Abstract
Background: Reliable diagnosis of cryptosporidiosis remains a major challenge in resource-limited settings, particularly among marginalised nomadic pastoralist populations with close human–animal interactions. Microscopy is widely used for routine diagnosis but is constrained by limited sensitivity and inability to differentiate Cryptosporidium species. Molecular techniques such as nested polymerase chain reaction (PCR) offer improved detection accuracy.
Objective: This study compared the diagnostic performance and agreement between modified Ziehl–Neelsen (mZN) staining and nested PCR targeting the 18S small-subunit ribosomal RNA (SSU rRNA) gene for the detection of Cryptosporidium spp. among nomadic pastoralists in Odeda Local Government Area, Abeokuta, Ogun State, Nigeria.
Methods: A cross-sectional study involving 105 stool samples was conducted intermittently between January 2021 and December 2022. Sample collection occurred during multiple field visits because nomadic pastoralist settlements are highly mobile and geographically dispersed. Participants were recruited through community-based engagement with settlement leaders and voluntary participation during each visit. Samples were analysed using mZN staining and nested PCR. A paired 2×2 contingency table was constructed. Agreement was assessed using McNemar’s exact test and Cohen’s kappa. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of mZN were calculated using PCR as reference, with 95% confidence intervals (CI).
Results: Modified Ziehl–Neelsen staining detected 65/105 (61.9%) apparent positive samples, whereas nested PCR detected 58/105 (55.2%). Twenty-seven samples were positive by both methods. McNemar’s exact test showed no significant directional bias between methods (χ2 = 0.5217, p = 0.4701). However, agreement between methods was poor (Cohen’s κ = −0.34). Using PCR as reference, modified Ziehl–Neelsen staining demonstrated sensitivity 46.6% (95% CI: 33.0–60.6%), specificity 19.1% (95% CI: 9.3–33.3%), PPV 41.5% (95% CI: 29.4–54.4%), and NPV 22.5% (95% CI: 11.8–36.6%).
Conclusion: Agreement between microscopy and nested PCR was poor. Although mZN staining remains useful as a low-cost screening tool in resource-limited settings, the substantial discordance observed in this study highlights the need for confirmatory molecular testing to improve diagnostic reliability and epidemiological accuracy.

Key words: Cryptosporidium spp.; modified Ziehl–Neelsen; nested PCR; diagnostic agreement; nomadic pastoralists; Nigeria.