This cross-sectional study was performed on high-risk patients to evaluate the prevalence of S. stercoralis infection using parasitological, serological, and molecular methods. Since previous studies have demonstrated the sensitivity of 90–97.5% for the agar plate culture [22, 23], it was considered the reference test in the present study. In this study, the prevalence of S. stercoralis was 8.7% with the ELISA test. The sensitivity and specificity of the ELISA test were 100% and 93.8%, respectively. The seroprevalence observed by the ELISA test was 3.2 greater than the agar plate culture. Among the parasitological methods, the formalin ethyl acetate concentration technique is more used in routine fecal examinations, because the results can be achieved more quickly [5]. However, it is difficult to recognize the dead larvae by the formalin ethyl acetate at low magnification [24]. Baermann funnel technique and agar plate culture are other diagnostic methods used in S. stercoralis detection. Although both techniques are more sensitive in detecting S. stercoralis than the direct method, they are cumbersome and time-consuming procedures [5]. Nevertheless, the agar plate culture is used as a more efficient technique in clinical laboratories to diagnose S. stercoralis [23]. In this study, prevalence of S. stercoralis with the agar plate culture (2.7%) was lower than the ELISA test (8.7%), but it was higher compared to the formalin-ether concentration technique (1.3%), Baermann funnel technique (2%), and direct smear examination (1%). The obtained results revealed that the direct smear examination had the lowest sensitivity in diagnosing S. stercoralis (37.5%). These findings match those observed in previous studies [3, 22, 25]. A single stool examination fails to diagnose in up to 70% of the strongyloidiasis cases [26]. Serial stool examinations in three consecutive days can increase the chance of diagnosis by direct smear examination. According to a study conducted by Khieu et al. (2013), prevalence of S. stercoralis infection was increased from 15.9–21.6% by analyzing three stool samples [27]. In chronic strongyloidiasis, sensitivity of single stool examination in detecting S. stercoralis in symptomatic patients is approximately 50%, while in asymptomatic patients probably is less [26, 28]. The low parasite load and the periodic excretion of Strongyloides larvae make it difficult to detect microscopically [29]. It should be noted that sampling in consecutive days, especially in patients with underlying diseases, will be very difficult and impractical, thus the solution is to use more sensitive methods.
In the present study, the ELISA test demonstrated a higher prevalence rate than the coproparasitological methods; however the prevalence observed was lower compared to those reported by Rafiei et al., in the same region [10]. This discrepancy might be related to the cross reactivity in patients with other STH infections and/ or the difference in the sensitivity of the ELISA kits. Another possible explanation for this result might be that we used NovaLisa Strongyloides, ELISA Kit (NovaTec Immunodiagnostica, Germany), which it uses a recombinant antigen (NIE). ELISA test has limitations of inability to identify between current and past infection and lower sensitivity in diagnosing strongyloidiasis in patients with hematologic malignancies and/ or infected with HTLV-1 [5]. To overcome these limitations, Strongyloides-specific recombinant antigens, such as NIE, SsIR, and rSs1a have been used in ELISA kits [5, 29, 30, 31]. The coproantigen ELISA has also demonstrated promising results in detecting current infections [32]
According to the obtained results (Table 3), the direct smear examination, formalin-ether concentration, and Baermann funnel technique were the tests with the highest specificity. Given that these tests showed the highest PPV, they could be used routinely for diagnosis of S. stercoralis larva or epidemiological studies. ROC analysis showed that ELISA test with an AUC 0.955 and sensitivity of 100% was the most sensitive test in diagnosing strongyloidiasis.
The prevalence observed in this study was not significantly different between males and females by the agar plate culture; however, males showed a higher prevalence (4.1%) than females (1.3%). This may be due to the fact that some men are more exposed to contaminants due to working outside the home. These findings are consistent with those of Luvira et al. [9] who found higher infection rates in males, but differ from those of other studies [10, 22]. Although we could not find a significant difference between rural and urban areas (p = 0.445), previous studies have reported that living in rural areas and prolonged exposure to contaminated soil may put humans at S. stercoralis infection risk [3, 33]. In Iran, migration from rural to urban areas has increased in recent years. It is possible that some patients who live in the urban areas were infected during their residency in the rural areas. In our study, of the eight positive cases by the agar plate culture, 75% had a history of prolonged exposure to soil. This result may be explained by the fact that walking barefoot is common among some rural residents of Khuzestan Province. Another possible explanation is the occupation exposure of some patients to contaminated soil. According to the occupational data in Table 4, a significant association was found between occupation and infection (p = 0.002). The highest infection rate (50.0%), belonged to the farmers. In the tropics, warm moist temperatures, lower socioeconomic, and poor hygienic conditions increase risk of S. stercoralis infection [34]. Age-related findings revealed that the most of the diagnosed cases by the agar plate culture (87.5%), and ELISA test (57.7%) were patients over 60 years old. These results differ from previous published studies [3, 10], but agree with the findings of other studies [35, 36, 37]. This result may be explained by the fact that the S. stercoralis has a unique life cycle that allows autoinfection [5]. Therefore, in elderly population that immune system is weakened [37], parthenogenesis by the adult female may lead to accelerated autoinfection and clinical presentations [5]. Clinical manifestations such as gastrointestinal and pulmonary symptoms were associated with the agar plate culture positive (p = 0.017), but not with the ELISA test (p = 0.311). The ability of the parasite to replicate might lead to persistence within a host for decades. This discrepancy between ELISA results and clinical symptoms could be attributed to the past S. stercoralis infection. Chronic strongyloidiasis is often asymptomatic, but in acute strongyloidiasis main complications are gastrointestinal and pulmonary symptoms. Peripheral eosinophilia is common during acute infection (75%-80%) but intermittent or in some cases up to 75% in chronic infection, and absent in severe strongyloidiasis and the immunocompromised patients [38, 39]. In our study, 87.5% of the positive cases by the agar plate culture had eosinophil count above 10.0%. The significant association between infection and eosinophilia (p = 0.001) might be related to the fact that S. stercoralis female worm live within the submucosa of the gut and thus the eosinophilic response may occurs at high level [8]. Should be mentioned that out of the seven positive cases who had eosinophilia, 5 patients had a history of asthma and COPD. Therefore, this finding might be related to their underlying diseases. In a study conducted by Ashrafi et al. (2010) from Gilan Province, Northern Iran, out of 150 patients with undiagnosed eosinophilia, 42% were diagnosed with strongyloidiasis by the formalin-ether and the Kato-Katz techniques [40].
Statistical analysis showed that the Baermann funnel technique had the highest agreement with the agar plate culture (0.854), so that it was detected 75% of the positive cases by the agar plate. This finding is in agreement with Hailegebriel et al. (2017) findings which showed 75% of S. stercoralis infection was diagnosed by either agar plate culture or Baermann funnel technique [22]. Our results show that the diagnostic methods had a significant difference with the agar plate culture in diagnosing S. stercoralis infection. Sensitivity of the agar plate culture compared to the Baermann funnel technique, formalin-ether concentration, and direct smear examination was 1.3, 2, and 2.6 folds, respectively. The results of Baermann funnel technique and direct smear examination are in keeping with previous studies [22, 41].
We observed that 75% and 34.6% of the positive cases by the agar plate culture and ELISA test were patients treated by steroids, respectively. The most common risk factors for fatal hyperinfection or disseminated infection in immunocompromised patients are immunosuppression caused by corticosteroids, HTLV-1, and HIV infections [38]. Corticosteroids by interfere with the Th2 response via binding glucocorticoid receptors in the CD4 + Th2 cells may change the regular mechanisms of immunity. In addition, corticosteroids may also lead to dissemination through increased number of filariform larvae [7]. Glucocorticoids, the most widely used immunosuppressive drugs prescribed, are the most specifically associated with altering chronic strongyloidiasis to hyperinfection [28].
Of the eight positive cases by agar plate culture, we could only follow-up two cases. In one case, the agar plate culture was positive 6 months after treatment and in the other case it was positive 8 months after treatment. Considering that the test results of both patients were negative in the first and second visits after treatment, it seems that chronic cases of the disease are not easily detectable by the culture. Therefore, the importance of treating ELISA-positive patients with ivermectin, particularly in endemic areas is raised.
This study had limitations that should be considered. First, only one stool sample was examined from each patient. Second, ivermectin was not available in the region and patients were treated with albendazole. Third, due to the limited budget, PCR was performed only on culture-positive samples. Forth, data on the CD4 and Viral load of the HIV patients, and the duration of treatment with corticosteroids was not available in all patients.