Prevalence, intensity and complications of Microsporidium spores amongst HIV-positive hospital patients in Ilorin, Nigeria

Background Microsporidiasis, which is of great concern for immunocompromised patients, is poorly studied in developing countries. Objectives A study was carried out amongst HIV-positive hospital patients and HIV-negative hospital controls in Ilorin, Nigeria, between January 2009 and July 2010 to determine the prevalence and intensity of Microsporidium spores and the complications associated with their presence. Method Stool samples from 750 HIV-positive patients and 375 HIV-negative patients were studied using the Chromotrope-2R staining technique. Determination of CD4+ count was performed on the Partec Cyflow SL-3 CD4/8 instrument. Intensity of spores was determined by counting the total number of the spores in a 10 µl stained smear of stool. Images were captured with Phenix Microimage Analysis Software and data obtained were analysed using the Statistical Package for the Social Sciences. Results The prevalence of Microsporidium isolates amongst the HIV-positive hospital patients was significantly higher (42.4%) than amongst the HIV-negative controls (19.2%) (p < 0.05). The intensity of microsporidial spores amongst HIV-positive hospital patients was also significantly higher than amongst the controls (p < 0.05). However, the difference in the intensity of spores amongst HIV-positive patients who were on antiretroviral therapy (n = 411) and those who were not (n = 339) was not significant (p = 0.236). Microsporidiasis in HIV infection infection was common amongst patients with with low CD4+ counts, diarrhoea, body rashes and cough. Conclusion Both the prevalence and intensity of Microsporidiasis are high amongst HIV-positive hospital patients; campaigns to promote awareness, prevention and control are required. Laboratory testing for microsporidia in HIV patients should be performed routinely so as to identify the organism for prompt medical attention.


Introduction
Microsporidiasis is recognised by the World Health Organization as being a globally-important medical, public health and socio-economic problem. 1,2 Microsporidia are obligate and opportunistic intracellular spore-forming parasites which belong to the phylum Protozoa. 3,4,5 Although there are more than 1000 known Microsporidium species and as many as 100 genera of the group affecting both vertebrates and invertebrates, 6 there are only 12 species, belonging to 8 genera, that are known to be pathogenic to humans. 7,8 The first well-documented human case of Microsporidiasis, where Encephalitozoon spp. was detected in a stool sample of a diarrhoea patient, was reported in 1959. 9 Since then, microsporidia have become increasingly better recognised as opportunistic human pathogens, 10,11,12 particularly in patients with suppressed immunity, including organ transplant patients and HIV patients. 5,13,14 Microsporidia can also infect people with competent immune systems, resulting in latent infections. 6,15,16 In HIV patients, microsporidia have been implicated in intestinal, ocular, pulmonary and renal diseases, and have been shown to cause diarrhea and other adverse health conditions. 17,18,19 Chronic diarrhoea is common in HIV-positive patients but the enteric pathogens are identified in fewer than 50% of cases. 5,7,8,20,21,22 The medical and public health implications of chronic diarrhoea in HIV-positive patients are problematic, especially with regard to the management of HIV with antiretroviral therapies. 23,24 With the detection of more cases of chronic diarrhoea in HIV-positive patients, 4,12,25,26 Enterocytozoonbieneusi (a species of Microsporidium) is increasingly suspected to be an aetiological agent, and has also implicated in the excessive morbidity and mortality. 18,26,27,28,29 In Portugal, Ferreira et al. 30 reported a 41.2% prevalence of microsporidial diarrhoea in HIV patients, calling for better management of diarrhoea in these patients.
There is a paucity of information on microsporidia, particularly in developing countries where no concerted effort has been made to ascertain the prevalence, intensity, mode of transmission and tissue pathology caused by Microsporidiasis, especially amongst HIV patients. In Nigeria, there is no documented information on human microsporidia and Microsporidiasis. The objective of this study was to determine the prevalence and intensity of Microsporidium spores in HIV-positive hospital patients in Ilorin, Nigeria, and to determine the association of Microsporidiasis with other medical conditions amongst these patients.

Description of the study area
This work was carried out at the University of Ilorin Teaching Hospital, a tertiary and referral hospital located in Ilorin, Kwara State, Nigeria.

Study design
A cross-sectional, hospital-based study was designed and implemented so as to determine which pathogenic microsporidia could be detected in both HIV-positive and -negative patients attending the University of Ilorin Teaching Hospital between January 2009 and July 2010. A sample size of 1125 was calculated using Fisher's formula and based on an estimated 47% prevalence of diarrhoea amongst HIVpositive patients. 5,31 The sample comprised 750 HIV-positive and 375 HIV-negative patients, matched 2-to-1 for age, sex and other socio-economic variables. Informed consent was obtained from participants before being enrolled for this study. A structured questionnaire was administered to each patient to collect demographic variables and ARV status, as well as information about other concomitant infections. The patients completed the questionnaire before the samples were collected.

Sampling method
Simple random-sampling by number randomisation was used to select HIV-positive patients for this study. HIV status of patients was determined based on rapid HIV testing kits. HIV-negative controls were selected by number randomisation amongst all possible matches.

Sample collection and processing
All participants were screened for HIV-status prior to stool collection. The screening and the collection of the stool samples were done by the researcher and two trained laboratory assistants. Three screening methods were used concurrently to test and confirm HIV-sero-status: (1) Antec HIV test (Antec International Ltd, U.K.), (2) Determine HIV 1/2 test (Inverness Medical, Beijing, China) and (3) Uni-Gold HIV test (Trinity Biotech, Ireland). Confirmation of HIV positivity occurred only when all three tests were reactive for HIV antibodies and confirmation for HIV negativity was when all three tests showed no reaction. For the purposes of this study, patients who showed reactions on one or two of the three test kits were retested, but were excluded if the results remained discordant.
Stool specimens were collected in sterile universal bottles from all patients. For the identification of Microsporidium spores, unconcentrated formalin-fixed stools were stained using the Chromotrope2R staining method. 5,32 Each stained smear was examined by light microscope under an oil immersion lens. 5 The Microsporidium photomicrographs and measurement of the spore size were taken according to the manufacturer's instruction, using Phenix Microimage Analysis Software (PMIAS 3.3), Microscope model ME 100. A positive control slide from the Division of Parasitic Diseases of the Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, reference No. UN3373, was compared against the positive samples in this study. In addition, three positive slides were sent to the CDC in Atlanta for confirmation. The intensity of the spores in the stool samples ( Figure 1) was determined by counting the total number of spores in a 10 µl stained smear of stool on a glass slide. 1 Counting and authentication of the spores was carried out manually by the lead investigator and three independent co-researchers after blinding on HIV sero-status. The average of the 4 results was taken to be the mean intensity of the spores per 10 µl of stool.

Determination of T-cell count
The CD4+ count was determined using a CD4/8 Cyflow machine (Partec Cyflow SL-3, Germany) according to the manufacturer's instructions. A 3 ml sample of blood in an Ethylenediaminetetraacetic acid (EDTA) tube was inserted into the machine and the CD4/CD8 count of each individual sample was determined.

Data analysis
The Statistical Package for the Social Sciences (SPSS version 16.00) was used for data anlysis. Prevalence of infection was given in percentages in line with the variables. Chi-square and 95% confidence interval analysis were the statistical Page 2 of 6

Spores
Spores Source: Experimental data from study FIGURE 1: Images of Microsporidium spores in stool samples from our study.

Spores
Spores Source: Experimental data from study FIGURE 1: Images of Microsporidium spores in stool samples from our study.

Spores Spores a b
Source: Experimental data from study FIGURE 1: Images of Microsporidium spores in stool samples from our study. tools used to determine significance at a cut-off value of 95% (p = 0.05).

Results
We The mean intensity of Microsporidium spores per 10 µl of stool amongst the HIV-positive patients who were on ARVs showed no significant difference when compared with those who were not on ARVs ( Table 3). The 52-61 age group demonstrated the highest intensity of Microsporidium spores (mean±standard deviation: 277±94.0) amongst HIV-positive patients who were on ARVs. Patients not on ARVs had the highest intensity of spores (234 ± 88.0) in the 32-41 age group. The difference in intensity amongst those patients on ARVs and those not on ARVs in relation to age was not statistically significant (p > 0.05). In both groups, intensity of Microsporidium spores was inversely associated with CD4+ count; age groups with low CD4+ had high intensity of Microsporidium (Table 3).
Amongst HIV-positive patients, those with diarrhoea had the highest prevalence of Microsporidiasis amongst all those with a single condition (95.6%) ( Table 4). This was followed closely by those with body rashes (91.7%). Amongst those with multiple conditions, those with diarrhoea and body rashes (with and without cough) had 100% prevalence of Microsporadiasis.

Ethical considerations Ethical clearance
Ethical clearance was sought and obtained from the Ethical Review Committee of the University of Ilorin Teaching Hospital with reference number, UITH/CAT/189/817.

Informed consent
The purpose of this study was explained in detail to all subjects during the study. Participation was voluntary and the confidentiality of information given for the purpose of this study was guaranteed.
All subjects were also apprised of the benefits of knowing their HIV status, CD4+ counts, and medical complications.

Data protection
All specimen collected were preserved according to international standards and kept under iced Hydrogen refrigeration in the data storage chamber of the University of Ilorin Teaching Hospital.

Trustworthiness
Independent researchers confirmed and counted the spores to address the issue of credibility and the average of the four counts was recorded. We also obtained a control slide of the organism from the CDC in order to ensure that the organism was identified correctly. To address the issues of dependability and confirmability, we relied on an average independent count and the control slide.

Discussion
Our findings revealed a high prevalence (42.4%) and significant intensity of Microsporidiasis amongst the HIVpositive patients, thus complicating morbidity and mortality  +ve; Positive for Microsporidiasis in areas where HIV is common. The infection was detected across genders and age groups indicating that microsporidia are ubiquitous as suggested previously. 6,16 Whilst prevalence increased with age amongst the HIV-positive patients, there was was not a clear trend amongst the HIV-negative controls. We observed that the intensity of spores was highest amongst the HIV-positive patients, especially in the very old and in those with very low (< 10) CD4+ counts, suggesting a relationship with the level of CD4+ counts in HIV-positive patients 19 . Previous studies have reported a high intensity of microsporidia in HIV-positive patients due to their low immune status, which created a condition for the rapid replication of Microsporidium spores 12 .
Amongst the HIV-positive patients, it was observed that 95.6% of those reporting with diarrhoea were positive for Microsporidium, which may implicate microsporidia as an aetiologic agent in the diarrhoea that is seen in these immunocompromised patients. This is in line with previous suggestions concerning the need for more investigations of the aetiologic agent of this diarrhoea amongst HIV-postive patients. 5,18 A 100% prevalence of Microsporidium spores was seen amongst HIV-positive patients who had both diarrhoea and body rashes (with or without cough). Other conditions associated with Microsporidium were body rashes only (91.7%), diarrhoea with cough (77%) and body rashes with cough (70.0%), stimulating interest about the involvement of Microsporidiasis in other complications amongst HIV patients. This is contrast with the findings of Nkinin et al. 16 who reported an unexpectedly high prevalence (67.5%) of Microsporidiasis in 126 immunocompetent patients in Cameroon in 2007, but found no clinical manifestations in these individuals.
Presently, there is no effective treatment for treating Microsporidiasis which has made control difficult, even in areas where there is knowledge regarding Microsporidium infections in humans. 23,24 Limitations of the study This study is subject to several limitations. First, measuring the intensity of spores in stool samples is somewhat subjective and variable; to reduce these issues, we had 4 experts independently count spores and averaged the results. Second, the study was hospital-based, limiting our   inference to the general population. Third, irregular power supply may have adversely affected some of our samples; this was reduced through the provision of an alternative power supply.

Recommendations
We recommend that routine laboratory screening be performed for microsporidia in the hospital setting and that positive isolates be made notifiable. More studies on the aetiology of Microsporidiasis (under light-and electron microscopy) should be undertaken, which should include histopathology study of different organs at autopsy.

Conclusion
We found a high prevalence and intensity of microsporidia amongst HIV-positive patients, especially amongst those with diarrhoea, body rashes and/or cough. These findings should provide base-line information for further studies and provoke interest in investigations of microsporidia that aim at determining the mode of infection, pathogenicity and treatment as well as prevention and control.
Health education campaigns to promote awareness, prevention and control of Microsporidiasis should be conducted regularly, particularly amongst HIV-positive patients as well as medical, public health and laboratory personnel. Positive results for microsporidia obtained through routine laboratory screening in hospitals should be notifiable. Finally, based on the prevalence and implications of microsporidial infection as observed in this study, there is a need for the development of an ideal drug for the treatment of Microsporidiasis.