Predictors of metabolic syndrome among people living with HIV in the Gedeo-Zone, Southern-Ethiopia: an unmatched case-control study

Background Metabolic syndrome (MS) among people with (PLHIVs) a global public However, there is no primary data about predictors of MS in the SSA and Ethiopia. Therefore, the aim of this study was to determine predictors of MS, among PLHIVs in the Gedeo-Zone,Southern-Ethiopia. Methods Unmatched case-control study approach, among PLHIVs who served at randomly chosen two hospitals and health centers in the zone, in between (December 29 th -2017 and January 22 nd -2019) was done. WHO-steps tools were used to gather the data, finally handled with (Epidata-V-3.1 and SPSS-V-22) software’s. Lastly, using a multivariable conditional-LR-model, 4-models with AOR (95%CI) were computed to arrived at the final model, and then variables accepted as significant at (p-value < 0.05)level.

(LMIC), with about 48% of deaths in these countries occurring before the age of 70, in 2015 [1][2][3]. In the Sub-Saharan Africa (SSA) region, over the next two decades, it is expected to escalate substantially [2]. In Ethiopia, it is estimated to account for 30% of total deaths, with 9% and 1% proportional mortality from CVDs and Diabetes, respectively [3].
Following the global implementation of highly active antiretroviral treatment (HAART), there achieved the reduction of 30 million new infections and nearly 8 million Acquired Immune Disease Syndrome(AIDS) associated deaths [2,4], and increase in life expectancy of people living with HIV (PLWH) worldwide. However, apart from the success achieved, works of the literature disclosed that antiretroviral therapy (ART) has contributed to forming an aging people living longer with the treatment, and thus to be at risk of developing None-communicable disease (NCD) [2,[4][5][6][7][8][9][10]. With this reasons, the HIV infection environment nowadays represented as a typical illustration of the interaction between infectious diseases and non-communicable diseases (NCDs) [2,4,[11][12][13].
The precise magnitude of MS in the HIV-infected population is still arguable; but the available data indicate the global prevalence of MS can be regarded as high, ranging from 11.2% up to 45.4% [4,13], and was also estimated to range from 13% to 58% in Africa [2,15]. In the sub-Saharan Africa (SSA), the prevalence of MS is not well established [4,18]. Adults with this problem will have a twofold as likely to die from and are three times as likely to have cardiovascular diseases (CVDs) and a five-fold greater risk of developing type 2 diabetes (T2DM) [2,7,11,14,19]. In this backdrop and its higher attendant economic and health system consequences of the identification and monitoring of comorbid health risks of HIV and ART [2,20], MS at present is a global public health issue [2], prominently in the SSA region and Ethiopia, the epicenter of HIV infection [2,11]. This signifies the importance of knowing what predicts the syndrome.
However, pieces of literature suggested as MS emergence is mostly linked with the interplay of various factors; while several studies revealed the existed direct association of MS with the individual-based factors [4,14,[21][22][23][24][25][26][27][28][29][30][31]. Inconsistently, some other studies also restated equivocally the explicit effects of all a ranges of the above factors with the incidence of the syndrome [4,14,15,18,32]. And this implicated the lack of clear evidence about the independent effect of each factor, like individual-based risk factors, HIV/AIDS-related factors, lifestyle or behavioral risk factors, and MS linked risk factors with MS occurrence [22,33,34]; which is precipitated with the scarcity primary studies in SSA [24,30], predominately in Ethiopia [28,35]; where the majority among the few studies were addressed crossectional [4,15,35,36], the foundation of this study.
Therefore, this study intended to determine predictors of metabolic syndrome among people living with HIV in the Gedeo-Zone, Southern-Ethiopia, with the employment of unmatched case-control study design, together with the adult treatment panel III criteria as a method to diagnose the subjects with or without the outcome of interest. The findings from this study have significance for public health practice and to establish baseline information for policy and program development. Notably, this study is an extension of the prior large study with multiples of objectives conducted for one year.
Hence, for the detail understanding of the methodological procedure employed, you can refer from the article published elsewhere with this citation [37].

Study design
A health facility-based unmatched case-control study was conducted. system (HMIS) reports, at time of the study, there were a total 3,597 adult PLHIVs (629 ART naive (370 female, 259 male) and 2968 current on ART (1813 female, 1155 male)). Among these, although (n=537 ART exposed and n=135 ART naïve) were registered in the (CHCCs) of the health centers, but (n=2395 ART expose and n=412 ART naïve/Unexposed) groups were enrolled and served under the (CHCCs) of the hospitals [37].
As per the current national consolidated guidelines for comprehensive HIV prevention, care and treatment guideline for Ethiopian, all HIV positives are eligible for ART irrespective of their WHO clinical staging and/or CD4 count and the ideal time for ART initiation depends on the clinical condition and readiness of the client, which should be offered on the same day to people who are ready to start. Rapid ART initiation (i.e. defined as the initiation of ART within seven days of HIV diagnosis, provided that there are no contraindications) ought to be offered to all people living with HIV following a confirmed HIV diagnosis, clinical assessment, and assessment of client readiness. As a priority, ART should be initiated in all adolescents and adults with severe or advanced HIV clinical disease (WHO clinical stage 3 or 4) and adults with a CD4 count of ≤350 cells/mm3. Over all, we have few options of drugs 1st line, 2 nd line, and 3rd line regimens. All of those regimens are based on the nucleoside reverse transcriptase inhibitors (NRTI), none nucleoside reverse transcriptase inhibitor (NNRTI) and a boosted Protease Inhibitor (PI). The preferred first-line regimen for adults and adolescents is TDF+3TC+DTG or TDF+3TC+EFV as a once-daily dose. Fixed-dose combinations and once-daily regimens are preferred for antiretroviral therapy [38].

The source and study population
The source population of the study was all adults age 18 years and above who were HIV positive and enrolled in the chronic HIV care clinics of the public health facilities (i.e. actively engaged in the delivery of HIV services), of Gedio zone at time of data collection arranged in the prior survey. A confirmed HIV-positive individual enrolled in the chronic HIV care clinics of the selected health facility chosen to conduct the prior study and identified by then as cases and controls, and fitted the eligibility criteria set for both groups were used as a study population.

Case identification procedure
Cases were identified whenever they came in to those randomly chosen public health institutions' chronic HIV care clinics (CHCCs) (either to take their routine services and or to be enrolled in to it for the first time), and then voluntarily engaged in to the prior study and go through with the physical and the biochemical data collection procedure using the WHO STEPS instrument during the data collection period. The physical measurements data encompassed the body weight, height, waist circumference (WC), and blood pressure data. The biochemical measurements data includes the fating plasma glucose levels and lipid profiles (Total cholesterol, Triglycerides, High and low density lipoprotein cholesterol) data.
It was accomplished through doing of laboratory tests, arranged in the next day or sometime after completion of the physical measurements of data collection process, but within 8-12 hour overnight fast, by drawing of 3-5 mL Venus blood. Often before analysis, the collected blood samples centrifuged with 5000 (rpm) for 5-10 minutes to separate the serum from the whole blood , and then storing of it to the refrigerator(adjusted at 2-8 0C or -200C), were performed. Following this, all the biochemical analysis arranged at Dilla University College of medicine and health science hospital clinical Diagnostic laboratory unit was run, using the BS-200E Clinical Chemistry analyzer through applying of a different technique. For instance, the enzymatic colorimetric assay technique was applied for the measurement of Total cholesterol (TC) (CHOD-PAP method) and triglyceride (GPO-PAP method). As well, while the glucose oxidase method (GOD-PAP), and the direct homogeneous enzymatic colorimetric assay methods were employed to measure the glucose, and the remaining the lipid profiles (High density lipoprotein (HDL-c) and Low density lipoprotein (LDL-c)), respectively. The biochemical analysis was made by laboratory technologists, guided with standard operating procedure (SOPs) that explains what to be done from sample collection to result releasing.
During analysis, the daily quality control samples together with the individual samples were run before running of the samples, and then the correct functioning of instruments, laboratory reagents, and technical performances were often verified. Lastly, all the physical and biochemical measurements data gathered blindly were used to ascertain and enumerated as a case or as a control, using the below designated case ascertainment method as a base after entry of the data in to the software designated in the analysis section.

Methods of case ascertainment
The cases were ascertained based on the revised National Cholesterol Education Program (NCEP)-Adult Treatment Panel three (ATPIII) criteria [13], by having at least three of the following traits: Fasting plasma glucose ≥ 6.1 mmol/L (110 mg/dl))). or previously diagnosed with type 2 diabetes mellitus or anti-diabetic treatment.

Eligibility criteria for the cases
Cases were considered in the study if and only if they became a confirmed HIV infected adult's patient, who meet the case ascertainment criteria in the prior study, and then signed the consent form to voluntarily participate in the current study. Cases were excluded if they were refused to participate in the study and they were unable to sign the consent or unable to follow an interview in their native language because of aphasia, reduced consciousness or other reasons such as pregnancy and severe illness.

Control selection method
For each identified cases the corresponding three consecutive controls were likewise randomly determined. They identified from the same sources where the cases were found, after gone through with the whole process that employed to identify the cases. The controls were eligible if they were found within the same age category, but not fulfill exactly the criteria established above to ascertain cases, and or diagnosed with either traits of MS and or has lower than two numbers of MS traits during the prior study time [37]. Controls were excluded if they refuse to participate in the study; we're unable to give informed consent or follow an interview in their native language because of aphasia, reduced consciousness, or other reasons.
The rationale to recruit the case and the control from the same sources ( i.e. in the health care institutions) than the community was due to the fact that the PLHIVs can easily be accessed whenever they enrolled and started to take services in the health care institutions; where they represented above the 95% of the PLHIVs in the Gedio zones.
Note that the identification and recruitment of cases and controls procedure was proceed with the assessment of each subject's suitability for the eligibility criteria set for the current study. It was accomplished consecutively whenever they returned back to the clinics for their subsequent routine care, sometime within the study duration. This action was undergone until all the predetermined and identified sample size for the study cases and controls attained. number of rooms available in the house for sleeping; the presence of any agricultural land, and the presence of any livestock, herds, other farm animals, or poultry) that measure household assets which is divided into quintiles to represent overall levels of household wealth, such as: quintiles 1-lowest, quintiles 2-second, quantiles3-middle, quintiles 4-fourth, quintiles 5-highest.
or recreational related physical activity.
Domain specific physical activity-mean: Mean minutes spent in work-, transport-and recreationrelated physical activity on average per day.
Total physical activity-mean: Mean minutes of total physical activity on average per day from work, transport and or recreations.

Levels of total physical activity according to former recommendations:
Percentage of respondents classified into three categories of total physical activity according to former recommendations.
Not meeting WHO recommendations on physical activity for health: Percentage of respondents not meeting WHO recommendations on physical activity for health (respondents doing less than 150 minutes of moderate -intensity physical activity per week, or equivalent).

Former recommendations for comparison purposes:
The three levels of physical activity suggested for classifying populations were low, moderate, and high. The criteria for these levels are shown below.
High: A person reaching any of the following criteria is classified in this category:-Vigorous-intensity activity on at least 3 days achieving a minimum of at least 1 ,500 MET-minutes/week OR -7 or more days of any combination of walking, moderate-or vigorous-intensity activities achieving a minimum of at least 3,000 MET-minutes per week.

Moderate:
A person not meeting the criteria for the "high" category, but meeting any of the following criteria is classified in this category:-3 or more days of vigorous-intensity tivity of at least 20 minutes per day OR-5 or more days of moderate-intensity activity or walking of at least 30 minutes per day OR -5 or more days of any combination of walking, moderate-or vigorous-intensity activities achieving a minimum of at least 600 MET-minutes per week.
Low: A person not meeting any of the above mentioned criteria falls in this category.

Metabolic Equivalent (MET):
is the ratio of a person's working metabolic rate relative to the resting metabolic rate. It allows us to calculate total physical activity. One MET is defined as the energy cost of sitting quietly, and is equivalent to a caloric consumption of 1 kcal/kg/hour. Therefore, for the calculation of a person's total physical activity using GPAQ data, the following MET values are used:  Biochemical measurements: elevated fasting plasma glucose, elevated triglycerides, and lower high density lipoproteins.

Data sources /Measurement
While the sources of data for the outcome variables were individuals physical and biochemical measurements, for the exposure variable were self-reported response from individual interview and the document review. The outcome was measured with the NCEP-ATPIII criteria [13]; which is a standardized techniques employed in the different literatures conducted in the globe, including the study country. The exposure variables were measured as per the standard set in the Ethiopia Steps Report On Risk Factors For NCDs And Prevalence Of Selected NCDs study [39]. This study employed a similar instrument that it gives reliable and valid measures. All the measurement techniques used to ascertain the outcome and exposure variables were comparable for the two study groups (cases and controls).

Study size
In our previously published article [37] , taking the MS as an outcome, we determined a sample size of 633 participants. Based on the inclusion and exclusion criteria we retrospectively selected 139 cases and 494 controls. This gives 1: 3 cases to control ratio.

Bias
In order to maintain the internal validity of the this study, a validated WHO steep instruments and document review data collection checklists were employed to gathered quantitative data. The WHO steep tool is considered a standardized tool that is freely flexible to investigate NCDs risks of various countries. The use of such a precision tool to collect data on history, physical and biochemical measurements reduces the likelihood of error in methods or interpretation and recording of data. The instruments were translated to the regional language (i.e. Amaharic), so that there were easy understanding between the data collectors and the participants and also data collectors obtain data in the same fashion. Additionally, pretesting of the tool was also carried out in adjacent health institutions, not selected for the main study, before the commencement of the data collection process to clarify any ambiguity and to verify the appropriateness of the tools.
Actions against threats of potential biases were addressed accordingly. For example, to eliminate recall bias, we have reducing the period for past events to the last few days. And, cases and controls were interviewed when they came to the health institutions to take their consecutive care arranged after a month of the prior cares. To minimize bias related to measurements, the standardized Adult treatment panel three (ATPIII) criteria was used to identify overall metabolic syndrome cases. Controls were also explicitly defined in the method part of this dissertation. To avoid selection bias due to a difference in the number and quality of the service delivered in each health facilities, stratification of these health institutions based on their levels of service delivery and daily patient flow was first made. Although, a continuative sampling technique was used to randomly selected ART exposed and ART naive PLWHs who were receiving their routine care at randomly chosen health centers and Hospital; a total patient flow under each institution for the two comparison groups were first determined, and the required adequate samples from each institution were proportionally drowning.

Quantitative variable
During data collection, both the case and the controls participants were assessed for their risks using the first step of WHO instrument and the checklist prepared for review of documents. The WHO first step tool gather data on from each individuals on, history of raised blood pressure, diabetes, raised cholesterol and/or CVDs, and lifestyle advice. The checklist was consists of questions regarding HIV related factors such as duration diagnosed with HIV, ART status, ART drug types, duration of ART use, CD4+ levels, RNA levels, OI's status and other illness associated risk factors. Later, all data gathered from each individuals cases and control with the same fashion indicated in the above paragraph, were entered into template formed using Epidata (version 3.1) software with the help of two data clerk; eventually validation was performed by using the original data as references. Last of all these, the data were transformed into Statistical Package for Social Sciences (SPSS -Version 22) for analysis.

Statistical methods
Before application of any statistical methods for the tranformed SPSS data, all the necessary data exploration techniques were employed to further cleaning of the data.
Next to this, a-ranges of descriptive data summarization statistical techniques such as proportion, mean, standard deviation was run, and then the results were presented using tables. between all the significant variables in the model were investigated by using VIF, and no correlation between independent variables was seen (VIF <10).

Ethical clearance
Since this study is an extension of the published article, information about the ethical procedure can be referred from the published article [37].

Discussion
To our knowledge, this is one of the few primary studies in the sub-Saharan Africa region, conducted using an integrated system of data collection (WHO stepstools and document review), to examine the predictors of metabolic syndrome among relatively larger number PLHIVs using a strong case-control study design, under one centralized clinical laboratory biochemistry unit.. Whereas, previous studies have examined the associated risk factors of MS using crossectional study approaches among a little number of individuals by employing nonstandard tools. Overall, the study concluded that the age, education, occupation, ART status, WHO-staging, physical activity state, previous history of blood sugar measured, raised BMI, raised WC, elevated fasting blood glucose, elevated triglycerides, and low HDL-cholesterols were found to be an independent predictor of metabolic syndrome. While, education, occupation, ART status, WHO-staging, and physical activity variables were found to be indirect predictors of MS, the rest of the variables were determined as the direct predictors of the syndrome.
It had several limitations, which were emerged from the nature use of a case-control study design, potentially liable for several biases, such as: selection biases, recall bias, information bias and social desirability biases, along with the other problems associated with the current study contexts like the absence of complete matching of it with an important variable (i.e. sex, age, and self-rated wealth), a strict adherence to the ATP III standardized criteria, and the variability's of the studies used for comparison purposes.
As it has been stated above, the odds MS was more likely increased among older age groups than a younger age. Although not consistent on the age groups, but a marked disturbance in the MS with aging has also been reported by several epidemiological studies done worldwide [15,18,21,23,25,29,35,40,41]. The observed comparability among the studies might be because, regardless of the difference in the target population, age is a common non-modifiable risk factor that equally likely predisposes all to such health mater. In the reverse, Bosho, et al (2018 ) problems in general and the syndrome in advance for those PLHIVs aged with the virus will be a vital aspect. Additionally, inconsistent with our finding, while several studies [15,26,35,40,[45][46][47][48] noted the direct link of sex with MS incidence, some other studies were also found a similar link of the place of residence with MS [40,41].
As well, the relation between education and the risk of MS is worth further discussion. Notably, our finding also pinpointed that the odds of MS was less likely among un-employed but worked as a home-maker and able to work than those employed in the government institution.
Inconsistent with this result, Mashinya et al (2015) [41] study stated that the high unemployment rate was a direct predictor of the syndrome. This unlikeness may be due to the difference in the design used by the former and the later studies. This could be justified due to similar details discussed overhead. As per Mashinya et al (2015) [41] study report, the high unemployment rate might be precipitated with stigma emerged from the viral infection, which may predispose the PLHIVs to high levels of stress; which eventually leads them to develop MS. However, in our view, the observed inverse association may not be out of the domains of differences income earned between the employed and un-employed PLHIVs. This means that, in our country contexts, the unemployed individuals are often earning a lower income that they cannot able to purchase any goods necessary for them to lead a qualified life that it might spontaneously help them to be protected from such highquality lifestyle-related health problem. Overall, the finding implicated that apart from the actions aimed at design of occupations among HIV infected people might so play an essential part by easing stress-related with unemployment.in indirectly, but still the planning of awareness creation intervention to all, regardless of their employment status may correspondingly play an essential role in the prevention of the risk of acquisition of MS.
Also, our study as well showed that compared with ART naïve a group, the risk of MS was less likely in the ART exposed PLHIVs. It was consistent with several previous studies [26,35,40,49]. Furthermore, specific with the ART regimen and in the contrary to our report, a few other studies besides notified the existence of significant differences across ART-regimen [15,21,26]. In this respects, still studies have shown inconsistencies' amongst each other about the definite ART regimen there were no consistencies in between. While few studies have agreeable output on the second line regimen, (primarily the protease inhibitors (PI)) as a predictor for the incidence of MS [21,35,51], however, few other studies were disclosed that the first-line ART regimen, mainly of the Nucleoside Reverse Transcriptase Inhibitors (NRTIs) and the Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) as predictor of MS incidence [15,26,35,42,51]. In relation to this regimen, studies were further revealed that in patients using Stavudine (d4T)[42, 52,53] alone, and NRTI as a whole As per the self-reported lifestyle associated factors, although the following factors such as alcohol consumption, smoking habits, 'Khat chewing habits, frequency of consumption of fruit and vegetable, and fat and oil showed no significant association with the presence of MS; however, physical activity was found to be indirect predictors of MS. Studies with this respects are inconsistent with some reporting significant association for smoking habits [41,50,60], alcohol consumption [41,50,61], and physical activity [41,62] while others and results of the same study reported no association for 'Khat chewing habits [42], alcohol use [35,42], physical activity [35,42,61], smoking habits [15,35,42,61,62]. This might be partly explained due to the lower number of individuals grouped under each category that might affect the analysis. Another possible justification the nature of the data relies on self-reporting, which is liable to response bias. With regards to diet, unrelated to our finding, Jantarapakde, et al (2014) study astonishingly discovered the presence of a direct association between food insecurity (OR 1.8, 95%CI 1.0-3.3, p = 0.05) [40]. This could be justified partly due to the time variation, and inconsistent approaches employed by the former and the latter studies.
In the contexts of MS related risks, the current finding notified that raised BMI and WC were directly associated with the incidence of MS. Correlating with our finding, and regardless of the standard criteria employed, several studies were reported similar [45,61,63] Further, in relation with the above contexts, while Jantarapakde et al (2014) [40] study noted that an overall fat redistribution (i.e. resulted either from "lipoatrophy and or hypertrophy", which is collectively called as "lipodystrophy") was a significant predictor of MS (OR 1.8, 95%CI 1.0-3.0, p = 0.032), largely in the ART exposed groups (P<0.005  [64]. The discrepancies observed in between the former and the current studies might be an attribute of the expiations given before, along with the differences observed by the studies on the application of standard diagnostic criteria and the cut of point of each of the biochemical profiles to be decided.
In light of these drawbacks, to increase the generalizability of the findings of this study, a much larger number of sample sizes than required were included in the analysis. The consideration of more than the minimum sample size required has increased the power of the study. On top of that, to see the result of chance, we besides computed the 95% CI as a measure of association between exposure and outcome variables. The confidence interval for most of the variables used in the current study was not wide enough suggesting the adequacy of the sample size. Furthermore, the predictors identified in those health care institutions of the current study can represent HIV-infected people found in the zone. This is because the PLHIVs flows in those health institutions represent the majority of them residing in the Gedio zone. Therefore, the findings of this study can be generalized to the study area and other similar settings in particular.
Finally, this study was funded with Addis Ababa University, college of Medicine and school of Health science, in collaboration with DIlla University College of medicine and health science, for the partial fulfillment of Ph.D in Public health. All the above funding organizations have no role in conducting this study as whole on preparing the manuscript, except the financial delivery and the supporting supervision.

Conclusion And Recommendations
Generally, the study concluded that while age, previous history of blood sugar measured, raised BMI, WC, IFG, TGL_c, and low HDL_c levels were the direct predictors of MS; education, occupation, ART status, WHO-staging, and physical activity were found to be an indirect predictors of MS. Implicated that the predictors that lead to developing MS components and MS are not yet shown difference with the general populations. It thus, essential to plan routine health education programs to be given to these PLHIVs, in the order to bring behavioral change on modifying their lifestyles. In addition, reorientate and realizing an integrated care plan that address both the routine care given to PLHIVs and a regular early screening of MS to be underway in the primary health care system, so as to optimize the prevention and management of it, and ultimately to reduce future epidemic cost, in the era of increased population, aging with ART, increased urbanization, and obesity, which have been observed in the SSA region, including Ethiopia. Finally, as a short term intervention the researchers' needs to evaluate the cost benefits of an integrated care plan that address both the routine care and the regular MS early screening program, in the meantime to design a longitudinal studies to further investigating the natural course of MS incidences among cohorts of PLHIVs , as a long-time action.

List Of Abbreviations
Abbreviations, Numbers and SI Units: AIDS: Acquired Immunodeficiency syndrome, ART: Furthermore, the official letter was produced and delivered by the author to the respective Southern Nations Nationalities Regional health bureaus, Gedeo zone and Woreda health bureaus and all of the institutions selected to conduct the study. Data were collected unlinked anonymously, without any personal identifiers. Each individual was enrolled entirely voluntary after written consent was obtained. Any information obtained during the study was retained with the greatest confidentiality.
Physical measurement was done by performing measurements at an ART clinic room that has been screened off to maintain the individual's privacy. All biochemical analysis was performed free of charge, and results were provided to the clinicians for further investigation, and possible management [37].
Consent for publication: Not applicable 3. Table 3 Proportion and Bivariable association in between behavioral risk factors and MS 4. Table 4 Proportion and Bivariable association in between MS related risk factors and MS 5. Table 5 Factors associated with metabolic syndrome in model one, two, three and four