To the best of our knowledge, this is the first study to report on the prevalence and predictors of intestinal microsporidiosis among HIV/AIDS patients in Yemen. It revealed the prevalence of intestinal microsporidiosis among 14.2% of HIV/AIDS patients receiving treatment in the ART clinic of the NACP in Sana'a city. The prevalence of intestinal microsporidiosis in the present study is comparable to the pooled prevalence of 13.0% in Eastern Europe and Central Asia, 14.4% in Western and Central Europe and North America and 15.4% in sub-Saharan Africa [25]. Compared to the present study, prevalence rates of 11.3–16.6% among HIV/AIDS patients were reported from Mali, Venezuela, Addis Ababa of Ethiopia, Benin city of Nigeria, Chandigarh city of India and Burkina Faso (using microscopy), from Kerman city of Iran, Tunisia and Guangxi region of China (using PCR), Russia (using serological testing) and Argentina using TEM [16, 17, 30–38]. However, much lower prevalence rates of 0.8–8.5% were reported among HIV/AIDS patients from the United States of America, France, the Democratic Republic of the Congo (using PCR), Shiraz city of Iran, Kinshasa city of Cameroon, Lucknow city of India (using PCR), Malaysia, the Abeokuta and Niger states of Nigeria and Hunan province of China [39–48]. On the other hand, higher prevalence rates of 21.3–85.2% were reported among HIV/AIDS patients from Uganda, Thailand, Lagos city of Nigeria, Buea and Limbe cities of Cameroon, Malawi (using PCR) and Mexico [49–54]. Variations in the prevalence of intestinal microsporidiosis could be attributed to differences in sociodemographic, environmental and behavioral risk factors. Besides, the use of different microscopical, immunological and molecular techniques plays a role in such variations. Therefore, the prevalence of intestinal microsporidiosis among HIV/AIDS patients in the present study should be compared and interpreted cautiously against the prevalence rates reported elsewhere.
The absence of a significant association between intestinal microsporidiosis and gender in the present study (18.0% vs. 13.1% for females and males, respectively) is in agreement with the finding reported among HIV-positive patients from Chandigarh city of India [17] and HIV/AIDS patients from Guangxi region of China [36]. In contrast, a significant association between intestinal microsporidiosis and gender of HIV-infected patients was reported from the Democratic Republic of the Congo, Benin city of Nigeria and Lucknow city of India [34, 41, 44]. On the other hand, the absence of a significant association between intestinal microsporidiosis and age in the present study is in agreement with the findings reported among HIV-infected patients from India, China and France [17, 36, 55]. In the present study, residence and household size were not significant predictors of intestinal microsporidiosis among HIV/AIDS patients. In contrast, a significant association was found between living in slum or rural areas and intestinal microsporidiosis among HIV-infected patients from Zimbabwe and New Delhi city of India [56, 57].
The absence of a significant association between intestinal microsporidiosis and the literacy status of HIV/AIDS patients in the present study is consistent with that reported among Chinese HIV/AIDS patients [36] and Nigerian HIV-infected patients [47]. Nevertheless, the absence of a significant association between intestinal microsporidiosis and the employment status of HIV/AIDS patients in the present study is inconsistent with that reported among Chinese HIV/AIDS patients, where the prevalence of intestinal microsporidiosis was significantly higher among farmers [36].
Having < 200 CD4 cells/µl was a significant independent predictor of intestinal microsporidiosis among HIV/AIDS patients in the present study, increasing the likelihood of infection by three-fold. Therefore, physicians should consider microsporidiosis in HIV/AIDS patients with such low CD4 cell counts, particularly diarrheic patients. This finding agrees with that found among HIV/AIDS patients from the Democratic Republic of the Congo, Cameroon, Lucknow city of India, and the Abeokuta and Niger states of Nigeria [41, 44, 46, 47, 52]. In contrast, intestinal microsporidiosis was not significantly associated with CD4 cell counts among HIV/AIDS patients from the Guangxi region of China [36]. Severe immunosuppression with below 200 CD4 cells/µl increases the viral load that affects the immune system, posing patients to the risk of infection with microsporidia and other opportunistic pathogens [11]. The protective role of T-cells against microsporidia has been well documented in animal models, where mice infected with microsporidia died of the disease but survived with T-cell transfers from sensitized donor mice [58].
Diarrhea was the only gastrointestinal feature significantly associated with intestinal microsporidiosis among HIV/AIDS patients in the present study, where patients with diarrhea were 3.4-fold more likely to have intestinal microsporidiosis. Similarly, intestinal microsporidiosis was significantly associated with diarrhea among HIV-infected patients from Lucknow city of India [44]. Intestinal microsporidiosis has therefore to be considered when investigating Yemeni HIV/AIDS patients for the causes of diarrhea, considering that routine stool examination does not include the detection of microsporidia. In contrast, intestinal microsporidiosis was not significantly associated with diarrhea among HIV-infected patients from Venezuela and the Abeokuta state of Nigeria [31, 46]. In another context, intestinal microsporidiosis could not be detected using PCR among HIV-patients with unexplained diarrhea in Denmark [59]. The absence of a significant association between intestinal microsporidiosis and nausea, vomiting or abdominal pain among HIV/AIDS patients in the present study is in line with that observed among HIV-infected patients from Thailand [50].
The source of drinking water was not significantly associated with the acquisition of intestinal microsporidiosis in the present study, showing that HIV/AIDS patients might be more exposed to spores from other sources such as contaminated soil. However, there is a need to assess the occurrence of microsporidian spores in different water sources. Likewise, the absence of such an association was found among HIV-infected patients from France and New Delhi city of India [55, 57]. In contrast, drinking unpiped or unfiltered water was found to be significantly associated with intestinal microsporidiosis among HIV-infected patients from Lucknow city of India and Zimbabwe [44, 56].
The present finding that bathing and/or swimming outdoors was not a significant predictor of intestinal microsporidiosis HIV/AIDS patients disagrees with the significant association between pool swimming and intestinal microsporidiosis observed among HIV-infected patients from France [55]. Nevertheless, the present study revealed that the likelihood of infection increased by three-fold and four-fold, respectively, among patients not washing hands after contact with soil and those eating without washing hands. It is noteworthy that microsporidia are ubiquitous, and their spores can survive and remain infective for up to six months in dry conditions [60]. Soil contaminated with spores can be a major source of infection transmission through unwashed hands after contact with soil and before eating. Therefore, hand hygiene with soap and water after contact with soil and before meals is an important practice to be delivered to HIV/AIDS patients as part of educational awareness-raising materials to reduce their exposure to many foodborne infections, including intestinal microsporidiosis.
The significant association between intestinal microsporidiosis and consuming unwashed raw produce among HIV/AIDS patients in Sana’a city is consistent with that reported among Peruvian HIV/AIDS patients consuming watermelon in Lima city [61]. Microsporidia are potentially transmitted via food-borne ingestion [62], and this could partially explain why those consuming unwashed raw produce in the present study were about twice and a half more likely to have intestinal microsporidiosis compared to their counterparts. In this context, microsporidian spores have been detected on fresh fruit and vegetables, and vegetables contaminated with spores have been associated with foodborne outbreaks elsewhere [63, 64]. HIV/AIDS patients should therefore be educated about the importance of proper washing of raw produce, such as fruit and vegetables, before eating.
Microsporidia are not host-specific, infecting a wide range of invertebrate and vertebrate animals. Nevertheless, the importance of zoonotic transmission is still not fully studied, particularly in developing countries. In the present study, the presence of domestic animals was not significantly associated with intestinal microsporidiosis among HIV/AIDS patients. Consistent findings were reported among HIV/AIDS patients from France and HIV-infected patients from Lucknow city of India and Nigeria [44, 47, 55]. In contrast, the presence of domestic animals was significantly associated with intestinal microsporidiosis among HIV/AIDS patients from Zimbabwe, Lima city of Peru, New Delhi city of India and the Democratic Republic of the Congo [56, 57, 61, 65]. However, the presence of domestic animals does not mean direct exposure or contact with them, and frequent occupational contact with animals can increase the risk of infection.
The absence of indoor latrines in the present study was a significant independent predictor of intestinal microsporidiosis, where the likelihood of infection was approximately six-fold higher among those having no latrines. This finding is consistent with that observed among HIV/AIDS patients from Peru, where the lack of flush toilets in houses was significantly associated with E. bieneusi infection [61]. Likewise, the absence of indoor toilets and using public toilets were significantly associated with intestinal microsporidiosis among HIV-infected patients from New Delhi city of India [57]. In another context, using public and individual pit toilets was significantly associated with intestinal microsporidiosis among HIV/AIDS patients from the Democratic Republic of the Congo [65]. In contrast, the absence of latrines was not significantly associated with intestinal microsporidiosis among Nigerian HIV-infected patients [47]. Because most infections occur by human-to-human transmission through the fecal-oral route [62], the transmission of spores while using public toilets could occur through touching door handles, water taps, or using the containers that are kept in public toilets for cleaning purposes. It is noteworthy that Yemenis usually use water cups or containers instead of toilet paper for such purposes, exposing them to the risk of infection. On the other hand, indiscriminate defecation was not significantly associated with intestinal microsporidiosis in the present study. In contrast, defecation in open fields was significantly associated with intestinal microsporidiosis among HIV-infected patients from New Delhi city of India [57]. Nevertheless, indiscriminate defecation outdoors can contribute to indirect risk of infection. Defecation in open fields could lead to contact with soil or exposure to recreational waters contaminated with spores from urine and stool of infected animals and humans [66].
The present study is limited by the use of light microscopy of stained smears for the detection of microsporidia, making their identification to the species level impossible. However, this study aimed to detect microsporidia rather than their speciation, and future molecular studies are required for the speciation and genotyping of microsporidia. Therefore, this is a pioneer study in uncovering the burden of intestinal microsporidiosis among HIV/AIDS patients in Sana’a. Another issue is that the prevalence of intestinal microsporidiosis could be higher than that found in the present study because of the lower specificity and sensitivity of light microscopy compared to molecular techniques [18]. Therefore, molecular studies are recommended to determine the prevalence of intestinal microsporidiosis among HIV/AIDS patients after controlling for the possible disadvantages of such techniques, such as the difficult extraction of spore nucleic acids and the presence of PCR inhibitors in stool samples. To reach as an accurate estimate of the prevalence of intestinal microsporidiosis as possible, the GCK staining technique was used for detecting microsporidian spores because of its high specificity and sensitivity levels compared to the chromotrope 2R-based WMT [24]. This technique is an improved modification of the quick-hot Gram-chromotrope technique [23], which was developed for rapid and good differentiation of microsporidian spores from the background fecal materials. From a diagnostic perspective, the diagnostic accuracy of the GCK staining technique needs to be evaluated compared to PCR to assess its utility in the diagnosis of intestinal microsporidiosis among HIV/AIDS and other immunocompromized patients in resource-limited countries, including Yemen, where the cost and applicability of PCR are still impractical.