Diagnostic performance of the 'Benzamin-Mujib diarrhea stool card' for dysentery and cholera in children under 5 years: a pilot study at a tertiary-care hospital in Bangladesh

¹Department of Paediatrics, Gastroenterology and Nutrition, Sylhet MAG Osmani Medical College Hospital, Sylhet, Bangladesh, ²Department of Paediatrics, Sylhet MAG Osmani Medical College, Sylhet, Bangladesh, ³Department of Microbiology, Sylhet MAG Osmani Medical College, Sylhet, Bangladesh, ⁴Department of Paediatrics, Jalalabad Ragib Rabeya Medical College Hospital, Sylhet, Bangladesh, ⁵Dr.Benzamin’s Pediatric Liver Research Center and Nutrition Clinic, Sylhet, Bangladesh

^&Corresponding author
Md Benzamin, Department of Paediatrics, Gastroenterology and Nutrition, Sylhet MAG Osmani Medical College Hospital, Sylhet, Bangladesh

Introduction    

To date, diarrhea is a significant public health issue and is the third leading cause of death in under-five children worldwide [1]. Despite a significant (79.2%) drop in mortality since 1990, acute diarrhea remains prevalent, particularly in low- and middle-income countries like Bangladesh. In 2021, diarrhea killed 56, 200 children under the age of five in Southeast Asia and 443, 832 globally [2]. Diarrhea causes approximately 7% of all under-five deaths in Bangladesh [3]. Acute invasive diarrhea characterized by bloody stool and, when accompanied by high fever, is called dysentery. In immunocompetent children, dysentery is most commonly caused by Shigella species, followed by Campylobacter jejuni and non-typhoidal Salmonella, while enteroinvasive Escherichia coli (EIEC) is a less frequent cause. In contrast, acute invasive diarrhea without fever is mainly due to Entamoeba histolytica and Shiga toxin-producing E. Coli (STEC). For watery diarrhea, Rotavirus, Adenovirus, Norovirus, Vibrio cholerae O1 and O139 (V. cholerae), Enterotoxigenic E. Coli (ETEC) are common organisms; among them, V. cholerae causes distinctive rice-watery stools with a fishy odor [4]. For resource-limited settings, the World Health Organization (WHO) and Integrated Management of Childhood Illness (IMCI) guideline suggest a symptom-based analysis and treatment of acute diarrhea without routine laboratory investigations [5,6]. Administering antibiotic is recommended only for suspected cholera and dysentery [5,6], but 58%-86% of patients with acute diarrhea received antibiotics in different healthcare settings in Bangladesh [7-11].

Although physicians mostly prescribe antibiotics in suspicion of cholera or dysentery, these conditions account for only 2.1%-14% and 1.8%-14.2% of acute diarrhea cases in Bangladesh, respectively [12-15]. Misdiagnosis and inappropriate antibiotic use contribute to substantially increasing treatment costs, persistent diarrhea, antimicrobial resistance, and various drug-related side effects [16-18]. Inappropriate information from the mother, a lack of low-cost investigations/easy-to-use diagnostic tools, and poor confidence in the clinical diagnosis of cholera and dysentery are the main causes of this malpractice and expensive therapy [11]. As fever is not included as a diagnostic criterion of dysentery in WHO/ IMCI guideline, antibiotics are also used in STEC-associated bloody diarrhea, which may increase the risk of hemolytic uremic syndrome (HUS) [19]. In light of this, Benzamin et al. [11] created the ‘Benzamin-Mujib Diarrhea Stool Card’ (BM diarrhea stool card), a visual decision-support tool that contains characteristics, a stool image with distinctive stool colour, and a few symptoms that point to the suspected organism of acute diarrhea and give confidence in the clinical diagnosis of cholera and dysentery. It also includes fever, with an axillary temperature of ≥101°F, as a criterion for the diagnosis of dysentery to avoid antibiotic use in STEC-associated diarrhea. This stool card was found to be a useful tool for judicious antibiotic use in the management of acute diarrhea, particularly in settings with limited resources [11]. However, its diagnostic accuracy and predictive value have not yet been evaluated. Therefore, this study aimed to determine the diagnostic performance of the ‘BM diarrhea stool card’ as a visual decision support tool for clinical diagnosis of cholera and dysentery among children under five years of age with acute diarrhea.

Methods     

Study design and setting: this prospective observational pilot diagnostic-accuracy study was conducted in the Department of Pediatrics, Sylhet M. A. G. Osmani Medical College Hospital, Bangladesh, from January to May 2025. The facility is a tertiary-care teaching hospital providing inpatient and outpatient care. The study was designed and reported in accordance with the Standards for Reporting Diagnostic Accuracy Studies (STARD) 2015 guidelines.

Study population: the study population comprised children aged 6 months to 5 years who were hospitalized in the pediatric inpatient department with acute diarrhea during the study period. Children with acute diarrhea accompanied by pneumonia, bronchiolitis, severe acute malnutrition, acute kidney injury, immunodeficiency, or heart disease, as well as those who had received antibiotics before hospital admission, were excluded. A total of 75 children were screened for eligibility, of whom 27 were excluded because of predefined comorbidities or prior antibiotic use, leaving 48 participants for the final analysis (Figure 1). The eligible participants were recruited consecutively during the study period. As this was an exploratory pilot study, the target sample size of 48 participants was based on pilot-study recommendations rather than a formal precision-based diagnostic-accuracy sample-size calculation [20].

Data collection: the BM diarrhea stool card (Figure 2) was briefly explained to all physicians working in the pediatric diarrhea unit before patient recruitment and feedback were taken. Following enrollment, the mother or caregiver was asked to identify the image on the card that most closely matched the color and appearance of the child's stool (Figure 3). A single freshly passed stool was directly inspected by the attending physician and compared with the card when the caregiver was uncertain. During subsequent follow-up, changes in stool appearance and associated symptoms, including blood in the stool and fever, were also documented using the card. A fresh stool specimen was collected in a sterile plastic container and transported to the microbiology laboratory as soon as possible, preferably within 1 hour of collection. Initial processing included microscopic examination and culture using routine culture media. Residual stool specimens were stored at -80°C and subsequently processed using multiplex reverse-transcription polymerase chain reaction (RT-PCR). Nucleic acids were extracted directly from stool specimens using the Bio-Speedy® Rapid Nucleic Acid Extraction Kit (Bioeksen; Cat. No. ZFNAE01), followed by testing with the Gastroenteritis RT-qPCR MX-24L panel for a broad range of bacterial, viral, and parasitic enteric pathogens. All stool examinations, including culture and RT-PCR, were performed by an experienced microbiologist who was blinded to the BM diarrhea stool card assessment. Reverse-transcription polymerase chain reaction and culture results were used as the reference standard for evaluating the diagnostic performance of the stool card. Participants were managed according to the IMCI guidelines and subsequent laboratory findings.

Definitions: diarrhea was defined as the passage of three or more loose or watery stools within 24 hours, and acute diarrhea as an episode lasting less than 14 days [4]. Dysentery was defined operationally as acute diarrhea with visible blood in the stool with an axillary temperature ≥101°F. A BM Diarrhea Stool Card result was considered suggestive of cholera when the stool appearance corresponded to subfigure b of (Figure 2) (BM diarrhea stool card). A BM diarrhea stool card result was considered suggestive of dysentery when the stool appearance corresponded to subfigure d of (Figure 2) (BM diarrhea stool card) and the child had an axillary temperature ≥101°F. Bloody stool without fever ≥101°F and stool appearances not corresponding to subfigures b or d of figure 2 (BM diarrhea stool card) were classified as non-dysentery and non-cholera, respectively. For the diagnostic-accuracy analysis, the reference-standard-positive dysentery was defined by detection of one or more of the following pathogens: Shigella/EIEC, Salmonella spp., or Campylobacter spp. by either RT-PCR or culture. Detection of Vibrio cholerae by either RT-PCR or culture was defined as reference-standard-positive cholera. Clinical diagnosis based on the BM diarrhea stool card was used as the index test, and cases were classified as either index-test positive or index-test negative. True-positive, false-negative, false-positive, and true-negative classifications were determined by comparing the BM diarrhea stool card classification with the corresponding reference-standard result.

Statistical analysis: all collected data were analyzed using Statistical Package for the Social Sciences (SPSS) version 26. MedCalc statistical software [21], was used to calculate the diagnostic performance of the BM diarrhea stool card against the reference standard. For dysentery, cholera, and the combined target condition, standard 2x2 contingency tables were constructed, and sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), accuracy, positive likelihood ratio (PLR), and negative likelihood ratio (NLR) were calculated with corresponding 95% confidence intervals. Positive predictive value and NPV were calculated directly from the observed study data rather than using an assumed population prevalence. Diagnostic estimates were interpreted cautiously because of the small pilot sample and the very small number of participants with laboratory-confirmed cholera. No external validation was performed because this was a single-center pilot study.

Ethical considerations: the study was conducted in accordance with the ethical standards of the Institutional Review Board, Sylhet M. A. G. Osmani Medical College Hospital, Bangladesh, under approval number SOMC/2023/2854. Written informed consent was obtained from the parent or legal guardian of each participating child before enrolment. The study was conducted in accordance with the ethical principles of the Declaration of Helsinki.

Results     

The mean age of the study population was 23.13 ± 16.9 months, and the male-to-female ratio was 2.7:1 (35 males and 13 females). None of the participants had received rotavirus vaccination. Severe dehydration was observed in 3 children (6.3%): one with cholera and two with rotavirus diarrhea. All children were discharged without significant adverse events.

Pathogen Identification: out of 48 stool samples, 15 (31.3%) tested positive for a single pathogen, while 30 (62.5%) were positive for multiple pathogens; 3 (6.25%) samples were negative. The most frequently identified organisms were Enteroaggregative Escherichia coli (EAEC) and Rotavirus, each detected in 16 cases (33.3%), followed by EPEC (10, 20.8%), Shigella/EIEC (8, 16.7%), and STEC (6, 12.5%). Adenovirus, Norovirus (GI/GII), and ETEC were each detected in 4 cases (8.3%), and pathogens detected less frequently included Campylobacter, Sapovirus, Cryptosporidium, Cyclospora, Giardia, and Plesiomonas shigelloides, with 2 cases each (4.2%). The least common was Vibrio cholerae, isolated in 1 case (2.1%).

Clinical features by diarrhea type: among the cohort, 16 children had pathogens associated with invasive bacterial diarrhea detected by RT-PCR. However, eight of these children did not meet the operational definition of dysentery because they lacked either bloody stool or the required fever. Notably, all six STEC cases (37.5%) presented without fever ≥101°F; only one had bloody stool. Clinical features in this group included high-grade fever in 10 (62.5%), abdominal pain in 13 (81.25%), bloody stool in 9 (56.25%), and tenesmus in 8 (50%). In contrast, children with non-invasive diarrhea exhibited low-grade fever in 22 (68.75%), vomiting in 17 (53.1%), and bloody stool in 4 (12.5%-two with rotavirus and two with rotavirus plus EAEC). Microscopic examination revealed numerous fecal leukocytes and red blood cells in children with invasive/bloody diarrhea, whereas non-invasive cases showed minimal or no leukocytes. Two cultures were positive for Shigella, and both were concordantly positive by RT-PCR; all other cultures were negative.

Diagnostic performance of the BM diarrhea stool card: among the 48 children included in the analysis, the BM diarrhea stool card classified 12 participants as suggestive of dysentery and 2 as suggestive of cholera. According to the reference standard, 10 participants were positive for dysentery and 1 for cholera. For dysentery, the BM diarrhea stool card showed a sensitivity of 80% (95% CI: 44.39-97.48), specificity of 89.5% (95% CI: 75.20-97.06), PPV of 66.7% (95% CI: 42.94-84.17), NPV of 94.4% (95% CI:83.04-98.33), PLR of 7.60 (95% CI:2.86-20.20), NLR of 0.22 (95% CI: 0.06-0.78) and accuracy of 87.5% (95% CI:74.75-95.27) (Table 1). For cholera, the BM diarrhea stool card showed a sensitivity of 100% (95% CI: 2.50-100.00), specificity of 97.9% (95% CI, 88.71-99.95), PPV of 50.0% (95% CI:12.58-87.42), NPV of 100% (95% CI: 92.29-100.00), PLR of 47.00 (95% CI: 6.76-326.75), NLR of 0.00, and accuracy of 97.9% (95% CI: 88.93-99.95). However, only one participant was positive for cholera according to the reference standard, while two were classified as cholera-positive by the stool card, including one false-positive result. Therefore, these estimates, particularly the sensitivity and PPV, should be interpreted with substantial caution (Table 2). For the diagnosis of dysentery and cholera combined, the BM diarrhea stool card showed a sensitivity of 81.82% (95% CI: 48.22-97.72), specificity of 86.49% (95% CI:71.23-95.46), PPV of 64.29% (95% CI:43.20-80.99), NPV of 94.12% (95% CI: 81.94-98.26), PLR of 6.05 (95% CI: 2.56-14.33), NLR of 0.21 (95% CI: 0.06-0.74), and overall accuracy of 85.42% (95% CI: 72.24-93.93) (Table 3).

Discussion     

This pilot study evaluated the diagnostic performance of the BM diarrhea stool card for clinical diagnosis of dysentery and cholera among children aged 6 months to 5 years with acute diarrhea. The card showed 80.0% sensitivity, 89.5% specificity, 66.7% PPV, and 94.4% NPV for dysentery. For the combined dysentery and cholera diagnosis, sensitivity was 81.8%, specificity 86.5%, PPV 64.3%, and NPV 94.1%. For cholera, the sensitivity estimate of 100% had a very wide 95% CI (2.5-100%), reflecting substantial uncertainty because two were classified as cholera by the card, while one participant was reference-standard-positive. Thus, the dysentery findings are more informative than the cholera estimates. The PPV of 66.7% and 64.29% for diagnosis of dysentery and combined dysentery-cholera, respectively, also indicates that a positive card classification did not invariably correspond to a reference-standard-positive condition and should therefore not be regarded as laboratory confirmation. For dysentery and the combined dysentery-cholera diagnosis, the PLRs were 7.60 (95% CI: 2.86-20.20) and 6.05 (95% CI: 2.56-14.33), respectively, while the NLRs were 0.22 (95% CI: 0.06-0.78) and 0.21 (95% CI: 0.06-0.74), respectively, indicating moderately good diagnostic performance. A positive result substantially increased the probability of disease, while a negative result substantially reduced the likelihood of disease. For cholera, the card showed a markedly higher PLR of 47.00 (95% CI: 6.76-326.75), indicating strong evidence for cholera when the test result was positive.

The NLR was 0.00, suggesting that a negative test result was highly effective in reducing the likelihood of cholera in this study population. However, the wide confidence interval for the PLR indicates some uncertainty around this estimate. The potential clinical implications of the BM diarrhea stool card should also be interpreted cautiously. The relatively high NPV observed for dysentery, cholera, and the combined cholera- dysentery suggests that the card may have value as a clinical decision-support or rule-out aid in populations with a similar prevalence of the target conditions. Its potential contribution to antibiotic stewardship should be evaluated prospectively by measuring prescribing decisions alongside diagnostic performance and clinical outcomes. The etiological distribution illustrates the complexity of childhood diarrhea in this setting. Enteroaggregative Escherichia coli (EAEC) and rotavirus were the most frequently detected pathogens, followed by EPEC and Shigella/EIEC, while 62.5% of participants had multiple pathogens detected. Regional and international variation in diarrheal etiology has been reported, reflecting differences in sanitation, vaccination, healthcare access, and local epidemiology [21-31]. Polymicrobial infection has also been observed in other studies but to a lesser extent [23-25]. Because mixed infections are common, it is difficult to attribute specific clinical features to a single pathogen unless characteristic symptoms such as bloody stools or rice-watery stools are present.

In the clinical dysentery group, 33% of cases were linked to rotavirus and EAEC. A similar finding was observed by Fonseca-Romero et al. [31]. Among the study population, 12.5% had STEC-associated diarrhea, and the BM diarrhea stool card distinguishes them from dysentery, due to the lack of either bloody stool or fever ≥101°F. However, this criterion requires validation in larger cohorts before its clinical implications can be established. Previous prediction and decision-support models for childhood diarrhea have incorporated seasonal and anthropometric variables [32], smartphone-based applications [23,33], electronic decision-support systems [34,35], blood investigations [36], or biomarkers such as fecal calprotectin and lactoferrin [37]. These approaches may provide useful predictive information but differ substantially in complexity, resource requirements, and intended settings. In Bangladesh, stool culture tests for Salmonella, Shigella, and Vibrio cost $21, comprehensive gastrointestinal pathogen panels cost $120, and rotavirus PCR costs $38 [38]. However, these tests are not widely available. The BM diarrhea stool card does not require electronic devices, specialized laboratory equipment, or additional investigations, which may make it feasible in resource-limited settings.

Based on the findings of this pilot study and the intended use of the card in resource-limited settings, the BM diarrhea stool card was subsequently modified to improve practical usability, including the addition of selected stool images and simplified Bengali-language instructions (Figure 4), particularly to facilitate use by village doctors and other frontline healthcare providers. However, the modified version was not evaluated in the present study, and therefore its diagnostic performance remains to be established. The study has several strengths, including its prospective design, consecutive recruitment, predefined clinical classification, laboratory-based reference testing, blinded microbiological assessment, and reporting of standard diagnostic-accuracy measures with confidence intervals. However, the small sample size, single tertiary-care setting, and inclusion of only hospitalized children limit generalizability. Small reference-standard-positive cases for cholera makes the cholera estimates particularly unstable. Observer reliability and external validation were not evaluated.

Conclusion     

The BM diarrhea stool card showed promising preliminary diagnostic performance as a visual decision-support tool for clinical diagnosis of dysentery and cholera. Its relatively high negative predictive values suggest potential usefulness as a clinical decision-support or rule-out aid. We recommend larger multicenter studies including primary care and community settings to assess the generalizability of the findings, observer reliability after standardized training, and the impact on antibiotic prescribing and patient outcomes before widespread implementation.

Competing interests     

The authors declare no competing interests.

Authors' contributions     

Conception and study designed: Md Benzamin. Data collection: Tarun Kanti Paul, Md Hadayet Hussain, Tuhin Barua Tamal, Pranto Chakroborty. Data analysis, and interpretation: Md Benzamin. Manuscript drafting: Md Benzamin. Manuscript revision: Akhlaq Ahmed, Tarun Kanti Paul, Md Tarek Azad, and Md Ziaur Rahman Chowdhury. Guarantors of the study: Md Benzamin and Md Ziaur Rahman Chowdhury. All the authors have read and approved the final version of this manuscript.

Acknowledgments     

We sincerely acknowledge all the children and their parents, along with hospital staff, who co-operated conducting this research.

Tables and figures     

Table 1: performance of BM diarrhea stool card for diagnosis of the dysentery

Table 2: performance of BM diarrhea stool card for diagnosis of the cholera

Table 3: performance of BM diarrhea stool card for diagnosis of the cholera and dysentery combined

Figure 1: participant flow and diagnostic classification of children with acute diarrhea according to the BM diarrhea stool card and reference-standard testing (note: Invalid index test =0, tnvalid reference test=0)

Figure 2: Benzamin-Mujib diarrhea stool card: a) rotavirus diarrhea; b) cholera; c) enterotoxigenic E. Coli (ETEC) diarrhea; d) dysentery

Figure 3: mother matching the stool appearance of her baby on the BM diarrhea stool card

Figure 4: the modified Benzamin-Mujib diarrhea stool card: A1, A2, C and any of figure D1, D2, D3 without fever or <101°F: non cholera, non dysentery diarrhea; no need of antibiotic; figure B1or B2: presumptive cholera; need antibiotic; figure D1 or D2 or D3+fever ≥101°F ± abdominal pains,tenesmus: presumptive dysentery; need antibiotic

References