Prevalence and predictors of anemia among adults on highly active antiretroviral therapy in Northeast Ethiopia: A retrospective cohort study.

BACKGROUND: Although antiretroviral therapy has significantly altered the natural history of human immunodeficiency virus infection and improved the quality of life of patients, there are conflicting reports regarding its impact on hematological outcomes. Thus, this study aimed at investigating the prevalence and predictors of anemia among adults on antiretroviral therapy in Northeast Ethiopia.
MATERIALS AND METHODS: A retrospective cohort study was carried out among adults who began antiretroviral treatment between September 2005 and January 2019 at two governmental hospitals in Dessie town. Data were collected from patients' medical records using a pretested data extraction instrument. Anemia was the primary outcome variable of the study. It was defined based on WHO criteria after adjustment for altitude and smoking status of measured values. Data were entered and validated using EpiData Version 3.1 and then exported to SPSS Version 20.0 for analysis. Descriptive analysis was done for prevalence and binary logistic regression was carried out to assess whether covariates were associated with experiencing anemia. Statistical significance has been considered at p-value <0.05.
RESULTS: Medical records of 392 patients (mean age: 35.58 ± 9.46 years) were reviewed. Of the total 392 patients, 218 (55.6%) were females, 261 (66.6%) were categorized under WHO clinical stage III/IV and 134 (34.2%) had a baseline CD4 cell count of <100 cells/mm3. The mean baseline CD4 cell count was 179 cells/mm3 (range: 2 to 853 cells) and 230 (58.7%) of the participants were on zidovudine-based regimen. Anemia was diagnosed among 162 (41.3%) patients. After adjustment for other confounding factors, risk of anemia was significantly associated with low baseline CD4 cell count (AOR 1.80, 95% CI 1.05-3.06) and tenofovir based regimen (AOR 2.05, 95% CI 1.31-3.21). On the other hand, being educated was found to be protective (AOR 0.40, 95% CI 0.21-0.78).
CONCLUSION: In this research, the prevalence of anemia was relatively high. Low baseline CD4 cell count and tenofovir based regimen were independent predictors of anemia; while being educated was protective. Treatment programs should focus on early diagnosis and treatment of HIV as well as routine screening and proper treatment of anemia.

Introduction

The introduction of highly active antiretroviral therapy (HAART) in 1996 and the increasing access to these potent antiretroviral drugs has dramatically improved the rate of HIV disease progression and mortality [1]. However, adverse drug events like hematologic toxicities remain as common causes of morbidity and mortality in people receiving HAART [2, 3]. Anemia is the most commonly reported hematologic abnormality with substantial impact on quality of life and clinical outcomes like HIV disease progression [4-7].

Studies have consistently shown that the prevalence of anemia is high among people living with HIV, particularly among those with advanced disease stage [8, 9]. The prevalence of anemia during the HAART era greatly varies depending on region, clinical settings and threshold used to define anemia [5, 10]. The estimated prevalence of anemia among adults receiving HAART was 35% in Europe and North America [11]; 39.2% in China [12] and 66.7% in Nepal [13]. Other reports also revealed that the prevalence of anemia ranges from 23 to 50% globally and 24 to 58% in Africa [3, 14].

Review of the literature demonstrated high prevalence of anemia among HIV infected adults in Ethiopia with pooled prevalence of 31% [15]. Studies conducted at different times from different parts of the country reported that the prevalence of anemia ranges from 11.4 to 70.1% [16-21]. Accordingly, it was estimated and reported as 23 to 70.1% in Northwest Ethiopia [21]; 36.5 to 53.3% in Southern Ethiopia [17]; 11.4 to 34.6% in Addis Ababa [16, 18, 19]; and 41.2% in Eastern Ethiopia [20].

Anemia in HIV-infected individuals has been associated with reduced quality of life, rapid disease progression, and increased mortality [4, 5, 22–25]. Analysis of a multi-centered data in Europe demonstrated that a 1 g/dL reduction in Hb raised the hazard of death by 57%, after controlling viral load and CD4 lymphocyte count. Accordingly, the latest hemoglobin value was an independent prognostic factor for death [23]. Mortality was higher in the first year following initiation of HAART, with anemia being one of the associated factors suggesting the need for routine screening and treatment of anemia [23, 26]. Anemia has also been associated with rapid progression to AIDS and included in the disease-progression scoring system for patients on HAART [5, 25].

Although HAART has generally been accepted as the gold standard in HIV care [27], there are inconsistent reports regarding its impact on hematological complications [28]. Whereas a number of studies reported that highly active antiretroviral therapy could reduce anemia, others reported no improvement or even anemia as an adverse drug reaction of the antiretroviral therapy [27, 29–34].

Anemia in HIV infection has been reported to be multifactorial being the result of a number of interactive factors making it difficult to determine causality [35, 36]. Antiretroviral and other drugs are also known to cause anemia [36-38]. Factors often reported to increase risk anemia include ART regimens containing zidovudine, more advanced disease stage or low CD4+ count, nutritional status or body mass index <18.5 kg/M2, female sex, duration of HAART, presence of opportunistic infections, and low baseline hemoglobin level [8, 12, 13, 15–18, 21, 30, 39–42].

Previous studies investigated the prevalence and predictors of anemia among HIV infected patients in general. Moreover, its prevalence and predictors vary by setting and population among others. However, there is insufficient data about anemia after initiation of HAART among adults in northeast Ethiopia. Thus, this study aimed at filling this gap by investigating the prevalence and predictors of anemia among adults on HAART.

Materials and methods

Study design, area and period

A retrospective cohort study was carried out from September 2018 to January 2019 among adults on a standard antiretroviral treatment at ART clinics of Boru Meda and Dessie Referral Hospitals. These are the only governmental hospitals in Dessie town serving majority of clients from the Northeast part of the country. Dessie is located 400 km to the north of the capital Addis Ababa with an altitude between 2,470 and 2,550 meters above sea level. Besides testing and treatment of HIV/AIDS, the hospitals also provide general outpatient and inpatient services including medical, surgical, pediatric, psychiatric, Ophthalmic, Emergency, Gynecology and Obstetrics care.

Study population and eligibility criteria

All HIV-positive patients aged 15+ who began ART between September 2005 and January 2018 and were treated for at least 3 months with pre- and post-ART hemoglobin values were eligible for study enrolment. On the other hand, patients who were already anemic at the time of ART initiation, transfer in/out cases, lost to follow up and those with incomplete medical records were excluded.

Sample size determination and sampling procedure

Sample size was computed using single population proportion formula based on the following assumptions: true population proportion or estimated prevalence (P) = 0.5, absolute precision (d) = 0.05, and 95% confidence level. Because there was no previous similar study in the area, the true proportion of the population was assumed to be 50%. To account for incomplete medical records, a 2% contingency was added. Thus, the final sample size was 392. Participants were enrolled based on their refill appointment; thus, simple random sampling technique was used.

Study variables

Anemia following at least three months of antiretroviral treatment, was the primary outcome variable of the study. Anemia was defined based on WHO criteria as a hemoglobin concentration of <12 g/dl for non-pregnant women and <13 g/dl for men after adjustment for altitude and smoking status of measured values [43]. Accordingly, 0.16 g/dL and 0.13 g/dL were subtracted from measured values for smokers and non-smokers, respectively. It was further classified as mild (11–11.9 g/dl for women and 11–12.9 g/dl for men), moderate (8–10.9 g/dl) and severe (<8 g/dl in both genders). Socio-demographic characteristics (age, gender, educational level), nutritional status, tuberculosis coinfection, WHO clinical stage, baseline CD4 cell count, ART regimen, adherence, isoniazid and co-trimoxazole preventive therapies were the predictor variables.

Data collection procedure

Socio-demographic characteristics, baseline and follow up clinical and laboratory data, as well as treatment outcomes were extracted from patients’ medical records using a structured and pretested data abstraction format. Unique ART numbers were used for the identification of individual participants. The data abstraction tool has been adopted from ART registry and follow up forms of ART clinics and pretested on transferred-in cases before use. Patient ART registers, follow up medical records and the electronic dispensing database were used as data sources. Laboratory test reports were also used to collect hematologic, virologic and immunologic results. World health organization (WHO) cut off values of hemoglobin were taken after adjustment to altitude and smoking status to define and classify anemia. Study participants were retrospectively followed from the date of enrolment to treatment initiation.

Data quality assurance

Pretest for the applicability of the data collection tool was done and training was given for the data collectors and supervisors before the commencement of the data collection procedure. Data were checked for completeness daily, coded, cleaned and verified before entry.

Data processing and analysis

Data were entered using EpiData Version 3.1 and exported to SPSS Version 20.0 for analysis. Descriptive statistics were used for the analysis of baseline sociodemographic and clinical characteristics. Descriptive analysis was done for prevalence and binary logistic regression models were used to identify the predictors of anemia. The following variables were assessed as potential predictors: gender, age, residence, educational status, nutritional status, WHO clinical stage, tuberculosis coinfection, ART regimen, and baseline CD4 cell count. Odds ratios (OR) with the 95% confidence intervals were reported and statistical significance was considered at p-value <0.05.

Ethical considerations

The study was carried out following approval by the institutional review committee of Wollo University and a subsequent permission from each hospital. Informed consent was waived by the Institutional Review Committee of the University and the hospitals due to the retrospective nature of the study, as all the data were collected from routine medical records. Confidentiality was guaranteed by omitting names or any personal identifiers. In addition, data were kept secured via out the research process to limit accessibility to a third party.

Results

Patient characteristics at enrollment

Medical records of 392 patients (mean age: 35.58 ± 9.46; range: 15–80) years were reviewed. Of the total 392 patients, 218 (55.6%) were females; 200 (51%) were married; 152 (38.8%) completed primary school; 214 (54.6%) were rural dwellers; 130 (33.2%) had a body mass index of <18.5 kg/m2; 62 (15.8%) use social drugs like Khat, alcohol, or cigarette; 59 (15.1%) were tuberculosis coinfected; and 233 (59.4%) of them were on their initial ART regimen.

Clinically, 261 (66.6%) patients were categorized under WHO stage III/IV disease and 134 (34.2%) of them had a baseline CD4 cell count of <100 cells/mm3 with mean cell count of 179 cells/mm3 (range:2–853). Regarding standard prophylaxis against common opportunistic infections, 354 (90.3%) and 275 (70.2%) of the participants were treated with cotrimoxazole and isoniazid preventive therapies, respectively. The predominant ART regimen was Zidovudine + Lamivudine + Nevirapine and 230 (58.7%) of all participants were on Zidovudine based regimen (Table 1).

Table 1

Baseline characteristics of patients on HAART in Northeast Ethiopia: 2005–2019.

		Antiretroviral therapy Regimen				Total
		AZT/3TC/NVP	AZT/3TC/EFV	TDF/3TC/EFV	TDF/3TC/NVP
Sex	Male	52	44	64	14	174 (44.4%)
	Female	108	26	57	27	218 (55.6%)
Age; mean: 35.58	≤35 years	103	27	64	21	215 (54.8%)
	>35 years	57	43	57	20	177 (45.2%)
Marital Status	Never Married	30	17	23	11	81 (20.7%)
	Married	75	35	69	21	200 (51%)
	Widowed	18	9	13	3	43 (11%)
	Divorced	37	9	16	6	68 (17.3%)
Education Level	Illiterate	50	19	23	13	105 (26.8%)
	Primary school	60	33	45	14	152 (38.8%)
	High school	41	16	33	6	96 (24.5%)
	College/above	9	2	20	8	39 (9.9%)
Residence	Rural	98	37	58	21	214 (54.6%)
	Urban	62	33	63	20	178 (45.4%)
Social drug use	None	138	58	98	36	330 (84.2%)
	Yes	22	12	23	5	62 (15.8%)
Tuberculosis test	Negative	138	52	105	38	333 (84.9%)
	Positive	22	18	16	3	59 (15.1%)
Experience to ART	Naïve*	90	48	86	9	233 (59.4%)
	Non-naïve	70	22	35	32	159 (40.6%)
WHO Clinical Stage	Stage I	11	2	16	4	33 (8.4%)
	Stage II	43	16	28	11	98 (25.0%)
	Stage III	95	43	61	24	223 (56.9%)
	Stage IV	11	9	16	2	38 (9.7%)
CD4 cell count in cells/ml3; (Mean: 179)	>200	43	24	49	14	130 (33.2%)
	100–200	56	18	38	16	128 (32.7%)
	<100	61	28	34	11	134 (34.2%)
Body Mass Index	<18.5 kg/m2	65	22	35	8	130 (33.2%)
	18.5–25 kg/m2	83	48	79	30	240 (61.2%)
	>25 kg/m2	12	0	7	3	22 (5.6%)
Cotrimoxazole Prophylaxis	No	8	10	13	7	38 (9.7%)
	Yes	152	60	108	34	354 (90.3%)
Received Isoniazid	No	43	26	36	12	117 (29.8%)
	Yes	117	44	85	29	275 (70.2%)
Total		160 (40.8%)	70 (17.9%)	121 (30.9%)	41 (10.5%)	392

Abbreviations: NVP, nevirapine; EFV, efavirenz; AZT, zidovudine; TDF, tenofovir; 3TC, lamivudine;

* were on their initial ART regimen

Interim treatment outcomes

Of the total 392 study participants, 164 (41.8%) had experienced at least one adverse drug reaction; 326 (83.2%) had good adherence to their prescribed medication; 314 (80.1%) achieved their virologic goal with undetectable viral load and 162 (41.3%) became anemic; of which 11 (6.8%) patients had experienced severe anemia requiring regimen change. The mean duration of follow-up on HAART was 76 ± 32.5 months (range: 7 to 150 months) (Table 2).

Table 2

Interim treatment outcome among adults on HAART in Northeast Ethiopia: 2005–2019.

		Antiretroviral therapy Regimen				Total
		AZT/3TC/NVP	AZT/3TC/EFV	TDF/3TC/EFV	TDF/3TC/NVP
Adherence	Good	131	52	106	37	326 (83.2%)
	Fair/poor	29	18	15	4	66 (16.8%)
Adverse Drug Reaction	No	89	45	84	10	228 (58.2%)
	Yes*	71	25	37	31	164 (41.8%)
Recent Retroviral Load	Not done	19	6	7	2	34 (8.7%)
	Undetectable	123	53	99	39	314 (83.1%)
	Detectable**	18	11	15	0	44 (11.2%)
New onset Anemia	No	98	49	59	24	230 (58.7%)
	Yes	62	21	62	17	162 (41.3%)
Duration of follow up in months (mean:76)	<61	28	12	79	12	131 (33.4%)
	61–90	64	12	30	25	131 (33.4%)
	>90	68	46	12	4	130 (33.2%)
Severity of anemia (n: 162)	Mild	38	10	38	4	90 (55.6%)
	Moderate	23	10	20	8	61 (37.6%)
	Severe	1	1	4	5	11 (6.8%)

* Lipodystrophy, anemia, Central Nervous System disturbance, and/or rash,

** >1000 copies/ml

Predictors of anemia

Bivariate analysis of sociodemographic and clinical variables revealed that low baseline CD4 cell count (crude odds ratio (COR) 1.66, 95% confidence interval (CI) 1.01–2.74) and TDF based ART regimen (COR 1.69, 95% CI 1.12–2.54) were associated with the risk of anemia. On the other hand, having education level of high school was found to be protective (COR 0.46, 95% CI 0.26–0.83). Similarly, multivariate logistic regression analysis confirmed that baseline CD4 cell count of 100–200 cells/mm3 (adjusted odds ratio (AOR) 1.80, 95% CI 1.05–3.06 and TDF based ART regimen (AOR 2.05, 95% CI 1.31–3.21) were significant predictors of anemia; and being educated was found to be protective (AOR 0.40, 95% CI 0.21–0.78) (Table 3).

Table 3

Possible predictors of anemia among adults on HAART in Northeast Ethiopia: 2005–2019.

Covariates	Categories	Anemia frequency (%)	COR (95% C.I.)	AOR (95% C.I.)
Sex	Male	66 (37.9%)	1.00	1.00
	Female	96 (44.0%)	1.29 (0.86, 1.93)	1.22 (0.77, 1.92)
Age, mean: 35.58	≤35 years	93 (43.3%)	1.00	1.00
	>35 years	69 (39.0%)	0.84 (0.56, 1.26)	0.77 (0.49, 1.20)
Body mass index	18.5–25 kg/m2	102 (42.5%)	1.00	1.00
	<18.5 kg/m2	52 (40.0%)	0.90 (0.58, 1.39)	0.88 (0.55, 1.40)
	>25 kg/m2	8 (36.4%)	0.77 (0.31, 1.91)	0.97 (0.36, 2.59)
Residence	Urban	71 (39.9%)	1.00	1.00
	Rural	91 (42.5%)	1.12 (0.74, 1.67)	0.96 (0.60, 1.53)
Education Level	Illiterate	52 (49.5%)	1.00	1.00
	Primary school	65 (42.8%)	0.76 (0.46, 1.26)	0.71 (0.41, 1.22)
	High school	30 (31.2%)	0.46 (0.26, 0.83) *	0.40 (0.21, 0.78) *
	College & above	15 (38.5%)	0.64 (0.30, 1.35)	0.46 (0.20, 1.07)
TB co-infection	No	136 (40.8%)	1.00	1.00
	Yes	26 (44.1%)	1.14 (0.65, 1.99)	1.01 (0.54, 1.90)
Experience to ART	Naïve	92 (39.5%)	1.00	1.00
	Non-naïve	70 (44.0%)	1.21 (0.80, 1.81)	1.08 (0.69, 1.69)
Baseline CD4 count	>200 cells/mm3	46 (35.4%)	1.00	1.00
	100–200 cells/mm3	61 (47.7%)	1.66 (1.01, 2.74) *	1.80 (1.05, 3.06) *
	<100 cells/mm3	55 (41.0%)	1.27 (0.77, 2.09)	1.43 (0.83, 2.47)
WHO clinical stage	Stage I or II	52 (39.7%)	1.00	1.00
	Stage III or IV	110 (42.1%)	1.11 (0.72, 1.70)	1.30 (0.80, 2.10)
ART Regimen	AZT Based	83 (36.1%)	1.00	1.00
	TDF Based	79 (48.8%)	1.69 (1.12, 2.54) *	2.05 (1.31, 3.21) *
Adherence	Good	132 (40.5%)	1.00	1.00
	Fair/Poor	30 (45.5%)	1.23 (0.72, 2.09)	1.24 (0.70, 2.19)
Isoniazid prophylaxis	No	55 (47.0%)	1.39 (0.90, 2.16)	1.59 (0.98, 2.57)
	Yes	107 (38.9%)	1.00	1.00
Co-trimoxazole prophylaxis	No	15 (39.5%)	1.00	1.00
	Yes	147 (41.5%)	1.09 (0.55, 2.16)	1.18 (0.58, 2.44)

* Significantly associated with anemia

Patients with baseline CD4 cell count of 100–200 cells/mm3 were about twice at risk of being anemic compared to those with CD4 cell count of >200 cells/mm3 (AOR 1.80, 95% CI 1.05–3.06). Surprisingly, the risk of experiencing anemia was two times (AOR 2.05, 95% CI 1.31–3.21) among individuals on TDF based ART regimen than those who were on AZT based ART regimen. Patients who were educated up to high school were about 60% less at risk of facing anemia compared to those individuals who didn’t attend school (AOR 0.40, 95% CI 0.21–0.78). However, no statistically significant association was found between anemia and the following covariates: sex, age, residency, body mass index, tuberculosis coinfection, WHO clinical stage, being naïve or non-naïve to ART, adherence, isoniazid and cotrimoxazole preventive therapies.

Discussion

In the HAART era, anemia is still common and independently associated with increased mortality. Correction of anemia was associated with reversal of this increased risk. Identification of individuals at risk is crucial for early detection and appropriate management of antiretroviral toxicities like anemia. This study was aimed to investigate the prevalence and associated factors of anemia among adults on highly active antiretroviral therapy.

According to this study, the prevalence of anemia was 41.3% (95% CI: 36–46%) showing that anemia is still prevalent among patients receiving HAART. This finding was comparable with reports from Southern Ethiopia 36.5% [39], Uganda 47.8% [44] and China 39.2% [12]. Our finding was significantly lower than that of prior studies conducted in Uganda 67.4% [45] and Nepal 66.7% [13]. However; this result was higher than reports from different parts of Ethiopia like Southern Ethiopia 34.8% [17], Addis Ababa 34.6% [16], Northwest Ethiopia 23–34% [21, 40, 46], and a national pooled prevalence of anemia 31.0% [15]. The observed discrepancy might be attributed to the differences in socioeconomic status, health literacy, study design, sample size, inclusion criteria, and most importantly the difference in cut-off values that might be used to define anemia.

Regarding the severity of anemia, majority of cases were mild (55.6%) with the remaining 37.6% to be moderate and only 6.8% of them were severe requiring regimen change. This finding was consistent with a report from rural China where 69.4%, 27.5% and 3.1% of all anemic patients were experiencing mild, moderate and severe anemia, respectively [12]. The prevalence of severe anemia was lower compared to that of previous studies conducted in Southern Ethiopia (21.8%) [17], Addis Ababa (14.5%) [16] and Nepal (17.8%) [13]. However, it was relatively higher than a report form Northwest Ethiopia 2.5% [46].

Multivariate logistic regression analysis shows that TDF based ART regimen and advanced disease (low baseline CD4 cell count) were independent predictors of anemia. Patients with baseline CD4 cells count of 100–200 cells/mm3 were approximately twice more likely to be anemic compared to those individuals with a CD4 count of >200 cells/mm3 (AOR 1.80, 95% CI 1.05–3.06). Similarly, studies conducted in Northwest Ethiopia [21, 40, 41, 46], Nepal [13], Uganda [44], and China [12] showed that low CD4 cell count was significantly associated with anemia. This was also supported by the result of a systematic review and meta-analysis done in Ethiopia [15]. The possible explanation for this association may be opportunistic infections, malnutrition, or HIV associated myelosuppression with advanced disease.

In this study, using Zidovudine (AZT) based ART regimen did not show significant association with anemia despite its frequent association with bone marrow suppression. Unusually, patients who took TDF-based ART regimens were twice more likely to be anemic than those who took AZT-based regimens (AOR 2.05, 95% CI 1.31–3.21). Although a similar finding was reported in Addis Ababa, Ethiopia [18], it is inconsistent with the finding of studies conducted elsewhere in Northwest Ethiopia [21, 40, 41]. Though it needs further investigation, it could be explained by anemia as a complication of TDF induced renal impairment.

This study also identified educational status as predictor of anemia among the study subjects. Patients who never attended school were more likely to suffer from anemia compared to those who completed high school. On the other hand, being educated up to high school was found to be protective (AOR 0.40, 95% CI 0.21–0.78) probably due to good health-seeking behavior or healthy diet patterns. The finding is supported by a report from Northwest Ethiopia [21].

However, no statistically significant association was observed between anemia and the following covariates: sex, age, residency, body mass index, tuberculosis coinfection, WHO clinical stage, being naïve or non-naïve to ART, adherence, as well as isoniazid and cotrimoxazole preventive therapies. These were inconsistent with the findings of previous studies done around the globe [12, 13, 15, 16, 21, 40, 41, 44–46]. Since the causes of anemia in HIV patients are multi-factorial, we recognize the inability of a single study to identify its specific causes exhaustively. Our study was not without limitation. The retrospective nature of this study might be a limitation as the accuracy of data depends on the completeness the records, and hence, information bias might have occurred because of underreporting/missing data elements.

Conclusions

In this study, the prevalence of anemia was relatively high though previous studies reported a significant association between HAART and increment in hemoglobin. Low baseline CD4 cell count and TDF based ART regimen were found to be independent predictors of anemia; while being educated was protective. ART programs should focus on early diagnosis and treatment of HIV as well as routine screening and proper treatment of anemia. Further studies are needed to identify prospectively patients at high risk and design interventions aimed to reduce this risk.

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10 Dec 2021

Prevalence and predictors of anemia among adults on highly active antiretroviral therapy in Northeast Ethiopia: a retrospective cohort study

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The duration of the study is between September 2005 to January 2018 in the abstract but its September 2005 to January 2019 in the methodology. Which is which?

It’s quite difficult to understand when the hemoglobin measurement was taken. The paper does not clearly state the timelines for hemoglobin measurements.

The stratification of the patients is not clear. The authors need to clearly define how their subjects were selected; what parameter was measured at what time. Was there baseline measurements and subsequent measurements of hemoglobin? Were all the subjects on ART at certain point? What was the point of including the naïve subjects when the work was clearly looking at effect of HAART on hemoglobin levels?

Anemia is multifactorial in nature. How were the authors able to control for other confounders which could affect the level of hemoglobin?

Why was the TDF based ART regimen associated with anemia in the study? No reason was assigned to this in the discussion.

Why was there no bivariate analysis between duration of HAART and the risk of developing anemia?

Reviewer #2: Reviewer Report to the author (s)

This MS might give important information about Prevalence and predictors of anemia among adults on highly active antiretroviral therapy for clinicians and for the scientific community. But the authors need to address the following points before publication:

�  The authors name in the cover page can be rewrite as “Teklehaimanot Fentie Wendie1*, Kassahun Bogale1, Getnet Mengistu1”

�  The corresponding author Email address in the cover page can be modifies as :

E-mail: tklhmntfnt5@gmail.com(TF) (Teklehaimanot.fentie@wu.edu.et)

�  In the abstract, method subsection, the authors need to indicate the specific study sites at the end of the study period: A retrospective cohort study was carried out among adults who began highly active antiretroviral treatment between September 2005 and January 2018 in ----------.

�  From the abstract, the authors need to avoid the use of abbreviations (Eg. HARRT, ART, TDF, etc) before indicating their full names.

�  In this MS, there is unnecessary excessive use of references throughout the document after single sentences. The authors need to be specific and better to use only recent and relevant one or two references after a sentence of paragraphs.

�  The introduction part needs some rearrangements for simplicity for readers. The authors first indicate the global burden of anemia on HIV positive individuals, then in African and Ethiopia and the study area particularly.

�  The title or the subtitles may not be necessarily indicated on separate pages. For example, the methodology part need to be indicated immediately after introduction part in the same page.

�  The title “Method and participants” can be modified as “Methodology” or “Methods and Materials”. Based on this modification, the subtitle “methods” in the abstract section can be modified.

�  In your methodology part, the study area is not well elaborated. In addition, the authors didn’t clearly indicate the reason why only two government hospitals were selected among the others.

�  Since your title focuses to see the condition of anemia among HIV positive individuals who are on HAART, it is not clear the reason why you include study participants started from the age of 15 years. In Ethiopian context, the word “adult” refers individuals’ grater or equal to 18 years. Please reconsider it.

�  Based on your sample size assumptions, the calculated sample size will be around 383(4). But as indicated in your result, the final sample size was 392. It needs justification.

�  The hemoglobin level was evaluated after adjusting the attitude and smoking status. But it is not clearly indicated in the methods part. It seems theoretical assumption.

�  It is better to indicate where the pretest of the data extraction tool was tested. It is not clearly indicated.

�  In your ‘ethical consideration” subtitle, the phrase “….in compliance with the Helsinki Declaration on the ethical principles of medical research involving human subject…” is not important. Better to be deleted.

�  Since your title is among adults on highly active antiretroviral therapy, how 233 (59.4%) of them were naïve to ART in your result? It is very confusing.

�  In result at the time of enrollment, what was the prevalence of anemia? It is not indicated. But is very important for comparison to see the prevalence of anemia before and after the initiation of ART.

�  It is better to insert the respective tables immediately after the text it refers.

�  Indicating the number of study participants (N=392) at the end of the titles of the table is not convincing for the reader; it is better to be deleted.

�  Since the authors already indicated the meaning of “COR” and “AOR” in “Predictors of anemia” subtitle, no need of indicating them at the end of table 3 as key word. This concept can also hold true for the other abbreviations indicated in the respective tables.

�  In your discussion part, second paragraph, the phrase “….defined as Hgb concentration <13 g/dl for males and <12 g/dl for females after adjustment for altitude and smoking status of measured values…” is not important. It is almost reputation. Better to be deleted.

�  When you compare results in discussion, you need to have 95% CI in your result. For example the prevalence of anemia in your result is 41.3%. But you didn’t indicate its 95%CI. Hence it is very difficult to say comparable, lower or higher than other reports. This can be true for other variables that you compare.

�  From your conclusion, avoid unnecessary explanations. For example avoid “(100 - 200 cells/mm3)”

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: Yes: Christian Obirikorang

Reviewer #2: Yes: Dr. Yitayih Wondimeneh

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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Submitted filename: Comments to PLOS One.docx

Click here for additional data file.

Submitted filename: Reviewer Report.docx

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23 Jan 2022

Author's Response to reviewers & editorial corrections

Title: "Prevalence and predictors of anemia among adults on highly active antiretroviral therapy in Northeast Ethiopia: a retrospective cohort study" (Manuscript ID: PONE-D-21-25369)

Authors:

Teklehaimanot Fentie Wendie (tklhmntfnt5@gmail.com)

Getnet Mengistu (mgetnet12@gmail.com)

Thank you for consideration of our manuscript for publication in your journal and our sincere gratitude goes to your esteemed reviewers. We have revised the above manuscript as per the comments given by the Reviewers and the requested Editorial corrections.

Journal Requirements:

�  Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

o We have prepared (edited) the manuscript using PLOS ONE style templates.

�  In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter.

o The raw data of the study was uploaded as Supporting Information file 1. This is stated in the data availability statement of the manuscript.

�  Regarding the occurrence of overlapping text with a previous publication and the need for language proof reading,

o A major revision was made as needed.

Reviewers' comments:

Reviewer #1:

�  The duration of the study is between September 2005 to January 2018 in the abstract but its September 2005 to January 2019 in the methodology. Which is which?

o Sorry for the editorial error! It is January 2019.

�  It’s quite difficult to understand when the hemoglobin measurement was taken. The paper does not clearly state the timelines for hemoglobin measurements.

o Baseline (at time of ART initiation) and recent follow up hemoglobin measurements were taken and adjusted for altitude and smoking status based on WHO recommendations to determine whether the patient became anemic or not.

�  The stratification of the patients is not clear. The authors need to clearly define how their subjects were selected; what parameter was measured at what time.

o Was there baseline measurements and subsequent measurements of hemoglobin? Yes

o Were all the subjects on ART at certain point? Yes

o What was the point of including the naïve subjects when the work was clearly looking at effect of HAART on hemoglobin levels? The expression is somewhat confusing. Actually, the intention was to mean “were not on any ART regimen other than the current regimen” not to mean that “they are not on ART”. This is restated in the main text as “were on their initial ART regimen”.

�  Anemia is multifactorial in nature. How were the authors able to control for other confounders which could affect the level of hemoglobin?

o As much as possible, we have tried to incorporate all potential predictors of anemia like gender, age, residence, educational status, nutritional status, WHO clinical stage, tuberculosis coinfection, ART regimen, and baseline CD4 cell count and binary logistic regression models were used to control possible confounders and identify the predictors of anemia.

�  Why was the TDF based ART regimen associated with anemia in the study? No reason was assigned to this in the discussion.

o This finding was also a surprise to the authors. Though it needs further investigation, it could be explained by anemia as a complication of TDF induced renal impairment.

�  Why was there no bivariate analysis between duration of HAART and the risk of developing anemia?

o Duration of HAART was assessed, but only those covariates with P-value of less than or equal to 0.25 were included in the multivariate analysis.

Reviewer #2: Reviewer Report to the author (s)

�  The authors name in the cover page can be rewrite as “Teklehaimanot Fentie Wendie1*, Kassahun Bogale1, Getnet Mengistu1”

o Rewritten as suggested.

�  The corresponding author Email address in the cover page can be modifies as :

E-mail: tklhmntfnt5@gmail.com (TF) (Teklehaimanot.fentie@wu.edu.et)

o Comment accepted

�  In the abstract, method subsection, the authors need to indicate the specific study sites at the end of the study period: A retrospective cohort study was carried out among adults who began highly active antiretroviral treatment between September 2005 and January 2018 in ----------.

o Inserted accordingly (….at two governmental hospitals in Dessie town)

�  From the abstract, the authors need to avoid the use of abbreviations (Eg. HARRT, ART, TDF, etc) before indicating their full names.

o Edited

�  In this MS, there is unnecessary excessive use of references throughout the document after single sentences. The authors need to be specific and better to use only recent and relevant one or two references after a sentence of paragraphs.

o Thank you very much for your valuable comments; we have tried to limit the number of references by retaining only the pertinent ones. As can be seen in the main text, the number of references is reduced from 72 to 46.

�  The introduction part needs some rearrangements for simplicity for readers. The authors first indicate the global burden of anemia on HIV positive individuals, then in African and Ethiopia and the study area particularly.

o The introduction is stratified by prevalence, impact and predictors of anemia and paragraphs are structured from global to local.

�  The title or the subtitles may not be necessarily indicated on separate pages. For example, the methodology part need to be indicated immediately after introduction part in the same page.

o Comment accepted

�  The title “Method and participants” can be modified as “Methodology” or “Methods and Materials”. Based on this modification, the subtitle “methods” in the abstract section can be modified.

o Modified as “Materials and methods”.

�  In your methodology part, the study area is not well elaborated. In addition, the authors didn’t clearly indicate the reason why only two government hospitals were selected among the others.

o We have revised this section. The reason why only two government hospitals were selected among the others is that HIV testing and treatment is mainly provided in government hospitals and these are the only government hospitals serving majority of the clients from the Northeast part of the country.

�  Since your title focuses to see the condition of anemia among HIV positive individuals who are on HAART, it is not clear the reason why you include study participants started from the age of 15 years. In Ethiopian context, the word “adult” refers individuals’ grater or equal to 18 years. Please reconsider it.

o Individuals grater or equal to 18 years are considered adults for “legal issues” but for “health perspectives” especially in ART programs, individuals greater or equal to 15 years are treated as adults per WHO and national ART guidelines.

�  Based on your sample size assumptions, the calculated sample size will be around 383(4). But as indicated in your result, the final sample size was 392. It needs justification.

o Right; the calculated sample size is 384. But, the final sample size was 392 because a 2% contingency was added to account for incomplete medical records.

�  The hemoglobin level was evaluated after adjusting the attitude and smoking status. But it is not clearly indicated in the methods part. It seems theoretical assumption.

o Measured haemoglobin concentrations were adjusted for altitude and smoking status based on WHO recommendations (1). Accordingly, 0.16 g/dL and 0.13 g/dL were subtracted from measured values for smokers and non-smokers, respectively. This is incorporated in the method. Remember that the study area has an altitude between 2,470 and 2,550 meters above sea level.

�  It is better to indicate where the pretest of the data extraction tool was tested. It is not clearly indicated.

o The data collection tool was pretested on transferred-in cases as already stated in the data collection procedure.

�  In your ‘ethical consideration” subtitle, the phrase “….in compliance with the Helsinki Declaration on the ethical principles of medical research involving human subject…” is not important. Better to be deleted.

o Comment accepted

�  Since your title is among adults on highly active antiretroviral therapy, how 233 (59.4%) of them were naïve to ART in your result? It is very confusing.

o “naïve to ART” is to mean “were not on any ART regimen other than the current regimen” not to mean that “they are not on ART”. This is restated as “were on their initial ART regimen”.

�  In result at the time of enrolment, what was the prevalence of anemia? It is not indicated. But is very important for comparison to see the prevalence of anemia before and after the initiation of ART.

o The intention of the study was to investigate the prevalence of anemia after the initiation of ART. So, Patients who were already anemic at the time of ART initiation were excluded as clearly stated in the eligibility criteria.

�  It is better to insert the respective tables immediately after the text it refers.

o Rearranged

�  Indicating the number of study participants (N=392) at the end of the titles of the table is not convincing for the reader; it is better to be deleted.

o Deleted

�  Since the authors already indicated the meaning of “COR” and “AOR” in “Predictors of anemia” subtitle, no need of indicating them at the end of table 3 as key word. This concept can also hold true for the other abbreviations indicated in the respective tables.

o Comment accepted and acted up on it.

�  In your discussion part, second paragraph, the phrase “….defined as Hgb concentration <13 g/dl for males and <12 g/dl for females after adjustment for altitude and smoking status of measured values…” is not important. It is almost reputation. Better to be deleted.

o Thank you very much for your critical comments. Deleted.

�  When you compare results in discussion, you need to have 95% CI in your result. For example the prevalence of anemia in your result is 41.3%. But you didn’t indicate its 95%CI. Hence it is very difficult to say comparable, lower or higher than other reports. This can be true for other variables that you compare.

o We have incorporated respective 95% confidence intervals.

�  From your conclusion, avoid unnecessary explanations. For example avoid “(100 - 200 cells/mm3)”

o Deleted as commented.

1. Organization WH. Haemoglobin concentrations for the diagnosis of anaemia and assessment of severity. World Health Organization, 2011.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

1 Mar 2022

Prevalence and predictors of anemia among adults on highly active antiretroviral therapy in Northeast Ethiopia: a retrospective cohort study

PONE-D-21-25369R1

Dear Mr Fentie,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Professor Kwasi Torpey, MD PhD MPH

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Comments adequately addressed

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Christian Obirikorang

18 Mar 2022

PONE-D-21-25369R1

Prevalence and predictors of anemia among adults on highly active antiretroviral therapy in Northeast Ethiopia: a retrospective cohort study

Dear Dr. Fentie Wendie:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

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on behalf of

Professor Kwasi Torpey

Academic Editor

PLOS ONE