The Open AIDS Journal




ISSN: 1874-6136 ― Volume 12, 2018
RESEARCH ARTICLE

Socio-Behavioral Risk Factors Associated with Cryptosporidiosis in HIV/AIDS Patients Visiting the HIV Referral Clinic at Cape Coast Teaching Hospital, Ghana



Yeboah K. Opoku1, 2, Johnson N. Boampong1, Irene Ayi3, Godwin Kwakye-Nuako1, Dorcas Obiri-Yeboah4, Harriet Koranteng5, George Ghartey-Kwansah1, 6, Kwame K. Asare1, 7, 8, *
1 Department of Biomedical Sciences, School of Allied Health Sciences, Collage of Health and Allied Science, University of Cape Coast, Cape Coast, Ghana
2 Biopharmaceutical Laboratory, College of Life Sciences, Northeast Agricultural University, Harbin150030, China
3 Department of Parasitology, Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana, P. O. Box LG 581, Legon, Accra, Ghana
4 School of Medical Sciences, University of Cape Coast, Cape Coast, Ghana
5 Jiamusi University No. 148, Xuefu Road, Jiamusi, Heilongjiang, China
6 Laboratory of Cell Biology, Genetics and Developmental Biology, College of Life Sciences, Shaanxi Normal University, Xi'an710062, China
7 Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University Sakamoto 1-12-4, Nagasaki852-8523, Japan
8 Leading Program, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan

Abstract

Objective:

To identify the socio-behavioral risk factors associated with cryptosporidiosis among HIV/AIDS patients with chronic diarrhea symptoms visiting the HIV referral clinic at Cape Coast Teaching Hospital, Ghana.

Methods:

A cross-sectional study was conducted among 50 HIV/AIDS patients with recurrent diarrhea. Questionnaires were administered to collect social and behavioral risk factors associated with Cryptosporidium and other opportunistic protozoan parasitic infections in HIV patients. Stool samples were collected for the diagnosis of enteric protozoan pathogens using modified Ziehl-Neelsen and acid-fast staining methods. CD4+ cells counts of study subjects were obtained from patients clinical records. The data obtained were analyzed using Pearson chi-square and multivariate-adjusted statistics tool on SPSS 16 for Windows.

Results:

Twenty-seven (54%) of the subjects were infected with enteric protozoan pathogens. The prevalences of Cryptosporidium, Cyclospora and Microsporidium infections were 46%, 32% and 16%, respectively. Cryptosporidium infection was significantly associated with drinking water (×2=13.528, p<0.001), Cyclospora was associated with the type of drinking water (×2=14.931, p<0.001) and toilet facilities used by the study subjects (×2=12.463, p<0.01), whiles Microsporidium infection was associated with hand washing behavior (×2=12.463, p<0.01). Enteric protozoans were frequently encountered among subjects with CD4+ T-cell count <200 cells/mm3. However, coinfection of Cyclospora spp & Cryptosporidium spp was not observed in CD4+ cell count <200 and >500 cells/mm3. Multivariate analysis showed that the risk factor for Cryptosporidium infection among HIV/AIDS patients was the source of drinking water (pipe borne water 76.2% prevalence: sachet water 25%; OR=0.10, 95%CI: 0.03-0.39, p<0.001).

Conclusion:

We report the risk factor for exposure of Cryptosporidium infection among HIV/AIDS patients for the first time in Ghana. The contamination of drinking water by protozoan parasites should be a public health concern. These results provide the stepping block to understand the transmission dynamics of Cryptosporidium and other opportunistic pathogens in HIV/AIDS infected patients in Ghana.

Keywords: Cryptosporidium oocyst, HIV/AIDS, CD4+, Risk factors, Sachet water, Chronic diarrhea, HIV referral clinic, Cape Coast, Ghana.


Article Information


Identifiers and Pagination:

Year: 2018
Volume: 12
First Page: 106
Last Page: 116
Publisher Id: TOAIDJ-12-106
DOI: 10.2174/1874613601812010106

Article History:

Received Date: 24/4/2018
Revision Received Date: 12/8/2018
Acceptance Date: 18/8/2018
Electronic publication date: 12/9/2018
Collection year: 2018

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© 2018 Opoku et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


* Address correspondence to this author at the Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University Sakamoto 1-12-4, Nagasaki 852-8523, Japan; Tel: (81)-95-819-7838; E-mail: kwamsare@hotmail.com




1. INTRODUCTION

Diarrhea related morbidity and mortality among HIV/AIDS patients are still a clinical challenge to both clinicians and affected individuals [1Deeken JF, Tjen-A-Looi A, Rudek MA, et al. The rising challenge of non-AIDS-defining cancers in HIV-infected patients. Clin Infect Dis 2012; 55(9): 1228-35.[http://dx.doi.org/10.1093/cid/cis613] [PMID: 22776851] , 2Deeks SG, Lewin SR, Havlir DV. The end of AIDS: HIV infection as a chronic disease. Lancet 2013; 382(9903): 1525-33.[http://dx.doi.org/10.1016/S0140-6736(13)61809-7] [PMID: 24152939] ]. This challenge emanates from the non-specificity of the etiological agents of chronic diarrhea in HIV/AIDs infection [3Kalichman SC. The Psychology of AIDS Denialism. Eur Psychol 2014.[http://dx.doi.org/10.1027/1016-9040/a000175] ]. Chronic diarrhea caused by opportunistic enteric pathogens is increasingly observed in HIV/AIDS patients with depleted CD4+ T lymphocytes cells in Ghana [4Tay SC, Aryee EN, Badu K. Intesti-nal parasitemia and HIV/AIDS co-infections at varying CD4+ T-cell levels. HIV/AIDS Res Treat Open J 2017; 4(1): 40-8.]. Cryptosporidiosis, chronic diarrhea caused by Cryptosporidium infection, is one of the life-threatening clinical conditions affecting HIV/AIDS patients [5Kotloff KL, Nataro JP, Blackwelder WC, et al. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study. Lancet 2013; 382(9888): 209-22.[http://dx.doi.org/10.1016/S0140-6736(13)60844-2] [PMID: 23680352] , 6Alemu A, Shiferaw Y, Getnet G, Yalew A, Addis Z. Opportunistic and other intestinal parasites among HIV/AIDS patients attending Gambi higher clinic in Bahir Dar city, North West Ethiopia. Asian Pacific J. Trop. Med. 2011 Aug 1;4(8):661-5. Lancet 2013; 382(9888): 209-22.[PMID: 23680352] ]. Although highly active antiretroviral therapy (HAART) has shown to increase CD4+ cell levels among HIV/AIDS patients, cryptosporidiosis still remains high in these immunocompromised individuals [7Nissapatorn V, Sawangjaroen N. Parasitic infections in HIV infected individuals: diagnostic & therapeutic challenges. Indian J Med Res 2011; 134(6): 878-97.[http://dx.doi.org/10.4103/0971-5916.92633] [PMID: 22310820] ]. In Ghana, the prevalence of cryptosporidiosis ranges from 12% to 28.6% among HIV/AIDS patients [8Adjei AA, Armah H, Rodrigues O, et al. Cryptosporidium Spp., a frequent cause of diarrhea among children at the Korle-Bu Teaching Hospital, Accra, Ghana. Jpn J Infect Dis 2004; 57(5): 216-9.[PMID: 15507781] , 9Adjei A, Lartey M, Adiku TK, et al. Cryptosporidium oocysts in Ghanaian AIDS patients with diarrhoea. East Afr Med J 2003; 80(7): 369-72.[PMID: 16167753] ].

Though there are increasing reports on the prevalence of cryptosporidiosis and its association with depleted CD4+ cells among HIV patients in Ghana, little is known about its transmission risk factors [10Boaitey YA, Nkrumah B, Idriss A, Tay SC. Gastrointestinal and urinary tract pathogenic infections among HIV seropositive patients at the Komfo Anokye Teaching Hospital in Ghana. BMC Res Notes 2012; 5(1): 454.[http://dx.doi.org/10.1186/1756-0500-5-454] [PMID: 22909315] ]. The only study which attempted to establish the socio-behavioral risk factors and transmission patterns of Cryptosporidium infection among HIV patients could not establish any significance between them [9Adjei A, Lartey M, Adiku TK, et al. Cryptosporidium oocysts in Ghanaian AIDS patients with diarrhoea. East Afr Med J 2003; 80(7): 369-72.[PMID: 16167753] ]. Several factors have been associated with Cryptosporidium infection in HIV/AIDS infections [11Smith HV, Cacciò SM, Cook N, Nichols RA, Tait A. Cryptosporidium and Giardia as foodborne zoonoses. Vet Parasitol 2007; 149(1-2): 29-40.[http://dx.doi.org/10.1016/j.vetpar.2007.07.015] [PMID: 17728067] -14Violari A, Cotton MF, Gibb DM, et al. Early antiretroviral therapy and mortality among HIV-infected infants. N Engl J Med 2008; 359(21): 2233-44.[http://dx.doi.org/10.1056/NEJMoa0800971] [PMID: 19020325] ]. A number of research findings have shown an association between Cryptosporidium and consumption of untreated water [15Said DE. Detection of parasites in commonly consumed raw vegetables. Alexandria J Med 2012; 48(4): 345-52.[http://dx.doi.org/10.1016/j.ajme.2012.05.005] , 16Moghaddam AA. Symptomatic and asymptomatic cryptosporidiosis in young children in Iran. Pak J Biol Sci 2007; 10(7): 1108-12.[http://dx.doi.org/10.3923/pjbs.2007.1108.1112] [PMID: 19070060] ]. The detection of Cryptosporidium spp. from potable water sources in Ghana suggested that improper treatment of drinking water could be a key transmission source for Cryptosporidium infection [17Duedu KO, Yarnie EA, Tetteh-Quarcoo PB, Attah SK, Donkor ES, Ayeh-Kumi PF. A comparative survey of the prevalence of human parasites found in fresh vegetables sold in supermarkets and open-aired markets in Accra, Ghana. BMC Res Notes 2014; 7(1): 836.[http://dx.doi.org/10.1186/1756-0500-7-836] [PMID: 25424391] , 18Anim-Baidoo I, Narh CA, Oddei D, et al. Giardia lamblia infections in children in Ghana. Pan Afr Med J 2016; 24: 217.[http://dx.doi.org/10.11604/pamj.2016.24.217.8012] [PMID: 27800072] ].

The transmission of Cryptosporidium is very complex especially in poor sanitation environments [19Harwood VJ, Staley C, Badgley BD, Borges K, Korajkic A. Microbial source tracking markers for detection of fecal contamination in environmental waters: Relationships between pathogens and human health outcomes. FEMS Microbiol Rev 2014; 38(1): 1-40.[http://dx.doi.org/10.1111/1574-6976.12031] [PMID: 23815638] ]. Cryptosporidium spp, Cyclospora spp, Microsporidium spp, and other microorganisms have been detected in plastic packaged drinking waters “sachet water” within Ghana [20Machdar E, van der Steen NP, Raschid-Sally L, Lens PN. Application of Quantitative Microbial Risk Assessment to analyze the public health risk from poor drinking water quality in a low income area in Accra, Ghana. Sci Total Environ 2013; 449: 134-42.[http://dx.doi.org/10.1016/j.scitotenv.2013.01.048] [PMID: 23416990] , 21Kwakye-Nuako G, Borketey P, Mensah-Attipoe I, Asmah R, Ayeh-Kumi P. Sachet drinking water in accra: The potential threats of transmission of enteric pathogenic protozoan organisms. Ghana Med J 2007; 41(2): 62-7.[PMID: 17925844] ]. Cryptosporidiosis is self-limiting diarrhea in immunocompetent individuals but becomes severe, chronic and life-threatening in children under five years and immunocompromised individuals especially those with profound malnutrition or wasting [22Stark D, Barratt JL, van Hal S, Marriott D, Harkness J, Ellis JT. Clinical significance of enteric protozoa in the immunosuppressed human population. Clin Microbiol Rev 2009; 22(4): 634-50.[http://dx.doi.org/10.1128/CMR.00017-09] [PMID: 19822892] ]. Host immunity which plays a protective role against severe cryptosporidiosis is boosted upon exposure to Cryptosporidium infection [23Mor SM, Tzipori S. Cryptosporidiosis in children in Sub-Saharan Africa: A lingering challenge. Clin Infect Dis 2008; 47(7): 915-21.[http://dx.doi.org/10.1086/591539] [PMID: 18715159] ]. In children under five years, clinical severity of cryptosporidiosis ameliorates with therapy from the onset of the disease [24Zu SX, Fang GD, Fayer R, Guerrant RL. Cryptosporidiosis: Pathogenesis and immunology. Parasitol Today (Regul Ed) 1992; 8(1): 24-7.[http://dx.doi.org/10.1016/0169-4758(92)90307-N] [PMID: 15463522] ]. The boosted acquired immunity protect such children against subsequent severe cryptosporidiosis upon infection. In HIV infections, the priming of CD8+ cell to kill CD4+ cells causes an imbalance between Th1 and Th2 cytokines and as a result predisposes the host small intestine to damage by inflammation [25van Wijk F, Cheroutre H. Mucosal T cells in gut homeostasis and inflammation. Expert Rev Clin Immunol 2010; 6(4): 559-66.[http://dx.doi.org/10.1586/eci.10.34] [PMID: 20594129] ]. Cryptosporidium infection augments the imbalance between Th1 and Th2 cytokines in an immunocompromised position leading to prolong chronic life-threatening diarrhea [26Bouzid M, Hunter PR, Chalmers RM, Tyler KM. Cryptosporidium pathogenicity and virulence. Clin Microbiol Rev 2013; 26(1): 115-34.[http://dx.doi.org/10.1128/CMR.00076-12] [PMID: 23297262] , 27Guerrant RL, DeBoer MD, Moore SR, Scharf RJ, Lima AA. The impoverished gut-a triple burden of diarrhoea, stunting and chronic disease. Nat Rev Gastroenterol Hepatol 2013; 10(4): 220-9.[http://dx.doi.org/10.1038/nrgastro.2012.239] [PMID: 23229327] ].

In our previous report, we showed that HIV/AIDS patients with CD4+ cell count below 200 cells/mm3 were frequently predisposed to cryptosporidiosis [28Kwakye-Nuako G, Boampong JN, Dong MK, et al. Modulation of cryptosporidiosis by CD4 levels in chronic diarrhea HIV/AIDS individuals visiting Tarkwa Municipal hospital, Ghana. Asian Pac J Trop Dis 2016; 6(10): 770-5.[http://dx.doi.org/10.1016/S2222-1808(16)61127-1] ]. Other researchers have also reported similar findings in other parts of Ghana [8Adjei AA, Armah H, Rodrigues O, et al. Cryptosporidium Spp., a frequent cause of diarrhea among children at the Korle-Bu Teaching Hospital, Accra, Ghana. Jpn J Infect Dis 2004; 57(5): 216-9.[PMID: 15507781] , 23Mor SM, Tzipori S. Cryptosporidiosis in children in Sub-Saharan Africa: A lingering challenge. Clin Infect Dis 2008; 47(7): 915-21.[http://dx.doi.org/10.1086/591539] [PMID: 18715159] ]. However, a few studies from Ghana have tried to identify an association between socio-behavioral risk factors and cryptosporidiosis among HIV infected patients. The zoonotic exposure of Cryptosporidium spp results in continuous circulation and transmission cycle in poor sanitation environments [29Putignani L, Menichella D. Global distribution, public health and clinical impact of the protozoan pathogen Cryptosporidium. Interdisciplinary perspectives on infectious diseases 2010.]. The identification of common exposure sources of Cryptosporidium infection among HIV patients could assist clinicians to offer the needed advice to this group of patients on how to reduce the risk of infection.

This study focuses on identifying social and behavioral factors that influence cryptosporidiosis infection in HIV patients visiting the HIV referral clinic in the Central region. This report has public health significance on the transmission of cryptosporidiosis in the immunocompromised host. The results being the first report to understand the transmission dynamics in Cryptosporidium spp and other opportunistic protozoan parasites in HIV/AIDS infection in Ghana could lead to policy formulation and rational approaches to control cryptosporidiosis and other opportunistic pathogens in HIV/AIDS infection.

2. MATERIAL AND METHODS

2.1. Study Site

A cross-sectional study was conducted at the HIV referral clinic in Cape Coast, Ghana. The clinic serves as a referral clinic for all the HIV clinics within seventeen districts in the region. The HIV referral clinic is located within the Cape Coast Teaching hospital situated at Cape Coast, the capital city of the region. The Central region is a governmental administrative area of size 9,826 km2 inhabited by about 2.2 million people. According to the 2016 HIV sentinel summary report, the HIV prevalence in the Central region stands at 1.8%.

2.2. Ethical Approval

Ethical clearance was obtained from the Ghana Health Service Ethical Review Committee on Research Involving Human Subjects (ERCRIHS), certificate number GHS-ERC: 24/01/12. Permission to interact and collect research samples from HIV infected clients assessing the HIV referral clinic was obtained from the administration of the hospital.

2.3. Selection of Study Subjects

The purpose of the study was explained to the potential subjects and those who accepted to take part in this research were enrolled in the study. The results of the participants were linked to their clinical data for prompt treatment if necessary with their consent and also their confidentiality assured that the data would not be released outside the health facility. In all fifty (50) subjects participated in the study from May 2012 to April 2013. All the participants were HIV/ AIDS patients who had been diagnosed with at least three days of recurrent diarrheal condition at the referral clinic. Unique identification (ID) numbers were given to each of the study subjects after accepting and signing the informed consent form. These unique ID numbers were also given to the stool samples and the administered questionnaires. These procedures boosted the confidence of the participants to effectively contribute to the study since participant names were not recorded in the research registrar. However, we were able to link the ID numbers to the patient’s clinical folder, which made it easier to report any enteric pathogenic infections to the clinician in charge of the referral clinic for prompt treatment.

2.4. Questionnaire Administration and Sample Collection

Structured and semi-structured questionnaires with translations in local dialects were administered to the subjects enrolled in the study. Guardians and parents of subjects below 12 years of age were assisted in answering the questionnaires. The questionnaires were used to obtain socio-demographic data such as age, sex, residence, educational status and possible behavioral risk factors associated with the transmission of enteric protozoan parasites such as hand and fruit washing practices, type of toilet facility, and source of drinking water. CD4 + cell counts of the participants were also obtained from their clinical folders.

Through the use of motion pictures and illustrations, all the fifty (50) participants were educated on how to collect stool samples without contamination. A sterile plastic container (labelled with subjects unique ID to match the questionnaire and clinical folder) with a tight-fitting lid which contained an applicator to scoop the stool samples were given to each subject. The stool samples collected at the clinic were stored at 4oC for a maximum of 12 hours and were transported on ice to the laboratory of the Department of Biomedical and Forensic Sciences, University of Cape Coast for pathogenic diagnostics and analysis.

2.5. Analysis of Stool Samples

The stool samples were immediately processed on arrival by wet mount, formalin-ethyl acetate sedimentation and microscopic observations were made to identify possible enteric pathogens in the samples. Four thin slides were made from each stool, air dried and fixed in absolute ethanol. The slides were then stained using Modified Ziehl-Neelsen and other acid-fast staining methods followed by microscopic identification of protozoan parasites [28Kwakye-Nuako G, Boampong JN, Dong MK, et al. Modulation of cryptosporidiosis by CD4 levels in chronic diarrhea HIV/AIDS individuals visiting Tarkwa Municipal hospital, Ghana. Asian Pac J Trop Dis 2016; 6(10): 770-5.[http://dx.doi.org/10.1016/S2222-1808(16)61127-1] ].

2.6. Statistical Analysis

The data collected from both questionnaires and stool samples were keyed into Microsoft Excel 2007 (Microsoft Corporation) and analyzed using SPSS statistical Software for Windows version 16 (SPSS Inc.). The prevalence of each protozoan pathogenic infection was estimated using proportions. All statistical associations in this study were tested using Pearson chi-square test; p-value ≤ 0.05 was considered statistically significant. Multivariate-adjusted statistics at 95% confidence interval (CI) was used as robust statistics to confirm risk factors associated with the exposure of HIV/AIDS patients to opportunistic pathogens.

3. RESULTS

Fifty (50) subjects with recurrent diarrhea attending HIV/AIDS clinic at the Cape Coast teaching hospital, Cape Coast were enrolled into the study. All the subjects provided stool samples and answered the questionnaires designed to investigate the possible risk factors for Cryptosporidium infection among HIV/AIDS patients and their CD4+ data were also obtained from their clinical records. The study subjects consisted of 14 (28%) males and 36 (72%) females. Twenty-seven (54%) of the enrolled subjects had enteric protozoans in their stool samples.

Three protozoan parasites Cryptosporidium (46%), Cyclospora (32%) and Microsporidium spp (16%) were identified from the stool samples. These protozoan parasites were stratified into tables with social demographic and behavioral characteristics to identify the risk factors for cryptosporidiosis and other protozoan infections among the study subjects. Among the subjects, 6 (42.9%) out of 14 males and 10 (27.8%) out of 36 females had Cyclospora infections (×2=1.053, p>0.05); 8 (57.1%) of the males and 15 (41.7%) of the females had Cryptosporidium infections (×2=0.972, p>0.05) whilst 4 (28.6%) and 4 (11.1%) had Microsporidium infections in males and females respectively (×2=2.286, p>0.05). There was no significant differences among the age categories for Cyclospora2=8.323, p>0.05), Cryptosporidium2=2.287, p>0.05) and Microsporidium2=4.772, p>0.05); for residence, Cyclospora2=0.006, p>0.05), Cryptosporidium2=2.061, p>0.05) and Microsporidium2=0.214, p>0.05) (Table 1). However, Cyclospora infections were significantly associated with the source of drinking water (×2=14.931, p<0.001) and the type of toilet facility used by the participants (×2=12.463, p<0.01), Cryptosporidium infection among the subjects in a similar trend was also associated with the source of drinking water (×2=13.528, p<0.001) whereas Microsporidium infection was associated with hand washing behavior of the subjects (×2=12.463, p<0.01). There was no significant association among the fruit washing behavior for Cyclospora2=0.146, p>0.05), Cryptosporidium2=1.567, p>0.05), and Microsporidium2=4.352, p>0.05) (Table 2).

Table 1
Socio-demographic characteristics and its associated risks with opportunistic pathogens in HIV infected patients.


Table 2
Behavioral risk factors and its association with opportunistic pathogens in HIV infected patients.


The frequencies of coinfection of enteric protozoans among the study subjects were classified as triple infection (Cyclospora, Cryptosporidium and Microsporidium) with a prevalence of 2 (14.3%) and 3 (8.3%) for males and females respectively; double infections of Microsporidium & Cyclospora were 2 (14.3%) and 1 (2.8%), and Cyclospora & Cryptosporidium were 1 (7.1%) and 5 (5.6%) for male and female subjects respectively; for single infections, the frequencies of Cyclospora infection were 1 (7.1%) and 1 (2.8%) whereas Cryptosporidium infection was 5 (35.7%) and 10 (27.8%) among male and female subjects respectively(Table 3).

Table 3
Prevalence of single and coinfections of protozoan pathogens among male and female HIV infected individuals.


The distribution of enteric protozoan infections was grouped with respect to CD4+ T-lymphocyte counts. In all, patients with CD4+ cell count lower than 200 cells/mm3 had 61.5% prevalence of enteric protozoans; 2 (12.5%) had triple infections, 1 (6.3%) of Microsporidium & Cyclospora double infection, 1 (6.3%) of Cyclospora and 7 (43.8%) of Cryptosporidium as single infections whilst 5 (31.3%) of the subjects in this category had no enteric protozoan infections. Triple infection 1 (4.5%), double infections 1 (4.5%) and 3 (13.6%) for Microsporidium & Cyclospora and Cyclospora & Cryptosporidium infections respectively, and no single Cyclospora infection was observed among patients with CD4+ cell count between 200-499 cells/mm3 (Table 4).

Table 4
Distribution of single and coinfections of protozoan pathogens among HIV infected individuals with respect to CD4 + cells counts.


The risk factors associated with Cryptosporidium and other enteric protozoan infections among the study subjects were robustly analyzed using multivariate-adjusted statistics. The significant risk factor for Cryptosporidium infection among HIV/AIDS patients with recurrent diarrhea in this study was the source of drinking water [sachet water (OR=0.10, 95%CI: 0.03-0.39, p<0.001) and borehole water (OR=0.11, 95%CI: 0.00-3.14, p=0.198) with pipe borne water as a reference]. Similarly, Cyclospora infection also had a source of drinking water as a risk factor [sachet water (OR=0.07, 95%CI: 0.02-0.33, p<0.001) and borehole water (OR=0.02, 95%CI: 0.01-5.77, p=0.356) with pipe borne water as a reference]. The kind of toilet facility used by subjects was observed to be a risk factor for Cyclospora infection by Pearson chi-square statistics (p<0.01). On the contrary, multivariate analysis showed no significant association with the kind of toilet facility used by study subjects [public toilet (OR=0.92, 95%CI: 0.22-3.83, p=0.905), household toilet (OR=0.33, 95%CI: 0.01-8.63, p=0.508), and open defecation (OR=0.44, 95%CI: 0.04-5.40, p=0.512) with personal water closet as a reference]. Hand washing behavior (p<0.05) was identified as a risk factor for Microsporidium infection among the study subjects. Multivariate analysis showed that study subjects who never or rarely wash their hands (OR=20.45, 95%CI: 0.93-449.12, p=0.05) were more prone to Microsporidium infection compared to the rest of the subjects enrolled into the study (Table 5).

Table 5
Multivariate-adjusted odds to test the strength of association between behavioral risk factors and protozoan pathogens among HIV infected individuals.


4. DISCUSSION

Gastroenteritis triggered by Cryptosporidium infection causes prolonged or chronic diarrhea which leads to malnutrition and wasting, a life-threatening condition in HIV/AIDS infections [27Guerrant RL, DeBoer MD, Moore SR, Scharf RJ, Lima AA. The impoverished gut-a triple burden of diarrhoea, stunting and chronic disease. Nat Rev Gastroenterol Hepatol 2013; 10(4): 220-9.[http://dx.doi.org/10.1038/nrgastro.2012.239] [PMID: 23229327] , 30Halliez MC, Buret AG. Extra-intestinal and long term consequences of Giardia duodenalis infections. World J Gastroenterol 2013; 19(47): 8974-85.[http://dx.doi.org/10.3748/wjg.v19.i47.8974] [PMID: 24379622] , 31Wilcox CM, Saag MS. Gastrointestinal complications of HIV infection: Changing priorities in the HAART era. Gut 2008; 57(6): 861-70.[http://dx.doi.org/10.1136/gut.2006.103432] [PMID: 18203808] ]. A substantial number of chronic diarrhea in HIV infection is as a result of cryptosporidiosis, however, it still remains a major challenge in HIV/AIDS clinical management due to late diagnosis [32Seddon J, Bhagani S. Antimicrobial therapy for the treatment of opportunistic infections in HIV/AIDS patients: A critical appraisal. HIV AIDS (Auckl) 2011; 3: 19-33.[PMID: 22096404] ]. The delay in diagnosis and treatment of cryptosporidiosis arises due to multiple causative agents of chronic diarrhea in HIV infections [33Tian LG, Chen JX, Wang TP, et al. Co-infection of HIV and intestinal parasites in rural area of China. Parasit Vectors 2012; 5(1): 36.[http://dx.doi.org/10.1186/1756-3305-5-36] [PMID: 22330320] ]. Growing number of studies have reported cryptosporidiosis in HIV infected patients in Ghana [8Adjei AA, Armah H, Rodrigues O, et al. Cryptosporidium Spp., a frequent cause of diarrhea among children at the Korle-Bu Teaching Hospital, Accra, Ghana. Jpn J Infect Dis 2004; 57(5): 216-9.[PMID: 15507781] , 28Kwakye-Nuako G, Boampong JN, Dong MK, et al. Modulation of cryptosporidiosis by CD4 levels in chronic diarrhea HIV/AIDS individuals visiting Tarkwa Municipal hospital, Ghana. Asian Pac J Trop Dis 2016; 6(10): 770-5.[http://dx.doi.org/10.1016/S2222-1808(16)61127-1] ], but little or no effort has been done to identify the risk factors for transmission of Cryptosporidium infection in this group of patients. Poor personal hygiene, lack of sanitation facilities, sexual behavior patterns, closeness to domestic animal and drinking water have been reported as risk factors associated with cryptosporidiosis [14Violari A, Cotton MF, Gibb DM, et al. Early antiretroviral therapy and mortality among HIV-infected infants. N Engl J Med 2008; 359(21): 2233-44.[http://dx.doi.org/10.1056/NEJMoa0800971] [PMID: 19020325] , 15Said DE. Detection of parasites in commonly consumed raw vegetables. Alexandria J Med 2012; 48(4): 345-52.[http://dx.doi.org/10.1016/j.ajme.2012.05.005] ]. Adjei et al could not establish any significant association between the risk factors and Cryptosporidium infection among HIV infected patients in Ghana [8Adjei AA, Armah H, Rodrigues O, et al. Cryptosporidium Spp., a frequent cause of diarrhea among children at the Korle-Bu Teaching Hospital, Accra, Ghana. Jpn J Infect Dis 2004; 57(5): 216-9.[PMID: 15507781] ]. In this study, we report the risk factors for Cryptosporidium, Cyclospora and Microsporidium infections in HIV/AIDS patients visiting HIV referral clinic in the Central region, Ghana.

The study showed that Cryptosporidiosis was the major diarrhea condition followed by cyclosporiasis in HIV/AIDS infected individuals. However, most of the cases of Cyclospora infections were frequently seen in a mix-infection with either Cryptosporidium or multiple infections of Cryptosporidium, Cyclospora and Microsporidium. The coinfection of Cryptosporidium and Cyclospora is increasingly being reported especially among HIV infected individuals [34Nsagha DS, Njunda AL, Assob NJC, et al. Intestinal parasitic infections in relation to CD4(+) T cell counts and diarrhea in HIV/AIDS patients with or without antiretroviral therapy in Cameroon. BMC Infect Dis 2016; 16(1): 9.[http://dx.doi.org/10.1186/s12879-016-1337-1] [PMID: 26754404] , 35Fletcher SM, Stark D, Harkness J, Ellis J. Enteric protozoa in the developed world: A public health perspective. Clin Microbiol Rev 2012; 25(3): 420-49.[http://dx.doi.org/10.1128/CMR.05038-11] [PMID: 22763633] ]. Previous reports have revealed that cyclosporiasis has a similar rate of complications in both immunocompromised and immunocompetent individuals [36Anane S, Attouchi H. Microsporidiosis: epidemiology, clinical data and therapy. Gastroenterol Clin Biol 2010; 34(8-9): 450-64.[http://dx.doi.org/10.1016/j.gcb.2010.07.003] [PMID: 20702053] ]. Despite the small number of male subjects enrolled in the study, higher prevalences of infection in all categories from coinfections to single infections compared to the female counterparts were observed. A larger percentage of female subjects had no protozoan infections compared to the male subjects. There are several possible explanations to this finding, among them are the low levels of personal hygiene [37Dwivedi KK, Prasad G, Saini S, Mahajan S, Lal S, Baveja UK. Enteric opportunistic parasites among HIV infected individuals: Associated risk factors and immune status. Jpn J Infect Dis 2007; 60(2-3): 76-81.[PMID: 17515636] ], less likely to seek medical care and may only do so when their clinical condition deteriorate [38Chesney MA, Smith AW. Critical delays in HIV testing and care: The potential role of stigma. Am Behav Sci 1999; 42(7): 1162-74.[http://dx.doi.org/10.1177/00027649921954822] ] may account for their frequent contact to these opportunistic pathogens compared to their female counterpart. However, a larger sample size is required to confirm this observation.

We did not observe any single microsporidiosis, a diarrhea condition cause by only Microsporidium infections but rather was always seen as coinfection with other pathogens in HIV/AIDS patients enrolled in this study. Microsporidiosis, an infection caused by Encephalitozoon species in HIV/AIDS infection has been associated with clinical symptoms such as keratoconjunctivitis, peritonitis, encephalitis, myositis and nephritis but more frequently chronic diarrhea and malabsorption resulting in reduced serum concentration of D-xylose after ingestion of mannitol [39Didier ES, Weiss LM. Microsporidiosis: Not just in AIDS patients. Curr Opin Infect Dis 2011; 24(5): 490-5.[http://dx.doi.org/10.1097/QCO.0b013e32834aa152] [PMID: 21844802] , 40Stentiford GD, Feist SW, Stone DM, Bateman KS, Dunn AM. Microsporidia: diverse, dynamic, and emergent pathogens in aquatic systems. Trends Parasitol 2013; 29(11): 567-78.[http://dx.doi.org/10.1016/j.pt.2013.08.005] [PMID: 24091244] ]. In this report, Microsporidium infection was always seen in coinfection with Cyclospora or with Cryptosporidium and Cyclospora infections. This observation suggests that Microsporidium infections may not be a major diarrhoea-causing pathogen among our HIV/AIDS infected subjects, although it has been frequently reported in several studies conducted in HIV/AIDS infected individuals [41Sokolova OI, Demyanov AV, Bowers LC, et al. Emerging microsporidian infections in Russian HIV-infected patients. J Clin Microbiol 2011; 49(6): 2102-8.[http://dx.doi.org/10.1128/JCM.02624-10] [PMID: 21450962] , 42Rabeneck L, Gyorkey F, Genta RM, Gyorkey P, Foote LW, Risser JM. The role of Microsporidia in the pathogenesis of HIV-related chronic diarrhea. Ann Intern Med 1993; 119(9): 895-9.[http://dx.doi.org/10.7326/0003-4819-119-9-199311010-00005] [PMID: 8215001] ]. Coinfection or multiple infections of Microsporidium with Cryptosporidium and Cyclospora infections may, however, act synergistically to invigorate the clinical conditions caused by Cryptosporidium or Cyclospora infections since these pathogens share similar disease-causing mechanisms [33Tian LG, Chen JX, Wang TP, et al. Co-infection of HIV and intestinal parasites in rural area of China. Parasit Vectors 2012; 5(1): 36.[http://dx.doi.org/10.1186/1756-3305-5-36] [PMID: 22330320] ].

CD4+ cells count lower than 200cells/mm3 have been associated with both cryptosporidiosis and cyclosporiasis [28Kwakye-Nuako G, Boampong JN, Dong MK, et al. Modulation of cryptosporidiosis by CD4 levels in chronic diarrhea HIV/AIDS individuals visiting Tarkwa Municipal hospital, Ghana. Asian Pac J Trop Dis 2016; 6(10): 770-5.[http://dx.doi.org/10.1016/S2222-1808(16)61127-1] ]. Individually, each of the opportunistic parasites has been shown to be associated with depleted CD4+ cells in immunocompromised patients [43Tuli L, Gulati AK, Sundar S, Mohapatra TM. Correlation between CD4 counts of HIV patients and enteric protozoan in different seasons - An experience of a tertiary care hospital in Varanasi (India). BMC Gastroenterol 2008; 8(1): 36.[http://dx.doi.org/10.1186/1471-230X-8-36] [PMID: 18713475] ]. Several other studies have also reported coinfections of these opportunistic protozoan parasites in HIV/AIDS infected individuals with lower CD4+ cells count [44Karp CL, Auwaerter PG, Auwaerter PG, Mayer KH. Coinfection with HIV and tropical infectious diseases. I. Protozoal pathogens. Clin Infect Dis 2007; 45(9): 1208-13.[http://dx.doi.org/10.1086/522181] [PMID: 17918086] , 45Kurniawan A, Karyadi T, Dwintasari SW, et al. Intestinal parasitic infections in HIV/AIDS patients presenting with diarrhoea in Jakarta, Indonesia. Trans R Soc Trop Med Hyg 2009; 103(9): 892-8.[http://dx.doi.org/10.1016/j.trstmh.2009.02.017] [PMID: 19327806] ].

In this study, we showed the prevalence of single and co-infection of Cryptosporidium, Cyclospora & Microsporidium with stratified CD4+ cells count. Although all groups of protozoan infections were observed among patients with CD4+ cell count lower than 200 or greater than 500 cells/mm3 with exception of Cyclospora & Cryptosporidium coinfections, it is not clear if this coinfection is protective among patients with CD4+ cell count <200 cells/mm3 and CD4+ cell count >500 cells/mm3. Nsagha et al [46Nsagha DS, Njunda AL, Assob NJC, et al. Intestinal parasitic infections in relation to CD4(+) T cell counts and diarrhea in HIV/AIDS patients with or without antiretroviral therapy in Cameroon. BMC Infect Dis 2016; 16(1): 9.[http://dx.doi.org/10.1186/s12879-016-1337-1] [PMID: 26754404] ], observed a similar phenomenon among HIV patients who were yet to be put on antiretroviral drugs. They recorded zero (0%) prevalence of C. cayetenensis among patients with CD4+ count <200 cell/mm3 and >500 cells/mm3. However, they observed 9.5% prevalence of C. cayetenensis among patients with CD4+ cells count between 200-499 cells/mm3. They also observed C. cayetenensis infection in all CD4+ cells count among patients who were already on antiretroviral drugs. Contrarily, Mitra et al [47Mitra S, Mukherjee A, Khanra D, Bhowmik A, Roy K, Talukdar A. Enteric parasitic infection among antiretroviral therapy naive HIV-seropositive people: Infection begets infection-experience from eastern India. J Glob Infect Dis 2016; 8(2): 82-6.[http://dx.doi.org/10.4103/0974-777X.182124] [PMID: 27293363] ] observed Cyclospora spp only in HIV-seropositive cases with CD4+ cells count <350 cells/μl but not in CD4+ cells count between 350-500 cells/μl or >500 cells/μl. Again, Cyclospora spp was only observed in acute diarrhea but not in chronic diarrhea or no diarrhea cases. Combining the observations made in this study and the previous two reports, the zero (0%) prevalence of Cyclospora & Cryptosporidium coinfections in CD4+ cells count <200 cells/mm3 and >500 cells/mm3 may not be due to chance but rather a phenomenon which requires further studies to understand.

In this study, we also delineated the social and behavioral risk factors associated with Cryptosporidium, Cyclospora & Microsporidium infections among the study subjects. Pearson chi-square statistics showed that Cryptosporidium infection is associated with drinking water. Cyclospora infection is also associated with the source of drinking water and the kind toilet of facilities used by the study subjects whilst Microsporidium showed association with the frequency of hand washing habits among the subjects. A more robust multivariate-adjusted statistics confirmed the source of drinking water as a risk factor for Cryptosporidium and Cyclospora infections among the HIV/AIDS patients attending the HIV referral clinic at Cape Coast.

The high occurrence of Cryptosporidium oocysts contamination in natural water sources coupled with its ability to withstand chlorine oxide, Alum-lime, chloramines and other treatment processes such as sedimentation, filtration or flocculation should make it a public health concern [17Duedu KO, Yarnie EA, Tetteh-Quarcoo PB, Attah SK, Donkor ES, Ayeh-Kumi PF. A comparative survey of the prevalence of human parasites found in fresh vegetables sold in supermarkets and open-aired markets in Accra, Ghana. BMC Res Notes 2014; 7(1): 836.[http://dx.doi.org/10.1186/1756-0500-7-836] [PMID: 25424391] , 48Addo KK, Mensah GI, Bekoe M, Bonsu C, Akyeh ML. Bacteriological quality of sachet water produced and sold in Teshie-Nungua suburbs of Accra, Ghana. Afr J Food Agric Nutr Dev 2009; 9(4)[http://dx.doi.org/10.4314/ajfand.v9i4.43874] , 49Robertson LJ, Campbell AT, Smith HV. Survival of Cryptosporidium parvum oocysts under various environmental pressures. Appl Environ Microbiol 1992; 58(11): 3494-500.[PMID: 1482175] ]. Cyclospora oocyst is also resistant to water treatment processes, however, shedding of oocysts by infected individuals including immunocompromised hosts is in very low numbers compared to Cryptosporidium [50ten Hove RJ, van Lieshout L, Brienen EA, Perez MA, Verweij JJ. Real-time polymerase chain reaction for detection of Isospora belli in stool samples. Diagn Microbiol Infect Dis 2008; 61(3): 280-3.[http://dx.doi.org/10.1016/j.diagmicrobio.2008.03.003] [PMID: 18424043] ]. Cryptosporidiosis is more aggressive in HIV/AIDS infections which requires meticulous efforts to reduce cryptosporidial associated morbidity and mortality.

In Ghana, contamination of water for irrigation, vegetable on sale at open-market, and plastic packaged drinking water “sachet water” by oocysts of Cryptosporidium, Cyclospora and other parasitic pathogens have been reported [49Robertson LJ, Campbell AT, Smith HV. Survival of Cryptosporidium parvum oocysts under various environmental pressures. Appl Environ Microbiol 1992; 58(11): 3494-500.[PMID: 1482175] , 51Keraita B, Drechsel P, Konradsen F. Using on-farm sedimentation ponds to improve microbial quality of irrigation water in urban vegetable farming in Ghana. Water Sci Technol 2008; 57(4): 519-25.[http://dx.doi.org/10.2166/wst.2008.166] [PMID: 18359990] ]. Multivariate analysis showed significant differences between HIV/AIDS patients with “sachet water” and those with pipe-borne water as the source of drinking water in both Cryptosporidium and Cyclospora infections. A closer look at the prevalence showed that 76.2% of the subjects who use pipe-borne water as their source of drinking water had Cryptosporidium infections compared to 25% of the subjects with sachet water as their source of drinking water. This indicates that the sources of drinking water in Ghana is heavily contaminated with parasitic pathogens due to poor environmental sanitation [52Nkrumah B, Nguah SB. Giardia lamblia: a major parasitic cause of childhood diarrhoea in patients attending a district hospital in Ghana. Parasit Vectors 2011; 4(1): 163.[http://dx.doi.org/10.1186/1756-3305-4-163] [PMID: 21859463] ]. Extra treatment systems such as UV water disinfection installation is required to complement the water treatment systems already in place in Ghana to improve potable water supply in the country [53Lui GY, Roser D, Corkish R, Ashbolt N, Jagals P, Stuetz R. Photovoltaic powered ultraviolet and visible light-emitting diodes for sustainable point-of-use disinfection of drinking waters. Sci Total Environ 2014; 493: 185-96.[http://dx.doi.org/10.1016/j.scitotenv.2014.05.104] [PMID: 24946032] ]. Until such installations are put in place to improve the quality of water in Ghana, it is advisable especially for HIV/AIDS infected individuals to boil their drinking water to minimize infections of opportunistic pathogens. Boiling water inactivate the oocysts of Cryptosporidium, & Cyclospora and other infectious pathogens making it safe for drinking [54Ashbolt NJ. Microbial contamination of drinking water and human health from community water systems. Curr Environ Health Rep 2015; 2(1): 95-106.[http://dx.doi.org/10.1007/s40572-014-0037-5] [PMID: 25821716] , 55Yoder JS, Harral C, Beach MJ. Cryptosporidiosis surveillance - United States, 2006-2008. MMWR Surveill Summ 2010; 59(6): 1-14.[PMID: 20535094] ].

In the case of Microsporidium infection, the possible risk factor for infection among the HIV/AIDS patients was identified in this study to be hand-washing behavior. Therefore, proper personal hygiene, improved sanitation and an extra treatment of drinking water would significantly reduce the exposure and infection of Microsporidium and other opportunistic pathogens [35Fletcher SM, Stark D, Harkness J, Ellis J. Enteric protozoa in the developed world: A public health perspective. Clin Microbiol Rev 2012; 25(3): 420-49.[http://dx.doi.org/10.1128/CMR.05038-11] [PMID: 22763633] ].

CONCLUSION

In conclusion, we report the risk factors for exposure of Cryptosporidium, Cyclospora and Microsporidium in HIV/AIDS infected individuals in Ghana for the first time. These findings should be of public health concern and measures should be put in place to improve the environmental sanitation and the quality of drinking water in Ghana. Clinicians should regularly offer advice to their HIV infected patients on how to improve their personal hygiene, their food source and an extra treatment of their drinking water to reduce opportunistic pathogenic infections.

AUTHORS’ CONTRIBUTION

All authors contributed equally

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

Ethical clearance was obtained from the Ghana Health Service Ethical Review Committee on Research Involving Human Subjects (ERCRIHS), certificate number GHS-ERC: 24/01/12.

HUMAN AND ANIMAL RIGHTS

No animals were used in this research. All research procedures followed were in accordance with the ethical standards of the committee responsible for human experimentation (institutional and national), and with the Helsinki Declaration of 1975, as revised in 2008.

CONSENT FOR PUBLICATION

Permission to interact and collect research samples from HIV infected clients assessing the HIV referral clinic was obtained from the administration of the hospital.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS

We want to express our profound gratitude to the Department of Biomedical and Forensic Sciences, University of Cape Coast, Ghana for partly sponsoring this study. Our gratitude also goes to the staff and clinicians at the HIV Referral Clinic and Cape Coast Teaching hospital, Cape Coast for giving us access to their health facility and their clients. Lastly, we are grateful to our study participants for their participation and support in making this study a success.

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