Factors associated with detecting pathogenic intestinal parasites in multiple stool microscopy samples: a retrospective cross-sectional study

Abstract

Determining the optimal number of stool samples for accurate parasite detection is crucial in healthcare. However, previous studies have reported conflicting results. This study investigated factors associated with the detection of pathogenic intestinal parasites in additional stool microscopy examinations. This retrospective cross-sectional study at a tertiary care hospital outpatient clinic included patients who submitted three stool samples between 2012 and 2021 and had at least one pathogenic intestinal parasite-positive stool sample. Diagnostic yields for one, two, and three stool specimens were the primary outcome. The secondary outcome was the identification of factors associated with the time to the first positive stool microscopy result, which was analysed using ordinal logistic regression. Among the 103 infected patients, compared with the infection detection rate in the first specimen, the rate increased with the second specimen and further increased with the third specimen, achieving a cumulative detection rate of 100%. Some parasites, such as hookworms, were easily detected in the first sample. However, more than half the patients infected with Trichuris trichiura and all patients infected with Isospora belli were missed if only one stool specimen was collected. Immunocompetent hosts were significantly more likely to have pathogenic intestinal parasites detected in later stool specimens (adjusted ordinal odds ratio = 3.94 [95% confidence interval: 1.34–14.05]). Collecting multiple stool specimens was associated with a higher likelihood of detecting pathogenic intestinal parasites, particularly in immunocompetent patients. Clinicians may request a second or even a third sample if the first is negative.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00436-025-08570-5.

Background

Intestinal parasitic infections are common chronic infections in humans, particularly in developing countries (Awasthi et al. 2003). The definitive diagnosis of intestinal parasites involves identifying eggs, cysts, or parasites in the stool, typically through stool microscopy. Various stool microscopy methods exist, each with different sensitivities for detecting intestinal parasites (Charoensuk et al. 2019). For example, the Kato-Katz method, recommended by the World Health Organization (WHO), has a sensitivity of approximately 0.52 (0.48–0.57) (Charoensuk et al. 2019). Owing to the low sensitivity of stool microscopy and intermittent excretion of larvae and eggs, the WHO and other sources recommend collecting multiple stool samples over consecutive days (Thomson et al. 1984; Marti and Koella 1993; Nazer et al. 1993; Hiatt et al. 1995; Cartwright 1999; Glinz et al. 2010; Tarafder et al. 2010; Staat et al. 2011). Studies suggest that collecting stool samples for 2 days is sufficient for detecting general intestinal parasites with a relatively low error rate (Cartwright 1999; Tarafder et al. 2010; Staat et al. 2011). However, this method may be less sensitive for detecting Strongyloides stercoralis, as its parasitic larvae are intermittently present. Previous research has shown that up to seven stool samples may be required for 100% sensitivity in detecting S. stercoralis (Arifin et al. 2019). Nevertheless, the American Society of Parasitologists, the Subcommittee on Laboratory Standards (American Society of Parasitologists 1978), and some studies have reported sufficient detection rates with a single concentrated stool specimen using the formalin-ethyl acetate concentration (FECT) method (Gyorkos et al. 1989; Senay and MacPherson 1989; Morris et al. 1992; Branda et al. 2006), particularly for protozoa that are regularly released in stool (Not et al. 2020).

Some patients have ova that are easily detectable via stool microscopy, whereas others require multiple stool samples for parasite detection. Few studies have explored the factors associated with increased diagnostic yield when collecting multiple stool samples. One study found that patients without diarrhoea and defecating fewer than three times per day had a significantly higher diagnostic yield when two or three specimens were submitted than when one specimen was submitted (Staat et al. 2011).

Existing evidence regarding the optimal number of stool samples for adequate parasite detection is conflicting (Thomson et al. 1984; Senay and MacPherson 1989; Morris et al. 1992; Marti and Koella 1993; Nazer et al. 1993; Hiatt et al. 1995; Cartwright 1999; Branda et al. 2006; Glinz et al. 2010; Tarafder et al. 2010; Staat et al. 2011), and few studies have investigated patient characteristics associated with increased diagnostic yield when collecting multiple samples from patients without diarrhoea (Staat et al. 2011). Our hypothesis posited that certain patient groups may be more likely to have parasites detected in a single stool specimen, whereas others may require multiple samples for accurate diagnosis. Understanding this could help minimise unnecessary stool specimen requests for specific patient groups, reducing the burden on patients and laboratory technicians and potentially lowering medical expenses. The aim of this study was to calculate the diagnostic yield for one, two, and three stool specimens and identify the factors associated with the detection of pathogenic intestinal parasites in additional stool microscopy examinations.

Methods

Study design and setting

This retrospective cross-sectional study was conducted in the outpatient clinic of Songklanagarind Hospital, affiliated with the Faculty of Medicine, Prince of Songkla University, Thailand.

Study sample

This study included the first outpatient visits where patients submitted three stool specimens for intestinal parasite testing between 1 January 2012 and 31 December 2021. Patients were included if all three specimens were collected within a 7-day period from the first specimen and at least one stool specimen tested positive. Hospital visits where doctors requested stool microscopy to investigate acute diarrhoea (duration < 14 days) were excluded, as parasitic infections are generally not considered the primary cause. Owing to limited existing research, the sample size was calculated based on a previous retrospective study at a tertiary care hospital in the USA (Cartwright 1999). This study reported detection rates of 19.8% (129/651) for a single specimen and 49.4% (357/723) for more than one specimen. Using a two-proportion comparison formula, the minimum required sample size was 76 participants, based on a power of 0.8 and a type I error rate of 0.05. A total of 103 eligible participants were included in the analysis.

Variables

Details of the variable lists and definitions are provided in Additional file 1. The dependent variable was stool microscopy results. At our hospital, we used a combination of Kato’s thick smear and direct smear techniques. Stool microscopy test results were categorized as either negative (no pathogenic intestinal parasites detected) or positive (pathogenic intestinal parasites detected), excluding Blastocystis hominis in asymptomatic immunocompetent patients (Roberts et al. 2014). The stool results included outcomes from three stool microscopy tests: ‘First positive’ (all pathogenic intestinal parasites detected in the first specimen), ‘Second positive’ (pathogenic intestinal parasite detected in the second specimen but not in the first or a new pathogenic intestinal parasite detected in the second specimen), and ‘Third positive’ (pathogenic intestinal parasites detected in the third specimen but not in the previous two or a new pathogenic intestinal parasite detected in the third specimen). The independent variables included diarrhoeal symptoms previously associated with the number of stool specimens required (Staat et al. 2011). Additional factors potentially associated with the outcome, such as age, immune status, and eosinophilia, were also included in the analysis.

Data management and analysis

Relevant data were entered into Microsoft Excel and analysed using R software (R Core Team 2022, Vienna, Austria). Diagnostic yield was calculated as the proportion of patients with parasitic infections among all patients. Pairwise comparisons of proportions with Bonferroni correction were used to compare the diagnostic yield of one, two, and three stool specimens.

Proportional ordinal logistic regression analysis was conducted to examine the factors associated with the sequence of positive stool results in infected patients. The analysis used the ‘polr’ function from the MASS library (Liang et al. 2020) and followed standard steps, including a literature review to identify relevant factors, univariable analysis, and model assumption testing. The four assumptions of the proportional ordinal logistic regression model—the dependent variable is ordered; one or more of the independent variables are either continuous, categorical, or ordinal; no multicollinearity; and the proportional odds ratio—were met. An initial model was constructed using factors with p-values < 0.2 from the univariable analysis, including immunocompetent status. Diarrhoea, a factor previously associated with the outcome, was then added to the initial model. Backward stepwise elimination, using the step function from the base library, was employed to refine the model and identify predictive factors. Both immunocompetent status and diarrhoea were retained in the final model. Statistical significance was determined using a p-value < 0.05.

Ethical considerations

The study protocol was approved by the Ethics Committee of the Faculty of Medicine, Prince of Songkla University (approval number REC.65-056-9-1). As the data were retrieved from the hospital information system and a retrospective medical record review was conducted, informed consent was not obtained from patients. However, patient confidentiality was maintained by anonymising the recorded information, and all results were reported in aggregate form.

Patient and public involvement statement

Patients were not involved in developing this research, which stemmed from challenges observed in the ordering practices of physicians for stool specimens. However, the findings will help physicians determine the optimal number of stool examinations to request and identify patient characteristics that may benefit from multiple samples. This can alleviate the burden on patients associated with collecting and transporting stool specimens as well as reduce the workload for laboratory technicians by minimising unnecessary tests.

Results

Over a 10-year period, 7290 patients submitted stool samples for microscopic tests, of whom 741 submitted three stool samples (Fig. 1). Among these, 107 were diagnosed with parasitic infections; however, four patients were excluded owing to the detection of B. hominis in asymptomatic immunocompetent patients. The final analysis included 103 patients (Table 1), approximately one-third of whom were older adults. The study sample was evenly distributed between males and females, and three-quarters of the patients were immunocompetent hosts. Clinical characteristics of patients did not differ based on the order of positive stool results.

Fig. 1.

Participant flow diagram

Table 1.

Baseline demographic and clinical characteristics of patients stratified by the order of positive stool results

Characteristic	Total (n = 103)	Positive in the first specimen (n = 63)	Positive in the second specimen (n = 25)	Positive in the third specimen (n = 15)	p-value
Age: ≥ 60 years	39 (37.9)	22 (34.9)	11 (44.0)	6 (40.0)	0.719a
Sex: Male	50 (48.5)	29 (46.0)	16 (64.0)	5 (33.3)	0.139a
Immunocompetent host	76 (73.8)	42 (66.7)	21 (84.0)	13 (86.7)	0.117a
Stool test for eosinophilia workup	22 (21.4)	14 (22.2)	7 (28.0)	1 (6.7)	0.271a
Having diarrhoea	11 (10.7)	6 (9.5)	3 (12.0)	2 (13.3)	0.731b
Having abdominal pain	7 (6.8)	3 (4.8)	4 (16.0)	0 (0)	0.15b
Having fever	5 (4.9)	3 (4.8)	1 (4.0)	1 (6.7)	1b

^aChi-square test

^bFisher’s exact test

Data are presented as n (%)

The diagnostic yields of different numbers of stool specimens were compared (Fig. 2). If only one stool specimen was collected, 61.2% (63/103) of patients with parasitic infections were diagnosed. Compared with collecting a single specimen, collecting two or three stool specimens provided significantly better diagnostic yields and number of pathogens (p < 0.001 for both). Regarding the patterns of stool microscopy results based on the order of collection, 24.3% (n = 25) and 14.5% (n = 15) of infected patients were diagnosed via the second and third stool specimens, respectively (Table 2).

Fig. 2.

Diagnostic yield comparison among one, two, and three stool specimens

Table 2.

Stool microscopy results sorted by the order of collection (n = 103)

+ positive; +  + greater detection of new pathogenic intestinal parasites than that from the previous specimen;—negative

 Blue indicates positive results in the first specimen group (n = 63, 61.2%), green indicates positive results in the second specimen group (n = 25, 24.3%), and yellow indicates positive results in the third specimen group (n = 15, 14.5%)

Regarding the details of pathogenic intestinal parasites identified in infected patients (Table 3), the three most commonly detected parasites were B. hominis in symptomatic patients, S. stercoralis, and hookworms. Nearly three-quarters of hookworm infections and two-thirds of Giardia lamblia and S. stercoralis infections were detected in the first sample. However, more than half the patients infected with Trichuris trichiura and all patients infected with Isospora belli would have been missed if only the first sample had been tested.

Table 3.

Pathogenic intestinal parasites in patients who submitted three stool specimens and had ≥ 1 parasite (n = 103 visits)

	Positive in the first sample (n = 63)	First detected in the second sample (n = 25)	First detected in the third sample (n = 15)	Total** (n = 107)
Blastocystis hominis*	29 (60.4)	10 (20.8)	9 (18.8)	48 (44.9)
Strongyloides stercoralis	17 (65.4)	7 (26.9)	2 (7.7)	26 (24.3)
Hookworm	13 (72.2)	4 (22.2)	1 (5.6)	18 (16.8)
Trichuris trichiura	4 (44.4)	4 (44.4)	1 (11.2)	9 (8.4)
Giardia lamblia	2 (66.6)	1 (33.4)	-	3 (2.8)
Isospora belli	-	1 (50.0)	1 (50.0)	2 (1.9)
Entamoeba histolytica	1 (100.0)	-	-	1 (0.9)

*In a symptomatic or immunocompromised patient

**Multiple parasites can be detected in each sample

Data are presented as n (%)

Regarding the factors associated with the order of positive stool results among infected patients (Table 4), multivariable analysis indicated that immunocompetent hosts were significantly more likely to have pathogenic intestinal parasites detected in later stool specimens than immunocompromised hosts, with an adjusted odds ratio of 3.94 (95% confidence interval 1.34–14.05).

Table 4.

Factors associated with the order of positive stool results (n = 103 visits)

Factor	Crude ordinal OR	p-value	Adjusted ordinal OR	p-value
Sex: male	1.07 (0.50–2.34)	0.490	-
Age: older adult	1.32 (0.60–2.90)	0.992	-
Immunocompetent host	2.80 (1.08–8.28)	0.044	3.94 (1.34–14.05)	0.020
Stool test for eosinophilia workup	0.74 (0.28–1.86)	0.539	-
Having diarrhoea	1.36 (0.38–4.44)	0.622	2.87 (0.69–12.09)	0.141
Having abdominal pain	1.40 (0.34–5.25)	0.620	-
Having fever	1.14 (0.15–6.57)	0.885	-

OR odds ratio

Discussion

Performing stool microscopy on a single sample may fail to detect parasitic infections, particularly for certain parasites. Collecting additional stool samples, such as a second or third, can improve the likelihood of detecting pathogenic intestinal parasites, especially in immunocompetent patients.

The diagnostic yield of pathogenic intestinal parasites from the first stool specimen in our study (61.2%) was lower than that reported in studies of adopted children in the USA (79.0%) (Staat et al. 2011) and at a university hospital parasitology laboratory in Hamilton (90.0%) (Senay and MacPherson 1989). This discrepancy may be due to differences in study settings, which could affect the prevalence of parasitic infections. Additionally, these previous studies used the FECT method, which is known for its higher sensitivity than Kato’s thick smear and simple smear method used at Songklanagarind Hospital.

Performing stool microscopy two or three times improves the detection rate of pathogenic intestinal parasites compared with using a single specimen, consistent with the findings of previous studies (Marti and Koella 1993; Cartwright 1999; Branda et al. 2006; Glinz et al. 2010; Tarafder et al. 2010; Staat et al. 2011). A negative result from a single specimen may represent a false negative, potentially owing to the low sensitivity of stool microscopic techniques (Charoensuk et al. 2019). Furthermore, the excretion of larvae and eggs in stool is irregular, as reflected in the varied patterns of stool positivity observed in our study. Some ova, such as hookworm eggs, may also be lost during sample preparation (Cools et al. 2019). However, certain parasites, such as G. lamblia and Ascaris lumbricoides, are often detected in the first stool specimen owing to their distinct morphology, as shown in previous studies (Marti and Koella 1993; Glinz et al. 2010). Our findings showed that immunocompetent hosts were significantly more likely to have pathogenic intestinal parasites detected in later stool specimens. This may be because immunocompromised patients are at greater risk of severe disease (Garcia et al. 2018), leading to a higher parasite burden that facilitates easier detection.

The strength of this study lies in its extensive 10-year retrospective study period, significantly longer than that of most previous studies, which were typically conducted over less than a year (Thomson et al. 1984; Nazer et al. 1993; Hiatt et al. 1995; Staat et al. 2011). Additionally, our study is one of the few to assess the factors associated with the timing of positive stool results using ordinal logistic regression. However, this study has some limitations. First, as a retrospective study, symptom information during stool collection was missing in approximately 30% of medical records. Assuming the absence of symptoms when not mentioned may introduce information bias. Second, the low prevalence of parasitic infections limited our ability to detect associations between patient characteristics and stool results. Third, Kato’s thick smear and direct smear techniques have lower sensitivities for detecting parasitic infections than concentration techniques (Charoensuk et al. 2018). In settings where higher sensitivity parasitological techniques, such as FECT, are used, one sample may be sufficient to detect the parasite, as shown in previous studies (Gyorkos et al. 1989; Senay and MacPherson 1989; Morris et al. 1992; Branda et al. 2006). Finally, as the study was conducted in a university hospital, the generalisability of the results to other settings may be limited.

A negative result from a single stool specimen does not definitively rule out parasitic infection. Collecting stool samples on multiple occasions increases the diagnostic yield, particularly in immunocompetent hosts, where additional samples have been shown to improve detection rates. However, physicians must consider the difficulty of obtaining multiple stool samples. To address this challenge, one sample may be initially requested. If the result is negative but the clinical suspicion of a parasitic infection remains high, additional samples may be requested. Moreover, methods should be combined during the analysis of one stool sample to increase diagnostic sensitivity (Chandrashekar 2013). Future research should address the low sensitivity of stool microscopy. As even three consecutive negative stool samples may miss certain parasitic infections, incorporating alternative methods with higher sensitivity, such as stool culture or polymerase chain reaction for parasites (Garcia et al. 2018), as a reference standard could enhance diagnostic accuracy and reliability.

Conclusions

Analysing a single stool sample may fail to detect a parasitic infection. Collecting multiple stool specimens improves diagnostic yield, particularly in immunocompetent patients. In clinical practice, it is common to start with a single sample and request additional second and third samples if the initial test result is negative.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

WHO

World Health Organization

FECT

Formalin-ethyl acetate concentration

OR

Odds ratio

Author contributions

TA, WS, and PS conceptualised and designed this study. TA analysed the data. All authors interpreted the results. TA drafted the manuscript. All authors read and approved the final manuscript.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

The study protocol was approved by the Ethics Committee of the Faculty of Medicine, Prince of Songkla University (REC.65–056-9–1). Informed consent was not obtained due to the retrospective medical record review study design.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.