Prevalence of Cryptosporidium species among HIV/AIDS patients in Sub Saharan Africa; Systematic Review and Meta-Analysis

Methods: A comprehensive search of electronic databases was conducted on PubMed, EMBASE, African Journal Online (AJOL) and advanced Google Scholar. The reference lists of all identified articles were searched for additional studies. Meta-analyses were carried out using review manager 5.3, comprehensive meta-analysis and R software version 3.6.1. Evidence for statistical heterogeneity of results was assessed using Cochrane Q x2 test and I2 statistic.

Cryptosporidium is primarily transmitted mainly through feco-oral route either by direct contact with an infected human or animal or indirectly via contaminated food or water [3]. The infectious dose of Cryptosporidium species is as low as 9-10 oocysts [4,5]. Oocysts of Cryptosporidium remain infectious in the environment for at least 6 months if kept moist and resistant to conventional water treatment such as chlorination [6,7].
Cryptosporidium species infection is common in many developing countries of sub-Saharan Africa due to poor sanitation, poor hygiene and unavailability of safe drinking water [8,9]. It was previously considered non-pathogenic or with transient pathogenic potential in immune-competent individuals, but nowadays becoming aggressive and cause debilitating illness in HIV/AIDS patients [10].
In patients with HIV/AIDS, prevalence of cryptosporidiosis vary widely, ranging from 0 to 100% with the higher rates reported in patients that have not started ART [11]. According to global disease burden report published in 2010, a prevalence rate of 2.6-21.3% has been documented in Africa. Cryptosporidiosis is one of the AIDS-defi ning illnesses and associated with an increased risk of death compared to other AIDS-defi ning illnesses [12]. The advent of highly active antiretroviral therapy (HAART) has reduced the prevalence of this disease in AIDS patients [13][14][15]. The emergence of drug-resistant HIV strain and failure of HAART has been associated with re-emergence of Cryptosporidium species infection in HIV/AIDS patients [15].
In 2013, globally an estimated 35.0 million people were living with HIV/AIDS. Sub-Saharan Africa accounts for 71% of the global burden of HIV infection [16]. AIDS-related death declined by about 42% from 2010 to 2017 in eastern and southern Africa, refl ecting that the rapid growth of treatment scale-up in the region [17]. About 80% of AIDS patients died from AIDS-related disease including intestinal parasites rather than HIV infection itself. Cryptosporidium species on the other hand is an emerging cause of chronic diarrhea with lifethreatening conditions in HIV/AIDS patients ]18].
Diarrhea occurs in about 90% HIV/AIDS patients in developing countries, including sub-Saharan Africa [19]. Cryptosporidium species is increasingly recognized as a leading cause of diarrheal disease, and its largest burden occurs in HIV/AIDS patients [20]. HIV/AIDS patients with CD4 count falls below 100 cells/ mm 3 the risk increases for severe disease accompanied by malabsorption, weight loss, and high case fatality [21].
In 2004, cryptosporidiosis was added to the WHO's 'Neglected Diseases Initiative' which includes diseases that occur mainly in developing countries [22]. Cryptosporidium co-infections and associated morbidities are common among people living with HIV/AIDS and have implications for their treatment and care. So, it is found mandatory to know the magnitude of the neglected Cryptosporidium infection among HIV/AIDS patients so as to develop strategies to prevent and control the disease. The main objective of this review was to summarize data on the prevalence of Cryptosporidium species infection among HIV/AIDS patients in sub-Saharan Africa.

Search strategy
In this review all studies published in the English language from 1990 to October 2019 were searched. The search strategy was conducted in three steps; initially, the search was conducted on PubMed followed by the analysis of text words contained in the title and abstract, and index terms used to describe the article. Secondly using all identified keywords, and mesh terms where each factor was combined using the "OR" operator and "AND" operator was used to search studies across PubMed, Embase, African Journal Online (AJOL) and Google scholar. Database search terms included were those that used to describe Cryptosporidium species and Human Immuno Virus (HIV) infection as well as terms that describe the context of this study. The search terms were: coccidian parasite, Cryptosporidiosis, Cryptosporidium parvum, opportunistic parasite, prevalence, and human immunodefi ciency virus, HIV, AIDS, HIV/AIDS, Africa, and Sub Saharan Africa). Full-text articles were retrieved after review of the title and abstract. Thirdly, the reference lists of all identified studies were searched for additional studies that are relevant to this study. An Endnote software version 5 was used to manage references in this review.

Inclusion and exclusion of studies
Inclusion criteria: To review the prevalence of Cryptosporidium species infections in HIV/AIDS patients, we considered the following criteria: i) Studies on the prevalence of Cryptosporidium among HIV /AIDS patients were included in the study. ii) Only a study conducted in sub-Sahara African countries were considered. iii) Studies published in English were selected. iv) No restriction on methods of diagnosis.
Exclusion criteria: Observational studies including case reports and case series were excluded.

Assessment of study quality
Studies selected for inclusion were assessed for methodological quality by two independent reviewers using standard critical appraisal instruments for prevalence study Condition, context and population (CoCoPop) from the Joanna Briggs Institute Meta-Analysis of Statistics Assessment for Review Instrument (JBI-MAStARI). A 10-point scoring system was used to rate the quality of the articles retrieved. Scoring was conducted by two independent investigators using a modifi cation of the Downs and Black checklist [23]. For inclusion in the review, both reviewers (E.A and K.D) agreed that a cut-off a score of five out of 10 was used to determine acceptable quality for inclusion. Disagreements were resolved by consensus.
The study quality assessments criteria were: objective of the study clearly described, study design clearly stated, the sample size representativeness of the population from which they were recruited, method of identifi cation of the parasite identifi cation clearly identifi ed, outcome assessed with the objective criteria, were confounders reported, were potential Citation: Awulachew  biases reported, was outcome clearly described, appropriate statistical analysis method used, and if whether the context of the study is sub-Saharan Africa (Table 1).

Data extraction
The two authors (E.A and K.D) extracted the data from included studies using a standardized form independently and checked the data together. A critical appraisal checklist for observational studies (prevalence study) adopted from JBI was used to assess the overall methodological quality of the included studies [24]. From each included studies, detail description of study subjects, report on the study area, year of publication, study design, sample size, method of Cryptosporidium species screening was extracted. Secondary outcomes for this study included clinical data including the presence or absence of diarrhea, whether the patient started ART treatment or not, a CD4 count of the patients, and distribution of Cryptosporidium species by age, sex of the patients ( Table 2).

Data analysis and data synthesis
Meta-analyses were carried out using review manager 5.3, comprehensive meta-analysis and R software version 3.6.1 with user contributed commands for meta-analyses: metan, metainf, metabias, and metareg [25]. The effect sizes and SEs of the studies were pooled using a random-effects model to calculate the pooled prevalence of Cryptosporidium species among HIV/AIDS patients. The association of prevalence of cryptosporidium species with CD4 count and with the presence and absence of diarrhea will be evaluated and odds ratio was used to present association.

Risk of bias and sensitivity analysis
The random-effects meta-analysis models were chosen because heterogeneity was demonstrated [26]. Evidence for statistical heterogeneity of results was assessed using Cochrane Q x 2 test and I 2 statistic. A signifi cance level of P<0.10 and I 2 >50% was interpreted as evidence of heterogeneity [27]. A potential source of heterogeneity was investigated by subgroup analysis and meta-regression analysis [28]. Where statistical pooling was not possible the fi ndings were presented in a narrative form including tables and fi gures to aid in data presentation where appropriate.
Sensitivity analyses were conducted to weigh up the relative infl uence of each individual study on the pooled effect size using user-written function, metainf [25]. The presence of publication bias was assessed informally by visual inspections of funnel plots [29].

Study selection
As shown in the fl owchart above ( Figure 1) the literature

Additional analysis
This review also extracted additional data on Cryptosporidium infection from the included study. Due to the variability of data quality and reporting system, we only extracted and analyzed the data on diarrhea, ART, and CD4 count. According to the Papers excluded after quality appraisal: -1 Figure 1: Flow chart of the search and study inclusion.  (Figure 3).

Heterogeneity and risk of bias
Subgroup analysis of fi ve studies showed the pooled prevalence of Cryptosporidium infection in HIV-infected patients was signifi cantly higher in patients with diarrhea (OR=1.779 95% CI: 1.057-2.994, p= 0.030) and the pooled prevalence of six studies in sub-Saharan Africa showed the highest prevalence Cryptosporidium infection in HIV/AIDS patients was signifi cantly higher in Naïve HIV/AIDS Patients (OR= 1.559; 95% CI= 1.100-2.209; P= 0.013).
The funnel plot helped us distinguish between publication bias and other causes of the asymmetry. It showed that small studies were found not only in the areas of statistical signifi cance. We demonstrated no publication bias (t= -1.3505, df= 19, p-value= 0.1927).

Summary of evidence
In sub-Saharan Africa high burden of Cryptosporidium infection in this review indicate the importance of routine testing for Cryptosporidium species in all HIV-infected people.
To our knowledge, this is the fi rst systematic review on prevalence of Cryptosporidium species among HIV infected people in sub-Saharan Africa. Our fi ndings demonstrated evidence for a high prevalence of Cryptosporidium species among HIV-infected patients in sub-Saharan Africa.
Due to missing data we did not analyze them.

Limitation
However, this systematic review came up with the prevalence of Cryptosporidium species among HIV/AIDS patients in sub-Sahara Africa, we acknowledge few limitations of the present meta-analysis, which may affect the results.
First of all four relevant studies which were identifi ed through our literature search were excluded due to unavailability for full-text review. Secondly, the majority of the studies were of moderate or low quality, as most of the data was from the conventional microscopic examination techniques; that have lower sensitivity than ELISA and polymerase chain reaction.
So, use of different diagnostic tests with varying diagnostic sensitivity is the other limitation of this study.

Conclusions
The results of our meta-analysis show a heavy burden of Cryptosporidium infection among HIV/AIDS patients in sub-