Introduction
Intestinal schistosomiasis, caused by the trematode Schistosoma mansoni, remains a significant public health challenge in many tropical and subtropical regions worldwide, including Brazil. Endemic areas, particularly in the Northeastern and Northern regions, continue to show high prevalence rates despite the substantial impact of control programs, which have markedly reduced morbidity and overall disease burden (Lindholz et al. Reference Lindholz, Favero, Verissimo, Candido, de Souza and Dos Santos2018; Santos et al. Reference Santos, Silva, Lima, Santos, Lima and Amor2017). However, the persistence of infection is often associated with a shift toward lower parasite loads, which complicates both accurate diagnosis and the evaluation of assessment strategies (Caldeira et al. Reference Caldeira, Teixeira, Silveira, Fries, Romanzini and Bittencourt2012; Gonçalves et al. Reference Gonçalves, Gonçalves Barreto, Maldonado, Maione, Rey and Soares2005; Menezes et al. Reference Menezes, Santos, Oliveira, Campos, Negrão-Corrêa and Geiger2023).
As parasite loads decrease in endemic regions, diagnostic challenges emerge, since disease detection has traditionally relied on the Kato-Katz (KK) method (Katz et al. Reference Katz, Chaves and Pellegrino1972). Although this method is widely used for populational screening, due to its simplicity and low cost, its sensitivity becomes limited, particularly in areas of low endemicity or among individuals with light infections – conditions commonly observed following long-term control interventions (Kongs et al. Reference Kongs, Marks, Verlé and Van der Stuyft2001). Several studies conducted in Brazil and other countries have demonstrated the reduced sensitivity of KK in detecting light infections (Bärenbold et al. Reference Bärenbold, Raso, Coulibaly, N’Goran, Utzinger and Vounatsou2017; Caldeira et al. Reference Caldeira, Teixeira, Silveira, Fries, Romanzini and Bittencourt2012; Fenta et al. Reference Fenta, Hailu, Alemu and Amor2020; Igreja et al. Reference Igreja, Matos, Gonçalves, Barreto and Peralta2017; Kongs et al. Reference Kongs, Marks, Verlé and Van der Stuyft2001; Menezes et al. Reference Menezes, Santos, Oliveira, Campos, Negrão-Corrêa and Geiger2023).
To improve diagnostic accuracy, alternative or complementary methods, such as the spontaneous sedimentation method (SSM), have been proposed. This method has proven useful for identifying infections missed by KK, particularly in cases of low-intensity cases (Bärenbold et al. Reference Bärenbold, Raso, Coulibaly, N’Goran, Utzinger and Vounatsou2017; Fenta et al. Reference Fenta, Hailu, Alemu and Amor2020). Although less frequently applied, SSM has demonstrated superior performance in specific epidemiological contexts, particularly when larger stool samples are analyzed, which improves egg recovery (Fenta et al. Reference Fenta, Hailu, Alemu and Amor2020). This characteristic becomes especially important in areas of low endemicity, where S. mansoni remains a public health concern despite the overall decline in prevalence (Gonçalves et al. Reference Gonçalves, Gonçalves Barreto, Maldonado, Maione, Rey and Soares2005).
In this context, the objective of this study was to compare the diagnostic performance of the KK and SSM methods for detecting intestinal schistosomiasis in three localities in the state of Sergipe, Northeastern Brazil. The study aimed to assess the efficacy of these methods under different epidemiological conditions, to improve the accuracy of prevalence estimates, and support enhanced diagnostic and control strategies for schistosomiasis, particularly in areas characterized by low parasite burdens.
Materials and methods
This study was conducted in two different populations from Sergipe (SE) and one locality in Alagoas (AL), in Northeastern Brazil, each characterized by varying degrees of schistosomiasis endemicity. Endemicity was defined by its two components (positivity and infection intensities) according to Brazilian control and epidemiological guidelines (Brasil, 2024). The study sites included: the village of Areia Branca, in the municipality of Pacatuba (SE), the rural settlement Marcelo Déda and a public school in the municipality of Malhador (SE), and the rural area of Feira Grande (AL).
A single stool specimen was collected from each participant using a clean plastic container labelled with a unique identification number. Stool collection took place in 2022 (Pacatuba), 2023 (Feira Grande), and 2025 (Malhador), involving voluntary residents from each community and schoolchildren. Each participant received a plastic container for stool collection. The samples were subsequently collected by local health agents and transported to the laboratory for analysis.
The inclusion criteria for the study population were residents of the study areas and families whose adult participants (aged ≥18 years) or legal guardians of children provided written informed consent. Children under the age of 18 were included through the Free and Informed Consent Form (FICF) signed by their parents or guardians. Exclusion criteria included individuals under antiparasitic treatment within the previous six months and children aged ≤2 years.
All procedures were approved by the Research Ethics Committees, under the Certificate of Presentation for Ethical Consideration (CAAE) numbers 55239522.3.0000.5149, 57431022.2.0000.5208, and 71027323.7.0000.5060. Parents, guardians, and participating children were informed about the study’s objectives and procedures. All stool samples were anonymized and coded to ensure confidentiality.
The participants were informed of their stool test results, and all diagnosed infections were treated with praziquantel. The dosage administered was 50 mg/kg for adults and 60 mg/kg for children, following the Brazilian technical guidelines (Ministério da Saúde 2018).
Stool samples were processed using two conventional parasitological methods: KK and SSM. The commercial KK kit (HelmTest®; Biomanguinhos/FIOCRUZ, Rio de Janeiro, RJ, Brazil) was used, and two slides were prepared for each sample, as recommended by the World Health Organization (WHO 2022). The number of eggs per gram of faeces (EPG) was calculated by averaging the egg counts from both slides and multiplying by 24.
The SSM (Lutz Reference Lutz1919) was also performed, based on the principle of spontaneous sedimentation, for diagnosing intestinal parasites. Briefly, approximately 2 g of stool were emulsified in water, filtered through gauze, and allowed to sediment in a conical tube for 2 hours. The supernatant was discarded, the sediment was resuspended, and the procedure was repeated twice. Drops of the final sediment were placed on slides, stained with Lugol’s iodine, and two slides were examined under light microscopy. To avoid interpretive bias, microscopists analyzing the KK slides were blinded to the SSM results, and vice versa.
Contingency table was used to compare the results and assess the agreement between methods. For each locality, cases were classified according to infection intensity: low, moderate, or high, following WHO guidelines (WHO 2022).
To evaluate the performance of each method, key diagnostic validity indicators were calculated: sensitivity, specificity, accuracy, area under the ROC curve (AUC), and Kappa coefficient. Sensitivity was defined as the proportion of true positive cases correctly identified by each method, relative to the total number of positives according to the reference standard (individuals were classified as positive when at least one method was positive and negative only when both tests were negative). Specificity was calculated as the proportion of true negative cases identified. Accuracy represented the total proportion of correct classifications (both positive and negative). AUC summarized the overall discriminative ability of each method, while the Kappa coefficient measured the level of agreement between methods beyond chance. The interpretation of Kappa followed Landis and Koch’s (Reference Landis and Koch1977) criteria: <0, poor agreement; 0 to 0.20, slight agreement; 0.21 to 0.40, fair agreement; 0.41 to 0.60, moderate agreement; 0.61 to 0.80, substantial agreement; and 0.81 to 1.0, almost perfect agreement.
All statistical analyses were performed using R software (version 4.5.0), and a significance level of 5% (p <0.05) was adopted for all comparisons.
Results and discussion
In Pacatuba, 234 individuals were examined, of whom 24 (10.3%) tested positive for schistosomiasis. Among these, 23 (95.8%) cases were identified by the KK, while 3 (12.5%) were detected by SSM. In Malhador (Marcelo Déda settlement), 95 (47.5%) out of 200 individuals tested positive, with 87 (91.6%) cases detected by KK and 39 (41.1%) by SSM. At Barrocão school in Malhador, the positivity rate was 23.0% (26/113), with 23 (88.5%) cases identified by KK and 16 (61.5%) by SSM. In Feira Grande, 15 positive cases were found among 343 individuals (4.4%), all of which were detected by KK (100.0%), while only one case was identified by SSM (6.7%).
In Malhador (Barrocão school), where the highest proportion of moderate or severe infections (42.3%) was observed, the SSM showed a sensitivity of 61.5%, an AUC of 0.81, and a Kappa coefficient of 0.71 – representing the best performance of spontaneous sedimentation among all evaluated sites (Table 1). Similar results were observed in the Marcelo Déda settlement, where the proportion of moderate/severe infections was 32.6%, and SSM achieved a sensitivity of 41.1%, an AUC of 0.71, and a Kappa coefficient of 0.42.
Performance metrics of Kato-Katz (KK) and spontaneous sedimentation method (SSM) for schistosomiasis diagnosis in four locations of Northeastern Brazil
a Both methods (KK and SSM) have 100% specificity.
Conversely, in areas with lower proportions of moderate/severe infections, the performance of SSM was substantially lower. In Pacatuba (SE), where only 4.2% of infections were moderate or severe, SSM sensitivity was 12.5%, with an AUC of 0.56 and a Kappa coefficient of 0.20. A similar pattern was observed in Feira Grande (AL), where only 13.3% of infections were classified as moderate/severe, and SSM sensitivity dropped to just 6.7%, with an AUC of 0.53 and a Kappa coefficient of 0.12.
Figure 1 shows that the sensitivity, area under the ROC curve (AUC), and Kappa coefficient of agreement of SSM progressively increased with infection intensity, indicating better diagnostic performance in scenarios with higher parasite burdens. However, even in these cases, KK consistently outperformed SSM across all parameters, with sensitivity ranging from 88.46% to 100%, AUC values between 0.94 and 1.00, and a Kappa coefficient always above 0.90.
Diagnostic performance of sensitivity (A), AUC (B), and Kappa coefficient (C) of the Kato-Katz and spontaneous sedimentation method as a function of the intensity of intestinal schistosomiasis infection in four locations of Northeastern Brazil.
This study compared the KK and SSM for the detection of S. mansoni in three locations in Northeastern Brazil. While KK showed superior sensitivity in all settings, the results indicate that, as infection intensity increased, SSM was able to detect additional cases, particularly in moderate- and high-intensity infections.
In Pacatuba (SE) and Feira Grande (AL) areas where infections with low parasite loads predominated, KK detected 95.8% and 100% of cases, respectively, whereas SSM detected only 12.5% and 6.7%. The low sensitivity of SSM in such contexts is consistent with previous reports, confirming that KK remains the preferred diagnostic method in low-intensity transmission settings.
In the Marcelo Déda settlement, where 47.5% of individuals tested positive, KK maintained its superiority, identifying 91.6% of cases, while SSM detected 41.1%. Although KK identified most of the cases, SSM detected eight infections missed by KK. This pattern aligns with the findings of Fenta et al. (Reference Fenta, Hailu, Alemu and Amor2020), who observed that SSM may demonstrate higher sensitivity in samples with higher parasite loads, although KK remains more efficient overall for detecting high-intensity infections.
At Barrocão school, both KK and SSM detected more cases. Kato-Katz identified 88.5% of infections, while SSM identified 61.5%. Moreover, SSM detected three cases that KK failed to identify. This underscores the complementary value of SSM in areas with moderate or severe infections, consistent with the findings of Bärenbold et al. (Reference Bärenbold, Raso, Coulibaly, N’Goran, Utzinger and Vounatsou2017), who reported higher sensitivity of SSM in cases of severe infections.
Although SSM processes a much larger stool volume, KK frequently achieves higher sensitivity due to intrinsic methodological advantages. The glycerine present in the KK thick smear clears faecal debris and enhances egg visibility, allowing efficient detection even with approximately 42 mg of stool. In contrast, sedimentation retains more debris and may lose eggs during repeated decantation steps, particularly in low-intensity infections. These factors help explain why KK consistently outperforms SSM despite the smaller amount of processed material.
While KK demonstrated higher sensitivity in all evaluated contexts, SSM played an important complementary role in detecting infections of moderate to high intensity. This likely reflects the larger quantity of stool processed and the improved egg recovery in such cases (Fenta et al. Reference Fenta, Hailu, Alemu and Amor2020; Kongs et al. Reference Kongs, Marks, Verlé and Van der Stuyft2001). These results suggest that SSM could serve as a valuable adjunct to KK, particularly in moderately endemic areas.
Previous literature comparing both methods also highlights the greater effectiveness of SSM in detecting moderate to high infections. Borel et al. (Reference Borel, Etard, Addo and Diakite1999) reported that SSM, by processing larger stool volumes, achieves superior sensitivity in high parasite load scenarios. Conversely, Kongs et al. (Reference Kongs, Marks, Verlé and Van der Stuyft2001) and Bärenbold et al. (Reference Bärenbold, Raso, Coulibaly, N’Goran, Utzinger and Vounatsou2017) observed that in areas of low infection intensity, KK performs better, corroborating the present findings that SSM is less effective in detecting light infections.
This study has some limitations that should be acknowledged. The sample was restricted to specific communities, and the number of participants, although representative of the surveyed areas, limits the statistical power for broader generalizations. Additional samples could improve diagnostic sensitivity, once a single stool sample was analyzed from each participant, which likely led to underestimation of prevalence due to daily variation in egg excretion, particularly for KK. Temporal variation in sample collection across study sites must be addressed. While environmental and control program dynamics could influence transmission over time, our analyses did not adjust for these potential time effects. Finally, the use of a composite reference standard based on the two index tests may introduce bias due to discrepant analysis (Hadgu Reference Hadgu1999; Lipman Reference Lipman and Astles1998; Miller Reference Miller1998), potentially affecting sensitivity and specificity estimates. Future studies should consider employing Bayesian latent class models as a more robust alternative for evaluating diagnostic accuracy without relying on an imperfect gold standard.
Despite these limitations, our findings remain relevant, as they demonstrate consistent differences in the diagnostic performance of KK and SSM across distinct epidemiological settings. These results underscore the importance of adopting complementary diagnostic strategies in endemic regions.
Although it has lower sensitivity and specificity, SSM remains operationally attractive due to its simplicity, minimal equipment requirements, and low cost. This makes it a feasible complementary method in rural and resource-limited settings where KK kits or trained microscopists may not be consistently available.
Future investigations should include larger sample sizes, multiple stool specimens per individual, and broader geographic coverage to confirm these observations and guide more effective surveillance and control strategies for schistosomiasis. Incorporating additional diagnostic approaches, including molecular or antigen-based tests, could also enhance accuracy and help identify residual transmission in low-endemicity areas.
In summary, the comparison between KK and SSM demonstrated consistently higher sensitivity of the KK method across all study sites. However, the detection of exclusive cases by SSM indicates that its combined use with KK may be advantageous, particularly in areas of low endemicity.
Based on our findings, the parallel application of both techniques could increase the overall detection rate by approximately 3%–8% in low-intensity areas and up to 10%–15% in moderate-to-high intensity settings, due to the identification of cases missed by KK alone. Although this complementary approach requires additional laboratory time and resources, the relatively low operational cost of SSM and its ability to detect otherwise overlooked infections support its inclusion in targeted surveillance activities and high-priority populations.
In endemic areas with heterogeneous infection intensities, the combined approach may improve prevalence estimates, optimize treatment coverage, and contribute to more effective interruption of transmission. These findings support the complementary use of KK and SSM to improve surveillance accuracy and guide evidence-based schistosomiasis control strategies.
Acknowledgements
SSD and CGT are research fellows (Bolsa de Produtividade em Pesquisa) from the National Council of Scientific and Technological Development – Brazil (CNPq).
Financial support
This research received no specific grant from any funding agency, commercial or not-for-profit sectors.
Competing interests
The authors declare that they have no competing interests.
Ethical standard
The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008.
