Severity and Determinants of Anemia in TB/HIV Coinfected Adults at Mekelle, Ethiopia: Hospital Based Retrospective Study

Background Anemia has up to 87% prevalence in high tuberculosis (TB) and human immunodeficiency virus (HIV) burden settings of the sub-Saharan Africa (SSA) including Ethiopia. It increases lost to follow-up (LTFU) rate, reduces quality of life, and shortens the survival of TB/HIV coinfected patients. However, there is limited information on severity level and determinants of anemia in TB/HIV coinfected adults in the study setting in particular. Therefore, this study is aimed to assess severity level and determinants of TB/HIV-associated anemia. Methods A hospital based retrospective study was conducted among 305 TB/HIV coinfected adults who enrolled for antiretroviral therapy (ART) from January, 2009 to December, 2016 in two public hospital of Mekelle, Ethiopia, by reviewing ART register. A multiple logit model was fitted to identify the baseline determinants of anemia using 95% confidence level or 5% level of significance for adjusted odds ratio (AOR). Results In the current study, the cumulative baseline prevalence of anemia was 59.0% (95% CI: 53.3%–64.6%). Considering severity level, the prevalence was 6.2%, 28.2%, and 24.6% for severe, moderate, and mild anemia, respectively. Being female (AOR = 0.380; 95% CI: 0.226–0.640), body mass index (AOR = 0.913; 95% CI: 0.836–0.998) reduces the odds of developing anemia whereas baseline ambulatory functional status (AOR = 2.139; 95% CI: 1.189–3.846), bedridden functional status (AOR = 2.208; 95% CI: 1.002–4.863), HIV clinical stage III (AOR = 2.565; 95% CI: 1.030–6.384), and HIV clinical stage IV (AOR = 2.590; 95% CI: 1.006–6.669) increased the odds of developing anemia for TB/HIV coinfected adults. Conclusions In the current study, significant TB/HIV-associated severe anemia was assessed which accounted for nearly one-ninth of all anemia cases while nearly half were moderate anemia. Therefore, curious attention has to be given for the management of TB/HIV-associated severe anemia in particular and anemia in general to reducing anemia associated bad outcomes most importantly death.


Introduction
According to the World Health Organization (WHO), anemia is defned as hemoglobin level's of <12.0 g/dl in women and <13.0 g/dl in men though other factors such as race, physiological status, and pregnancy status have to be taken into consideration [1]. Anemia is a global public health problem that afects an estimated one-fourth of the world's population, with an inconsistent discrimination of cases occurring in sub-Saharan Africa (SSA) [2,3]. Te burden of anemia increases the morbidity and mortality, decreases quality of life, and negatively afects productivity of women particularly [4].
Tough etiology of anemia is multifactorial, evidences have been showed that HIV and tuberculosis (TB) are both strongly signifcantly associated with anemia. HIV and its viral proteins as well as immune suppressions during the natural course of HIV infection were found to be responsible for bone marrow suppression which most likely leads to HIV-associated anemia [5,6]. Furthermore, tuberculosis (TB) plays a signifcant role in causing HIV-related anemia in SSA region including Ethiopia [7][8][9].
Anemia is frequently manifested among HIV patients of Antiretroviral Terapy (ART)-naïve in SSA, with a prevalence that ranged from 45% to 87% [10][11][12][13]. Anemia is one of the leading cause and frequent clinical complications of both HIV and TB diseases and it is associated with substantial morbidity and mortality in TB/HIV coinfected patients [14][15][16]. It is also related with markedly increased lost to follow-up (LTFU) rate of up to 23% in anemic TB/HIV coinfected adults as compared to nearly 10% among nonanemic adults at baseline in a study conducted in the same setting [17]. Reversely, anemia may serve as a useful biomarker for the HIV-associated TB diagnosis and clinical management of such patients [18]. On the other hand, anemia had reported to be strongly and consistently associated with HIV disease progression, suggesting the need for routine screening and treatment even in patients on highly active antiretroviral therapy (HAART) [19][20][21].
HIV-infected people with low CD4+ cell count, high erythrocyte sedimentation rate, and vitamin D defciency was identifed the main predictors of anemia [22,23]. Literature has revealed that the risk of death was reached up to 70% or greater in HIV-infected anemic patients compared with nonanemic counterpart which signifcantly afects the quality of life [24].
To the level of our understanding, only few studies were conducted on HIV-associated anemia in Ethiopia, but none were in TB/HIV coinfected patients. Consequently, these studies reported the prevalence of anemia that varied from 23.0% to nearly 43.0% with the highest contribution being not on ART. Rural place of residence, WHO clinical stage III/IV, presence of TB coinfection, and low baseline CD4 cell count were reported as positive predictors of HIVassociated anemia in one and/or more of these studies [25][26][27][28]. However, none of the studies were assessed the severity level and baseline determinants of TB/HIVassociated anemia. Tus, this study is aimed to generate evidence on TB/HIV-associated anemia and its determinants in the current study setting.

Study Setting, Design, and Population.
A baseline hospital based cross-sectional study was conducted among TB/ HIV coinfected adults who started ART from September, 2009-December, 2016 at two governmental hospitals namely-Mekelle hospital and Ayder comprehensive specialized hospital (ACSH)-Mekelle, Ethiopia. Mekelle is the capital city of the national regional state of Tigray which is 783 kilometers far from Addis Ababa, the capital of Ethiopia. ART service has been begun since 2004 in Mekelle Hospital and since 2009 in ACSH. To date, the ART clinics are active to provide ART services, and above tens of thousands of HIV patients utilized the service collectively in the two hospitals. Data were extracted from March 1 to April 31, 2017 by reviewing ART registers. All active TB and HIV coinfected adults who fulflled the eligibility criteria were included in the current study. TB/HIV coinfected adults who had no baseline measurement of hemoglobin were excluded from the current study analysis.

Sample Size Determination and Sampling Proportion.
We used the single population proportion formula to estimate the minimum sample size considering the following key assumptions: 95% confdence level, 5% acceptable level of precision (margin of error), and overall prevalence of anemia (23%) among HIV patients irrespective of TB status conducted in Debre-Tabor, Ethiopia [28]. (1) Eventually, 305 TB/HIV coinfected patient registers were reviewed in the current study using all consecutive sampling approach.

Study Variables, Tool, Data Quality, and Data Collection
Procedure. A well-organized data abstraction sheet (checklist) was prepared and then modifed considering the most important baseline socio-demographic and clinical characteristics of the target participants. After training on how to review data was given to data collectors, the following data elements were collected from the ART registers using a well standardized checklist: (1) Baseline socio-demographic characteristics of TB/ HIV coinfected adults (sex, age, educational status, marital status, religion, and place of residence). (2) Baseline clinical and laboratory characteristics of the participants, i.e., functional status, WHO clinical stage, Body Mass Index (BMI) in kg/m 2 , serum alanine transferase (ALT) level, serum aspartate transferase (AST) level, and CD 4 + cell count (in cells/mm 3 ). (3) Hemoglobin concentration (g/dl) of TB/HIV coinfected adults. In the current study, hemoglobin was considered as the proxy indicator of anemia to classify the study subjects as nonanemic (hemoglobin ≥12 g/dl for women and ≥13 g/dl for men) or anemic (hemoglobin <12.0 g/dl for women versus <13.0 g/dl for men). Anemia is further classifed in adult men as mild anemia (11.0-12.9 g/dl), moderate anemia (8.0-10.9 g/dl), and severe anemia (<8.0 g/ dl). On the other hand, the classifcation of anemia is slightly diferent in adult women as mild anemia (11.0-11.9 g/dl), moderate anemia (8.0-10.9 g/dl), and severe anemia (<8.0 g/dl) [1]. Tus, anemia status is the outcome variable labeled as 1 if anemic (mild, moderate, or severe) and 0 otherwise (i.e., nonanemic).

Journal of Tropical Medicine
Te variable place of residence is operationalized as urban (i.e., for patients from Mekelle) or rural (i.e., for patients living outside Mekelle). Tus, some participants who were from urban area might be classifed as rural considering the operational defnition.

Statistical Methods of Data Analysis.
First, data were entered, exported, and then cleaned in STATA version 14.2 statistical tool. Te participants were described using frequency and percent. Serum ALT and AST levels, age, and BMI were described using median and interquartile range (IQR). Te adequacy of cell count was also checked using cross-tabulation prior to run the binomial model. Te severity of anemia was presented using bar graph. Model ftness and diagnostics (i.e., collinearity) were checked prior to declare the best ftted model. Te logit model was ftted as a parsimonious model explaining the anemia data very well so that determinants of anemia were identifed according to the fnal logit model ftted in the current study. Finally, the strength of association of each statistically signifcant predictor was measured and interpreted based on the adjusted odds ratio (AOR) and its corresponding 95% confdence interval (CI). For the categorical predictors, the AOR was interpreted in comparison to the reference category for the odds of developing anemia. On the other hand, for the continuous predictor particularly baseline BMI, the AOR was interpreted as a unit increase in the continuous predictor BMI decreases the odds of developing anemia by (1 − AOR) * 100%.

Ethics
Approval and Consent to Participate. Ethics approval was issued by the Institutional Review Board (IRB) of Mekelle University-College of Health Sciences, and the consent to participate was fully waived by the IRB since secondary data were extracted from HIV registers. However, data were kept confdentially, and no personal or identifable information was used in the present study.

Te Severity of TB/HIV-Associated Anemia by Socio-Demographic
Characteristics. In the current study, the overall prevalence of anemia in TB/HIV coinfected males was 68.2% as compared to 50.2% for females. However, the prevalence of severe form of anemia was signifcantly higher in females (7.6%) than in males (4.7%). Te overall prevalence of anemia ranges from 52.1% (i.e., 8.4% severe anemia) in TB/HIV coinfected adults with secondary education to 66.7% (i.e., 5.6% severe) among adults with primary level of education. However, 75% of Muslim TB/HIV coinfected adults reported to have anemia with equal share of the three anemia levels-25% each. Tough there was insignifcant diference in the prevalence of anemia by place of residence, severe anemia had exceeded by 3% in patients from rural areas (Table 1).

Te Severity of TB/HIV-Associated Anemia by Baseline
Clinical Characteristics. Te prevalence of anemia was linearly increased from 46.1% in adults with working functional status to 72.2% in adults with bedridden functional status. Likewise, the severe form of anemia was similarly increased from 2.8% in TB/HIV coinfected adults with working functional status to 14.8% in patients with bedridden functional status. Te anemia prevalence was elevated as the baseline WHO clinical stage of HIV advanced from stage I or II (28.1%) to stage IV (67.6%) where the severe type of anemia was increased from 0% in adults with baseline stage I or II to 9.2% in stage IV. Te prevalence of anemia was almost twice in patients with baseline CD4+ cell count ≤200 cells/mm 3 as compared to patients with CD4+ cell count >200 cells/mm 3 (64.0% versus 36.4%) in which 6.8% of patients with low CD4+ cell count tend to develop severe anemia. Te median baseline BMI was 17.4 kg/m 2 while the median serum ALT and AST levels were 34.5 and 40 IU/l, respectively ( Table 2).

Determinants of TB/HIV-Associated Anemia.
According to the fnal multiple logit model ftted, the baseline determinants of anemia were as follows: sex (female), functional status (ambulatory and bedridden), WHO clinical stage (III and IV), and BMI (kg/m 2 ). Keeping other variables constant, female TB/HIV coinfected adults were protected by at least 35.6% and utmost 77.4% (62.0% on average) (AOR = 0.380; 95% CI: 0.226-0.640) from developing anemia than males. Reversely, the odds of developing anima among TB/HIV coinfected adults with baseline ambulatory functional status, bedridden functional status, WHO clinical stage III and IV were increased by

Discussion
Te current study was conducted to investigate the severity level and determinants of anemia in TB/HIV coinfected adults. Tus, it has been revealed that 59.0% (95% CI: 53.3%-64.6%) of adults coinfected with TB and HIV were anemic, and 6.2% had severe anemia at ART enrollment. Moreover, being female, baseline ambulatory functional status, baseline bedridden functional status, baseline WHO clinical stage III, baseline WHO clinical stage IV, and baseline BMI were identifed as statistically signifcant determinants of baseline anemia in the current study. In this study, the overall prevalence of anemia at baseline was found to be 59.0% (24.6% mild, 28.2% moderate, and 6.2% severe anemia) which almost exactly in line to fndings obtained across Europe though the target groups were HIV patients of HAART naïve [29]. Te current prevalence was higher than other studies that ranged from 18.9% in Uganda to 49.6% in Indonesia [25][26][27][28][30][31][32][33][34]. Reversely, the baseline prevalence was lower than other studies conducted among ART naïve HIV or TB/HIV coinfected patients in Ghana, Nigeria, and Gambia that have been reported 63.0%, 69.2%, and 67.0%, respectively [11,35,36]. Te reasons for the high prevalence of anemia at baseline in the current study are most probably related to the contribution of tuberculosis in speeding up the occurrence of HIV-associated anemia [7][8][9]. On the other hand, the low prevalence when compared to the other three studies could be the diference in operationalizing or defning anemia using hemoglobin level as proxy indicator and diference in ethnicity, geographic location, stage of the HIV infection, presence of other opportunistic infection, and feeding style of the population that might contributing a lot for the inconsistency of anemia prevalence.
In the current study, female sex is negatively statistically associated with the anemia prevalence in adult TB/HIV coinfected patients prior to ART initiation. Te fnding was in agreement with other studies conducted in Benin city (Nigeria) and Zewditu Memorial Hospital (Ethiopia) which have been reported all the low prevalence of anemia in females as compared to male HIV patients of ART naïve or ART experienced [11,25]. However, it is inconsistent with other similar studies that have been reported the direct efect of female sex to anemia [26,32,33,35]. Te main reason for the inconsistent fndings as compared to the current study fnding might be the diference in defnitions of anemia that used diferent cutof values of hemoglobin for males and females across the diferent studies. Of course, logically the prevalence of anemia was expected to be higher on adult HIV and/or TB coinfected women because of the contributions of blood loss during menstruation, pregnancy, and delivery that aggravate the prevalence of anemia [22,23]. Despite this fact, the prevalence of anemia was lower among adult TB/HIV coinfected women than men mainly due to (1) the cutof point for anemia classifcation used for both sexes was not uniform (i.e., a woman with hemoglobin level of 12 g/dl and above is considered as normal whereas a man with hemoglobin level of 13 g/dl and above was considered as normal) and (2) there were other important but uninvestigated variables that might be confounded with sex such as feeding style and iron-folic acid supplementation that prevent the occurrence of anemia. Furthermore, the treatment seeking behavior to anemia and good feeding practices Journal of Tropical Medicine might be well exercised by females than males that most likely enriched during antenatal care (ANC) and postnatal care (PNC) follow-ups. BMI (kg/m 2 ) is also signifcantly negatively predicting the odds of developing anemia among TB/HIV coinfected adults. A unit increase in baseline BMI tends to protect the odds of developing anemia by almost 9% in the current study. Tis fnding was consistent with other studies conducted in South Africa, Uganda, and Rwanda which all have supported the inverse relationship between baseline BMI and anemia [30,33,37]. However, the socio-demographic variable sex was fxed in the Rwandan study where the participants were only adult women. It is obvious that nutritional defciencies including iron, folic acid, and vitamin B 12 play a major role in causing nutrition-related anemia in HIV and/or TB infected/coinfected adults [5,6].
Both baseline ambulatory and bedridden functional status of adult HIV patients were statistically positively associated with the odds of developing TB/HIV-associated anemia. Tough literature on this variable was scarce, it is clear that patients with advanced functional status (i.e., ambulatory and bedridden) might tend to be afected by diferent opportunistic infections that could negatively afect iron concentration and absorption in which iron defciency anemia could account for the highest share of anemia in the current study.
Similarly, HIV adults of ART naïve with WHO clinical stage III and IV were more likely to develop TB/HIVassociated anemia than those with clinical stage I/II. Te result of the current study was supported by the fndings of other studies conducted in Uganda and South Africa which have been reported that the odds of developing anemia was increasing as patients clinical stage advanced from stage I/II to III and then IV [32,33]. Te key reasons could be as HIV infection was advanced from the early stages (I or II) to III/ IV-there would be marked weight loss (>10% of total weight), consistent diarrhea, opportunistic infections and then advancement to AIDS defning illness, and other complications [38]. Tus, all these advanced infections and complications during the advanced clinical stages (III and IV) might lead to severe anemia in particular and anemia in general following excessive fuid loss due to prolonged diarrhea in stage-III and occurrence of diferent opportunistic infections in stage-IV mostly that might suppress the immune system ultimately.
Despite an appropriate study designed, the current study was not free of limitations. Te study population were considered as TB/HIV coinfected adults at baseline regardless of the exact time of TB development. Te pregnancy status of women at baseline was not known, and thus, the anemia classifcation did not consider pregnancy status for women that could underestimate the prevalence of anemia in female participants. Most literature used in the current study was HIV-associated anemia irrespective of TB status at enrollment since studies assessed TB/HIV-associated anemia were scarce.

Conclusions
In the current study, approximately six in ten TB/HIV coinfected adults had laboratory evidence of anemia with almost one-ninth, and half of the anemia cases were severe and moderate anemia, respectively, at baseline. Low baseline BMI, ambulatory functional status, bedridden functional status, WHO clinical stages III and IV increased the odd of developing TB/HIV-associated anemia. Tus, curious attention has to be given for severe anemia in particular, and it has to be managed accordingly so as to limit anemia/severe anemia associated bad outcomes of TB/HIV coinfection most importantly death.

Data Availability
Te datasets used to support the fndings of this study are available from the corresponding author uponrequest.

Disclosure
A preprint has previously been published [39].

Conflicts of Interest
Te authors declare that they have no conficts of interest.