S, Shakya A, Lynch J, Karki K, Bajracharya DC, Sack DA, Debes AK. Enhancing national cholera surveillance using rapid diagnostic tests (RDTs): A mixed methods evaluation. 2025 May 6;19(5):e0013019
10. World Health Organization (WHO). Cholera 2018. Weekly Epidemiol Rec. 2019; 93:561–80.
11. World Health Organization. The Global Task Force for Cholera Control. Interim Technical Note. The Use of Cholera Rapid Diagnostic Tests. 2016. p. 1–5.
12. Chowdhury G, Senapati T, Das B, Kamath A, Pal D, Bose P, Paul S, Asish M, Dutta S, Ramamurthy T. Laboratory evaluation of rapid diagnostic tests for the detection of Vibrio cholerae O1 using diarrheal samples. 2021; 10(3): 260-75.
13. Kalluri P, Naheed A, Rahman S, Ansaruzzaman M, Faruque ASG, 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(1):49–55.