|Year : 2014 | Volume
| Issue : 2 | Page : 116-121
Microsporidia in diarrheic patients: detection and evaluation of intestinal inflammation and malabsorption
Amel A El-Sayed, Hend A El-Taweel PhD , Sahar A Abou Holw, Safia S Khalil
Department of Parasitology, Medical Research Institute, Alexandria University, Alexandria, Egypt
|Date of Submission||25-May-2014|
|Date of Acceptance||25-Jul-2014|
|Date of Web Publication||19-Jan-2015|
Hend A El-Taweel
91 Ahmed Shawky Street (7/13), Moustafa Kamel, 21523, Alexandria
Source of Support: None, Conflict of Interest: None
Early reports on microsporidial diarrhea involved mainly HIV-immunodeficient patients. More recent studies indicate that microsporidial spores may also be detected in immunocompromised persons not infected with HIV as well as in immunocompetent individuals. However, the exact mechanism of microsporidial diarrhea is not clearly defined.
The aims of this study were to evaluate the contribution of microsporidia toward the burden of diarrheal diseases and to investigate the occurrence of associated intestinal inflammation and malabsorption.
Patients and methods
Stool samples of 237 patients with diarrhea were examined for microsporidial spores using modified trichrome stain. Microsporidia-positive samples were examined for concomitant parasitic infections. Intestinal inflammation was evaluated in patients infected solely with microsporidia ( n = 30) by comparing the fecal lactoferrin level measured by an enzyme immunoassay with that of a control group of healthy, age and sex matched, parasite-free nondiarrheic individuals ( n = 15). Fecal pH and assessment of fecal fat using oil red O stain were used as indictors of carbohydrate and fat malabsorption, respectively.
Microsporidian spores were detected in 15.6% of diarrheic patients. Considering the median and range values, microsporidia-infected patients showed significantly higher fecal lactoferrin levels (median 49.75, range 2.8-220 μg/g, respectively) and lower fecal pH (median 5.82, range 5.12-6.98, respectively) compared with the control group (median 4.1, range 2.4-31 μg/g and median 6.7, range 6.3-7.16 μg/g, respectively). A significantly greater proportion of microsporidia-infected patients had elevated lactoferrin levels (>7.4 μg/g stool), markedly acidic stool (pH < 6), or increased fecal fat compared with the control group.
Infection with microsporidia is present in a considerable proportion of diarrheic patients and results in an intestinal inflammatory response as well as carbohydrate and fat malabsorption. Enteric microsporidiosis should be taken into consideration in the management of diarrheal diseases.
Keywords: diarrhea, lactoferrin, malabsorption, microsporidia
|How to cite this article:|
El-Sayed AA, El-Taweel HA, Abou Holw SA, Khalil SS. Microsporidia in diarrheic patients: detection and evaluation of intestinal inflammation and malabsorption. Parasitol United J 2014;7:116-21
|How to cite this URL:|
El-Sayed AA, El-Taweel HA, Abou Holw SA, Khalil SS. Microsporidia in diarrheic patients: detection and evaluation of intestinal inflammation and malabsorption. Parasitol United J [serial online] 2014 [cited 2017 Aug 16];7:116-21. Available from: http://www.new.puj.eg.net/text.asp?2014/7/2/116/149563
| Introduction|| |
Microsporidia are obligate intracellular spore-forming organisms that infect a wide range of hosts  . They are described by convention as protozoa, although phylogenetic studies indicate that they are more closely related to fungi , . In humans, early reports on microsporidiosis involved mainly HIV-immunodeficient patients, where microsporidial spores were discovered in their stool samples following the identification of HIV as the causative agent of AIDS  . More recent reports have shown that spores of microsporidia are also detected in immunocompromised persons not infected with HIV as well as in immunocompetent individuals , . Several species of microsporidia have been implicated in human infections. Enterocytozoon bieneusi and Encephalitozoon intestinalis, the most commonly encountered species, are transmitted through ingestion of food and water contaminated with the spores and infect the enterocytes of the small intestine, causing diarrhea  .
The pattern of microsporidial diarrhea is influenced by the host immune status; it persists in immunodeficient individuals, whereas it disappears within a few weeks in individuals with intact immunity despite persistence of infection for long periods  . In immunocompromised patients, microsporidia cause extensive damage, with villous atrophy, crypt hyperplasia, and impaired nutrient absorption, resulting in weight loss and wasting  . In immunocompetent individuals, the mechanism, impact, and public health significance of microsporidial diarrhea are not clearly defined.
An important initial approach in the management of diarrhea is to evaluate its type as this guides the need for further testing  . Although many enteric bacterial infections are associated with inflammatory diarrhea, viral infections have been found to induce subtle or no inflammation  . Some pathogens induce damage of intestinal architecture and impair nutrient absorption, leading to osmotic diarrhea  . Little is known about the role of intestinal inflammation and malabsorption in the pathogenesis of microsporidial diarrhea. A sensitive nonspecific method for the assessment of gastrointestinal inflammation is the estimation of fecal lactoferrin, which is a glycoprotein constituent of the secondary granules of neutrophilic leukocytes  . Lactoferrin is released at the site of inflammation following the flux and degranulation of neutrophils, where it participates in the host defense against various pathogens , .
The present study investigated the detection rate of microsporidiosis in patients with diarrhea and evaluated the contribution of inflammation and malabsorption toward its pathogenesis.
| Patients and methods|| |
Type of the study
Case control study.
The study included 237 patients with diarrhea. Their age ranged from 20 to 45 years. They were recruited from among those attending the Parasitology Department, Medical Research Institute, Alexandria University, during the period from May 2012 to November 2012. Patients were subjected to an assessment of history and a thorough clinical examination.
Detection of microsporidiosis
Patients enrolled in the study were asked to provide stool samples. After thorough mixing, thin fecal smears were prepared on glass slides, allowed to dry, and fixed in absolute methanol. The smears were stained using modified trichrome stain and then examined microscopically under oil immersion magnification (×1000)  . At least 30 microscopic fields were examined. Spores of microsporidia were identified with their characteristic morphology as ovoid pink bodies measuring ~1-2 μm in length with a clear uncolored vacuole and a pink polar body  .
Evaluation of intestinal inflammation and malabsorbtion
For evaluation of intestinal inflammation and malabsorption associated with enteric microsporidiosis, a case-control study was carried out. Cases (group I) included diarrheic patients with microsporidia-positive stool samples. Their medical history was recorded. Exclusion criteria included intake of intestinal antiseptics, antimicrobials, or NSAIDs within the previous 2 weeks, inflammatory bowel diseases, chronic diseases, for example, chronic kidney disease, chronic liver disease, malignancy or diabetes mellitus, and the presence of concomitant parasitic infections. The latter was determined by examining stool samples using direct saline smear, formol ether concentration technique, and modified Ziehl-Neelsen stain  . Fifteen age-matched and sex-matched healthy individuals with no detectable ova or parasites in their stool samples were included in the control group (group II). Portions of stool samples of cases and controls were kept frozen at −20°C for further testing.
Assessment of intestinal inflammation and malabsorption
Intestinal inflammation was assessed by measuring concentrations of lactoferrin in frozen fecal samples using the quantitative enzyme-linked immunosorbent assay IBD Scan (TechLab, Blacksburg, Virginia, USA)  . The assay was performed according to the manufacturer's instructions. With each batch of samples, a positive control (purified human lactoferrin) and a negative control (washing buffer) were included and lactoferrin concentrations were determined by comparison with a standard curve using purified human lactoferrin. Lactoferrin level 7.4 μg/g or less stool was used as a cut-off value as described by the kit manufacturer.
The pH of fresh fecal samples emulsified in distilled water was measured using a pH meter  . This was used to evaluate carbohydrate malabsorption  . An acidic level less than 6.5 was set as a classification criterion and marked acidity was defined by pH less than 6.
Fecal split fat content was estimated qualitatively using oil red O staining. Two drops of 36% (vol/vol) acetic acid, followed by drops of oil red O were added to stool smears. After mixing, the preparation was covered with a cover-slip and heated gently until it began to boil. While still warm, the slides were examined under low-power magnification (×100). The numbers of large fat globules (>20 μm) in 10 fields were recorded. Increased fecal split fat content, defined by fat globules 10 or more per field, was used as an indicator of impaired absorption of fat  .
Data analysis was carried out using the statistical package for social sciences (SPSS, version 17.0, 2006; SPSS Inc., Chicago, Illinois, USA). Normality of quantitative variables was tested using the Kolmogorov-Smirnov test. Fecal lactoferrin levels and pH values were presented as median and range. The nonparametric Mann-Whitney test was used to compare these two parameters in cases and controls and Spearman's correlation coefficient (r) was used to assess their correlation in the patient group. The χ2 -test was used to analyze categorical variables. P value less than 0.05 was considered statistically significant.
The study was reviewed and approved by the Local Ethical Committee of the Medical Research Institute and an informed consent was obtained from all participants.
| Results|| |
Out of 237 diarrheic stool samples, spores of microsporidia were detected in 37 (15.6%) samples. All patients examined had no apparent cause of immunosuppression. In all positive smears, the average number of spores was 10 or more per oil immersion field ([Figure 1]). Microsporidian spores were found alone in 30 samples and in conjunction with Giardia lamblia trophozoites and/or cysts in four cases, with Hymenolepis nana eggs in two cases and with Entamoeba histolytica cysts in one case. Among patients infected only with microsporidia (n = 30), symptoms other than diarrhea were abdominal discomfort (73%), fever (33%), and vomiting (17%).
|Figure 1 Arrows point to spores of microsporidia under oil immersion magnification in a modified trichrome-stained smear of a fecal sample obtained from a diarrheic patient.|
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[Figure 2]a and b shows the fecal lactoferrin level and pH values in patients and controls. Lactoferrin level was higher in the patient group (median 49.75, range 2.8-220 μg/g) compared with the control group (median 4.10, range 2.4-31 μg/g, respectively, P < 0.05). Patients showed lower fecal pH values compared with the control group (median 5.82, range 5.12-6.98 vs. median 6.7, range 6.3-7.16, respectively, P < 0.05). Among patients, no statistically significant correlation was found between fecal lactoferrin level and pH (r = 0.044, P > 0.05).
|Figure 2 Box plots showing (a) fecal lactoferrin levels (μg/g stool) and (b) fecal pH values in diarrheic microsporidia-infected patients (n = 30) and controls (n = 15). Mann– Whitney test.|
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The distribution of patients and controls according to the presence or absence of deranged test results is shown in [Table 1]. The majority of patients with microsporidial diarrhea (70%) had a lactoferrin level higher than 7.4 μg/g stool as compared with the control group (13.3%) (P < 0.05). Increased fecal fat was significantly more common in the patient group (63.3%) compared with only one individual in the control group (11 globules/field). Categorization of fecal pH values indicated a significant difference between patients and controls; marked stool acidity (<6) was detected in more than 50% of patients and mild acidity in 30%. Slight alteration in stool pH (6.3-6.5) was present in 20% of controls.
|Table 1 Distribution of microsporidia-infected diarrheic patients and controls according to fecal lactoferrin level, pH, and fat content|
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| Discussion|| |
Enteric microsporidiosis is underestimated especially in developing countries as its diagnosis is not typically included in routine examination of stool for ova and parasites. It requires special staining techniques that are rarely ordered because of technician inexperience and/or lack of physicians' awareness of its clinical significance , . Most cases of microsporidial diarrhea have been described in immunocompromised patients ,, . For individuals without immunosuppression, recent studies suggested that it might be more common than previously appreciated , .
In the present study, spores of microsporidia were detected in a considerable proportion (15.6%) of apparently immunocompetent individuals with diarrhea. Published reports showed the presence of microsporidium infection in 17% of elderly patients with diarrhea in Spain  , in 10% of Indian HIV-negative individuals with diarrhea  , and in 6% of patients presenting with digestive system complaints in Turkey  . Several environmental sources of microsporidia have been documented. The spores were recovered from drinking water despite meeting the European and national regulations on water sanitary quality  . Potential sources of human infection also include contaminated food and other infected individuals or animals  . Like other fecal-oral pathogens, infection with microsporidia is more common among poor individuals under impoverished settings  . Of concern, previous studies in Egypt have reported that fresh fruit juices, coins and banknotes are potential environmental vehicles for the transmission of microsporidial spores , .
We found elevated fecal lactoferrin levels in patients with microsporidial diarrhea, confirming the stimulation of an intestinal inflammatory response. An in vitro study showed that microsporidiosis induces the production of proinflammatory cytokines and chemokines, which are important initiators of polymorphonuclear cell recruitment to the intestinal mucosa  . A high lactoferrin level was confirmed in cryptosporidiosis and giardiasis and marked elevation is well documented for some bacterial infections ,, .
In contrast to the present study, Samie et al.  found that fewer HIV-negative individuals infected with E. bieneusi in South Africa had elevated levels of lactoferrin compared with noninfected individuals. This discrepancy may be attributed to a decreasing inflammatory response because of recurrent exposure in endemic areas  . Another explanation is the infection by different species and/or genotypes of microsporidia. It has been suggested that genotypes of E. bieneusi have different biologic characteristics and pathogenic potential as only some genotypes are linked to an increased risk of diarrhea in HIV-infected patients  . Data on genetic differentiation of microsporidia species associated with human infection in Egypt are limited. One study detected E. intestinalis, but no E. bieneusi in cancer patients  .
Lactoferrin shows antimicrobial activity against several pathogens  . With microsporidia, lactoferrin as well as other neutrophilic peptides were found to inhibit enterocytes infection by E. intestinalis and to inhibit spore germination of the insect microsporidian species, Anncaliia algerae  . Thus, neutrophil recruitment and release of their peptides at the site of infection play a significant role in controlling microsporidiosis.
In our study, evaluation for malabsorption in patients relied on the measurement of fecal pH and staining of fecal smear using fat stains. Although these tests are relatively simple, they have important implications in diarrheal illnesses. Fermentation of the malabsorbed carbohydrates by colonic bacteria results in more acidic luminal contents. A measured stool pH less than 5.3 (only three patients in the present study; data not shown) points to isolated carbohydrate malabsorption. Generalized malabsorption results in less acidity because of the buffering action of the malabsorbed amino acids. In most secretory states, fecal pH tends to be neutral because of excess bicarbonate content  . Staining of fecal fat droplets is a rapid tool often used to screen patients for fat malabsorption. It can be performed on a single stool sample, thus obviating the need to collect a large amount of stool for chemical analysis. At the same time, it is highly specific and adequately sensitive , .
In many intestinal infections, the associated pathological changes interfere with nutrient absorption  . Findings in the present study suggest the presence of an ongoing malabsorptive pathology in enteric microsporidiosis. A growing body of evidence has linked infection with this intracellular spore-forming organism to impaired absorption of d-xylose, fat, vitamin B 12 , and zinc in immunocompromised patients. Furthermore, clearance of infection was associated with normalization of the results of absorption tests  . Proposed mechanisms of impaired absorption include lactase deficiency, reduced villus height, and villus surface reduction  . In the present study, the lack of a correlation between fecal lactoferrin and fecal pH suggests that intestinal inflammation and malabsorption occurred independently. Apart from inflammation, parasite invasion and excess death of enterocytes may have contributed toward malabsorption  . It is noteworthy that the consequences of mucosal damage and malabsorption associated with microsporidiosis on nutritional health are likely to be severe in vulnerable groups. Clinically, it is well recognized that microsporidiosis causes weight loss in HIV-infected patients and in children with persistent diarrhea , .
It was argued that shedding of microsporidian spores in stool samples could be the result of subclinical infection, persistent shedding after symptoms have subsided, or ingestion of pathogens below an infectious dose  . In the present study, the implication of microsporidia in the etiology of diarrhea was confirmed by the ability to detect many spores in several microscopic fields of smears prepared from unconcentrated stool. Other techniques, such as molecular methods, can detect a very low number of spores that might be clinically insignificant , .
| Conclusion|| |
In conclusion, the current study showed the presence of microsporidian spores in a considerable proportion of diarrheic patients and highlighted the possible implication of an inflammatory component and impaired absorption in the pathogenesis of enteric microsporidiosis in apparently immunocompetent individuals. Testing for microsporidia should be part of patients' evaluation for diarrhea, particularly whenever inflammatory changes are suspected. Microsporidia should be included in the list of pathogens causing tropical malabsorption.
| Acknowledgements|| |
A.A. El-Sayed interviewed patients, collected and prepared stool samples. H.A. El-Taweel proposed the study topic and participated in the conception and implementation of work, interpretation of the results, and writing of the manuscript. S.A. Abou Holw participated in the design, conception and implementation of work, interpretation of the results, and writing of the manuscript. S.S. Khalil reviewed the presentation and interpretation of the results.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]