Prevalence of cytopenia and its associated factors among HIV infected adults on highly active antiretroviral therapy at Mehal Meda Hospital, North Shewa Zone, Ethiopia.

BACKGROUND: Cytopenias affect the outcomes of highly active anti-retroviral therapy that results in higher morbidity, mortality, and impaired quality of life. The purpose of this study was to assess the prevalence of cytopenia and its associated factors among HIV infected adults on highly active antiretroviral therapy at Mehal Meda Hospital, North Shewa Zone, Ethiopia.
METHOD: A cross-sectional health facility based study was conducted among 499 consecutively selected adult HIV infected patients taking HAART for at least six months from January to April 2018. The study participant's socio-demographic and clinical information was collected using a pre-tested questionnaire and reviewing of medical records by trained clinical nurses. Complete blood count and CD4 T cell count were determined by Sysmex KX-21 N and BD FACS count respectively. Bivariate and multivariate analysis was performed to identify the independently associated factors of cytopenia and prevalence ratios and their 95% confidence intervals were estimated using Poisson regression model with robust error variance to quantify the strength of statistical association. In all cases, a P value less than 0.05 was considered statistically significant. RESULT: Out of the total study participants, 39.9% had at least one form of cytopenia, 23.2% had anemia, 13.8% had leukopenia, 12.4% had thrombocytopenia, 11.62% had bi-cytopenias, and only 1% had pancytopenia. In multivariate analysis, cytopenia was independently associated with older age groups, male gender, ZDV based regimen, and CD4 count less than 200 cells/mm3.
CONCLUSIONS: In this study, the magnitude of any cytopenia was 40% among adult HIV infected patients taking highly active antiretroviral therapy and the prevalence increased as the CD4 count decreases. Therefore, these warrant the need for monitoring hematological parameters of HIV infected patients on HAART to reduce morbidity and mortality.

Introduction

Human immunodeficiency virus (HIV) continues to be a major global public health issue; 37.9 million people globally were living with HIV and 770,000 people died from acquired immunodeficiency syndrome (AIDS) related illnesses at the end of 2018. Of all people living with HIV, over two-thirds live in Africa [1]. In Ethiopia, an estimated 613,000 people were living with HIV in 2017, 74% of the peoples living with HIV are from Amhara, Oromia, and Addis Ababa [2].

HIV not only targets the immune system that leads to progressive immune dysfunction but also affects the hematopoietic system of the infected individuals that result in cytopenias [3]. Cytopenias are the most common hematological complications of HIV infection and may affect any of the major blood lineages leading to anemia, thrombocytopenia, and/or leucopenia. The causes of cytopenias in HIV infection are multifactorial, including a direct consequence of HIV infection, effects of medications, opportunistic infections, hepatitis B virus and hepatitis C virus co-infection, and others. The pathophysiology of cytopenias can broadly be classified as a bone marrow production defect and increased peripheral loss or destruction of blood cells [3-5]. The frequency and severity of cytopenias increase as CD4 count declines and HIV infection advances. Cytopenias can affect the outcomes of highly active antiretroviral therapy (HAART), resulting in higher morbidity, mortality, and a negative impact on the quality of life [6-8]. Anemia is the most frequent cytopenia in HIV infected patients, even in patients taking HAART. It has been independently associated with accelerated HIV disease progression, mortality and decreased quality of life [9-11]. In HIV patients, the prevalence of anemia varies significantly among studies ranging from 1.3% to 95% [9]. Although treatment with HAART decreases the prevalence of severe anemia [12], the overall burden of anemia in HAART treated HIV patients remains high. In China, anemia occurs in 39.2% HIV patients receiving HAART [13]; in Europe and North America the prevalence is 26% [14]. In Ethiopia, the prevalence of anemia among HIV infected adult individuals receiving HAART ranges from 11.4% to 43% [12, 15–19].

Leucopenia is one of the hematological abnormalities encountered commonly in patients with HIV infection. Neutropenia is the most common leucopenia that occurs in 5–30% of patients with early symptomatic HIV infection and up to 70% of patients at advanced stages of AIDS [8, 20, 21]. Like other cytopenias, the cause of HIV-associated neutropenia is multifactorial, including a direct consequence of HIV infection, autoimmune disorders, opportunistic infections, malignancies, and drugs used to treat HIV and opportunistic infections like antiretroviral therapy (particularly zidovudine-containing regimens), cotrimoxazole, and others, which are myelotoxic [20, 22, 23].

Thrombocytopenia is also commonly observed cytopenia in HIV-infected patients. It may occur in 4.1–40% of HIV patients and the prevalence and severity of thrombocytopenia increases as the disease stage advances [3, 20]. The causes of thrombocytopenia include immune-mediated platelet destruction, impaired megakaryocytosis/direct infection of megakaryocytes, hypersplenism, opportunistic infections, malignancy, and myelosuppression effects of medications [20, 24].

Cytopenia remains problematic in resource limited countries like Ethiopia. The diagnosis of cytopenia and its underlying mechanisms during antiretroviral therapy ensure optimal management of patients with HIV disease [25]. However, in our setting, there is scarce information on the magnitude and factors associated with cytopenias in patients under HAART. Therefore, the purpose of this study was to assess prevalence of cytopenia and its associated factors among HIV infected patients on HAART at Mehal Meda Hospital, North Shewa Zone, Ethiopia.

Materials and methods

Study design, setting and population

A cross-sectional study was conducted to assess the prevalence of cytopenia and its associated factors among HIV infected patients on HAART at Mehal Meda Hospital from January to April 2018. The Hospital is found in Mehal Meda town in Menz Gera Midir Woreda North Shewa Zone of the Amhara Region, Ethiopia. Mehal Meda town is about 280 km away from the capital city Addis Ababa and 148 km from Debre Brhan city. Mehal Meda Hospital provides comprehensive health care services including HIV/AIDS diagnosis, treatment and monitoring for a catchment population of more than 360,000.

A total of 499 consecutively selected adult HIV infected patients taking HAART for at least six months were enrolled from ART follow-up unit of Mehal Meda Hospital. The sample size was calculated using single proportion formula with an assumption of a proportion of cytopenia among HIV infected patients taking HAART of 50% since there is no study conducted in the area. The assumptions also include 95% confidence interval, 5% marginal error and 10% non-response rate. The calculated sample size become 422 but we included 499 subjects into the study. HIV-infected patients with known hematological disorders, pregnancy, postpartum period, blood transfusion (within three months) and patients younger than 18 years were excluded from the study.

Data collection procedure

The study participant’s socio-demographic and clinical information was collected using a pre-tested questionnaire and by reviewing of medical records by trained clinical nurses. Height and weight of the participants were measured by the data collectors.

Venous blood specimen (about 4 ml) in EDTA vacutainer tube was collected from each study participants by a senior laboratory professional for complete blood count and CD4 T cell count determination. Sysmex KX-21N (Sysmex corporation kobe, Japan) automated hematological analyzer were used to determine complete blood count (total white blood cell count (WBC), red blood cell count, platelet (PLT) count, and hemoglobin concentration level) and BD FACS count (Becton Dickenson and Company, California, USA) machine was used to measure CD4 T cell count of the study participants. Standard operating procedures and manufacturers’ instructions were strictly followed in each laboratory procedures to maintain quality of the laboratory results. Expiry date of reagents were checked and quality control materials were run along with patient samples to check precision of the instruments and accuracy of the results.

Operational definitions

Anemia was defined based on the World Health Organization (WHO) criteria: hemoglobin (Hgb) concentration <13 g/dl for males (15 years of age and above) and < 12 g/dl for females (non-pregnant women). Anemia was also categorized using the WHO classification into mild anemia (for men: Hgb level from 11.0–12.9 g/dl, for non-pregnant women: Hgb level from 11.0–11.9 g/dl), moderate anemia (Hgb level from 8.0–10.9 g/dl for both sexes), and severe anemia (Hgb lower than 8.0 g/dl for both sexes) [26].

As anemia, there is no generally accepted cut-offs for leucopenia and thrombocytopenia; we defined them as used in other studies [19, 27]. Leucopenia was defined as a total WBC < 4.0 × 103/μL, whereas thrombocytopenia was defined as a total platelet count < 150 × 103/μL. The platelet counts from 100 to150 × 103/μL, 50 to 100 × 103/μL, and less than 50 × 103/μL were considered as mild thrombocytopenia, moderate thrombocytopenia, and severe thrombocytopenia, respectively [28].

Any cytopenia was defined as presence of at least one form of cytopenia (anemia, thrombocytopenia, or leucopenia), bi-cytopenia was defined as presence of two forms of cytopenias (anemia, thrombocytopenia and leucopenia) and pancytopenia was defined as having all three forms of cytopenia simultaneously on the study participants.

Statistical analysis

All collected data were entered in to “Epi Data version 3.1” and exported to Stata® 12.0 (StataCorp, College Station, Texas) statistical software for analysis. Continuous variables were expressed as median and inter-quartile range (IQR) and categorical variables were reported as frequencies and percentages. Chi-square (×2) test for categorical variables was performed to assess their relation with the outcomes. Bivariate and multivariate analysis was performed to identify the independently associated factors of cytopenias and prevalence ratios and their 95% confidence intervals were estimated using Poisson regression model with robust error variance to quantify the strength of statistical association. Variables with p < 0.20 at bivariate analysis were included in the multivariable analysis. In all cases, a P value less than 0.05 was considered statistically significant.

Ethical considerations

Ethical approval was obtained after the study protocol was approved by Research and Ethics Review Committee of the Department of Medical Laboratory Sciences, Wollo University and a letter of permission to conduct the study was obtained from Mehal Meda Hospital. Individual written informed consent was obtained after the purpose and importance of the study were explained. To ensure confidentiality, participant’s identifiers were removed and only code numbers were used throughout the study. Any abnormal test results of participants were reported to the concerned body in the ART clinic of the hospital.

Result

Sociodemographic characteristics of study participants

A total of 499 HIV positive patients taking ART were included in this study. The median age of the study participants was 36 years (Interquartile Range: 29–43) and most of the study participants (n = 163; 32.7%) were within 30–39 years of age. Of the total 499 study participants, 260 (52.1%) were females, 301 (60.3%) were from urban setting, 394 (79.0%) had primary education and below, 240 (48.1%) were married and 163 (32.7%) were employed (Table 1).

Table 1

Sociodemographic characteristics of HIV positive patients taking ART at Mahal Meda Hospital, North Shewa, Ethiopia, 2018 (n = 499).

Variables	Category	Frequency (n = 499)	Percentage (%)
Age (in years)	18–29	138	27.7
	30–39	163	32.7
	40–49	149	29.9
	> = 50	49	9.8
Sex	Male	239	47.9
	Female	260	52.1
Residence	Urban	301	60.3
	Rural	198	39.7
Educational status	Primary school and below	394	79.0
	High school and above	105	21.0
Occupational status	Employed	163	32.7
	Unemployed	336	67.3
Marital status	Married	240	48.1
	Unmarried	259	51.9
Family size	Three and below	273	54.7
	Above three	226	45.3

Clinical and laboratory characteristics of the study participants

Among the study participants, 295 (59.1%) were in WHO clinical stage I and 226 (45.3%) were taking 1e (TDF-3TC-EFV) ART regimen. Majority of the study participants (n = 329; 65.9%) were used the ART regimen with in duration group of 12–59 months with median of 31.0 months (interquartile range: 17.0–60.0 months). One hundred twenty nine (25.9%) of the study participants had opportunistic infection (tuberculosis; 24 (4.8%), pneumonia; 34 (6.8%), oral candidiasis; 7 (1.4%), and diarrhea; 64 (12.8%)) and 30 (6%) of the study participants had chronic disease (diabetic mellitus 9 (1.8%) and hypertension 21 (4.2%)). The median body mass index of the study participants was 19.65 Kg/m2 (interquartile range: 17.96–21.33 Kg/m2) and 150 (30.1%) study participants were under weight (BMI<18.5 Kg/m2). Majority of the respondents 245 (49.1%) had CD4 cell count greater than 500 cell/μL while only 70 (14%) of the respondents had less than 200 cell/μL. The median CD4 count, hemoglobin concentration, white blood cell count and platelet count was 482.0 cell/μL (interquartile range: 290.0–669.0 cell/μL), 14.20 g/dL (interquartile range: 12.60–15.60 g/dL), 6.40 x103/μL (interquartile range: 4.80–7.90 x103/μL), and 242.0 x103/μL (interquartile range: 194.0–301.0 x103/μL), respectively (Table 2).

Table 2

Clinical and laboratory characteristics of HIV infected patients on HAART at Mahal-Meda Hospitals, Northern Shewa Zone, Ethiopia, 2018 (n = 499).

Variables	Category	Frequency (n = 499)	Percentage (%)	Median (IQR)
WHO clinical stages	Stage I	295	59.1	-
	Stage II	145	29.1	-
	Stage III	53	10.6	-
	Stage IV	6	1.2	-
Types of ART regimens	1C	163	32.7	-
	1D	89	17.8	-
	1E	226	45.3	-
	1F	10	2.0	-
	1H	11	2.2	-
Duration of ART regiment	<12 months	46	9.2	-
	12–59 months	329	65.9	-
	> = 60 months	124	24.8	-
Opportunistic infection	TB	24	4.8	-
	Pneumonia	34	6.8	-
	Oral candidiasis	7	1.4	-
	Diarrhea	64	12.8	-
	No	370	74.1	-
Chronic illness	Diabetic Mellitus	9	1.8	-
	Hypertension	21	4.2	-
	NO	469	94.0	-
BMI category	18.5–24.9	330	66.1	-
	<18	150	30.1	-
	> = 25	19	3.8	-
CD4 count (cells/μL)	<200	71	14.2	-
	200–499	183	36.7	-
	> = 500	245	49.1	-
Duration in months		-	-	31.0 (17.0–60.0)
BMI Kg/m2		-	-	19.65 (17.96–21.33)
CD4 count (cells/μL)		-	-	482.0 (290.0–669.0)
White blood cell count (x103/μL)		-	-	6.40 (4.80–7.90)
Hemoglobin concentration (g/dL)		-	-	14.20 (12.60–15.60)
Platelet count (x103/μL)		-	-	242.0 (194.0–301.0)

WHO = World Health Organization, IQR = interquartile range, 1c = ZDV -3TC-NVP, 1d = ZDV-3TC-EFV, 1e = TDF-3TC-EFV, 1f = TDF-3TC-NVP, 1h = ABC+3TC+EFV, ZDV = zidovudine, 3TC = lamuvidine, EFV = efaverenz, NVP = neverapine, TDF = tenofovir, ABC = abacavir, BMI = body mass index.

Prevalence and potential risk factors of cytopenias

Out of the total study participants, 39.9% had any cytopenias, 23.2% had anemia, 13.8% had leukopenia, 12.4% had thrombocytopenia, 11.62% had bi-cytopenias, and only 1% had Pancytopenia. The most frequent bi-cytopenias was anemia and thrombocytopenia combination (4.2%). Of the anemic study subjects (n = 116), 8.62% had severe anemia, 54.31% had moderate anemia, and 37.07% had mild anemia. Majority of the thrombocytopenic study participants had mild thrombocytopenia (87.1%) (Table 3).

Table 3

Prevalence of cytopenias among HIV infected patients on HAART at Mahal-Meda Hospitals, Northern Shewa Zone, Ethiopia, 2018 (n = 499).

Hematological abnormalities	Frequency (%)
Any cytopenia	199 (39.9)
Anemia	116 (23.2)
Leucopenia	69 (13.8)
Thrombocytopenia	62 (12.4)
Bi-cytopenia
Anemia and leucopenia	18 (3.6)
Anemia and thrombocytopenia	21 (4.2)
Thrombocytopenia and leucopenia	14 (2.8)
Pancytopenia	5 (1.0)
Anemia severity (n = 116)
Mild	43 (37.07)
Moderate	63 (54.31)
Severe	10 (8.62)
Thrombocytopenia severity (n = 62)
Mild	54 (87.10)
Moderate	4 (6.45)
Severe	4 (6.45)

Mild anemia = (Hgb level: 11.0–12.9 g/dl for men, 11.0–11.9 g/dl for women), moderate anemia (Hgb level 8.0–10.9 g/dl for both sexes), severe anemia (Hgb level <8.0 g/dl for both sexes), mild thrombocytopenia (platelet counts: 100 to150 × 103/μL), moderate thrombocytopenia (platelet counts: 50 to 100 × 103/μL), severe thrombocytopenia (< 50 × 103/μL).

The prevalence of any cytopenia (at least one form of cytopenia) was significantly associated with male patients, older age groups, rural dwellers, being unmarried by marital status, WHO clinical stage II, ZDV based regimen, taking ART regimen below 60 months, presence of opportunistic infection, and CD4 count less than 200 cells/mm3 and 200–499 cells/mm3on the bivariate analysis.

Using multivariate logistic regression analysis, cytopenia was independently associated with male gender (PR = 1.34, 95% CI: 1.07–1.67, P = .011), unmarried (PR = 1.36, 95% CI: 1.09–1.69, P = 0.007), ZDV based regimen (PR = 1.56, 95% CI: 1.24–1.96, P<0.001), taking ART regiment for 12–60 months (PR = 1.54, 95% CI: 1.16–2.06, P = 0.003), CD4 count less than 200 cells/mm3 (PR = 2.06, 95% CI: 1.54–2.75, P<0.001) and 200–499 cells/mm3 (PR = 1.36, 95% CI: 1.06–1.74, P = 0.015) (Table 4).

Table 4

Factors associated with cytopenia among HIV infected patients on HAART at Mahal-Meda Hospitals, Northern Shewa Zone, Ethiopia, 2018 (n = 499).

Variable	Any Cytopenia		Bivariable	P-value	Multivariable	P-value
	No, n (%)	Yes, n (%)	PR (95% CI)		PR (95% CI)
Age group (in years)
18–29	83 (60.1)	55 (39.9)	1		1
30–39	103 (63.2)	60 (36.8)	0.92 (0.69–1.23)	0.587	1.02 (0.75–1.37)	0.918
40–49	94 (63.1)	55 (36.9)	0.93 (0.69–1.24)	0.608	0.93 (0.68–1.28)	0.667
> = 50	20 (40.8)	29 (59.2)	1.48 (1.09–2.03)	0.012	1.33 (0.93–1.90)	0.116
Sex
Male	124 (51.9)	115 (48.1)	1.49 (1.19–1.86)	<0.001	1.34 (1.07–1.67)	0.011
Female	176 (67.7)	84 (32.3)	1		1
Residence
Urban	193 (64.1)	108 (35.9)	1		1
Rural	107 (54.0)	91 (46.0)	1.28 (1.04–1.59)	0.023	1.22 (0.98–1.52)	0.076
Educational status
Primary school and below	242 (61.4)	152 (38.6)	0.86 (0.67–1.10)	0.237
High school and above	58 (55.2)	47 (44.8)	1
Occupational status
Employed	105 (64.4)	58 (35.6)	1		1
Un employed	195 (58.0)	141 (42.0)	1.18 (0.93–1.50)	0.181	1.01 (0.80–1.28)	0.914
Marital status
Married	164 (68.3)	76 (31.7)	1		1
Unmarried	136 (52.5)	123 (47.5)	1.50 (1.19–1.88)	<0.001	1.36 (1.09–1.69)	0.007
WHO clinical stage
stage I	191 (64.7)	104 (35.3)	1		1
Stage II	77 (53.1)	68 (46.9)	1.33 (1.05–1.68)	0.016	1.13 (0.90–1.41)	0.295
Stage III and IV	32 (54.2)	27 (45.8)	1.29 (0.95–1.78)	0.108	0.81 (0.58–1.13)	0.221
Regimen
ZDV based	123 (48.8)	129 (51.2)	1.81 (1.43–2.28)	<0.001	1.55 (1.24–1.96)	<0.001
Non ZDV based	177 (71.7)	70 (28.3)	1		1
Duration
<12 months	24 (52.2)	22 (47.8)	1.60 (1.07–2.40)	0.022	1.49 (0.97–2.32)	0.071
12–60 months	189 (57.4)	140 (42.6)	1.43 (1.06–1.92)	0.019	1.54 (1.16–2.06)	0.003
>60 months	87 (70.2)	37 (29.8)	1		1
Opportunistic infection
Yes	59 (45.7)	70 (54.3)	1.56 (1.26–1.92)	<0.001	1.05 (0.84–1.32)	0.655
No	241 (65.1)	129 (34.9)	1		1
Chronic disease
Yes	14 (46.7)	16 (53.3)	1.37 (0.96–1.95)	0.083	0.97 (0.65–1.44)	0.878
No	286 (61.0)	183 (39.0)	1		1
BMI category
18.5–24.9	198 (59.8)	133 (40.2)	1
<18	88 (59.1)	61 (40.9)	1.02 (0.81–1.29)	0.875
> = 25	14 (73.7)	5 (26.3)	0.65 (0.31–1.41)	0.277
CD4 count category
<200	26 (36.6)	45 (63.4)	2.19 (1.68–2.85)	<0.001	2.06 (1.54–2.75)	<0.001
200–499	100 (54.6)	83 (45.4)	1.56 (1.22–2.01)	0.001	1.36 (1.06–1.74)	0.015
> = 500	174 (71.0)	71 (29.0)	1		1

WHO = World Health Organization, ZDV = zidovudine, PR = Prevalence ratio, CI = Confidence interval, 1.00 = reference group.

The prevalence of anemia was significantly higher in males than females (28% vs18.8%, P = 0.016). Anemia was also higher in HIV patients with older age group, rural residents, WHO clinical stage III and IV, ZDV based ART regimen, and opportunistic infection. The prevalence of anemia was decreased as ART regiment usage becomes longer. However, the prevalence of anemia increased as the CD4 T cell count decreased (Table 5).

Table 5

Factors associated with anemia among HIV infected patients on HAART at Mahal-Meda Hospitals, Northern Shewa Zone, Ethiopia, 2018 (n = 499).

Variable	Anemia		Bivariable	P-value		Multivariable	P-value
	No, n (%)	Yes, n (%)	PR (95% CI)			PR (95% CI)
Age group (in years)
18–29	106 (76.8)	32 (23.2)	1			1
30–39	126 (77.3)	37 (22.7)	0.98 (0.65–1.48)	0.920		0.98 (0.62–1.55)	0.926
40–49	121 (81.2)	28 (18.8)	0.81 (0.52–1.27)	0.362		0.84 (0.52–1.35)	0.463
> = 50	30 (61.2)	19 (38.8)	1.68 (1.05–2.66)	0.030		1.39 (1.05–2.32)	0.027
Sex
Male	172 (72.0)	67 (28.0)	1.49 (1.08–2.06)	0.016		1.22 (0.89–1.67)	0.210
Female	211 (81.2)	49 (18.8)	1			1
Residence
Urban	253 (84.1)	48 (15.9)	1			1
Rural	130 (65.7)	68 (34.3)	2.15 (1.56–2.98)	<0.001		2.05 (1.48–2.84)	<0.001
Educational status
Primary school and below	305 (77.4)	89 (22.6)	0.88 (0.60–1.28)	0.496
High school and above	78 (74.3)	27 (25.7)	1
Occupational status
Employed	131 (80.4)	32 (19.6)	1			1
Un employed	252 (75.0)	84 (25.0)	1.27 (0.89–1.83)	0.191		0.88 (0.61–1.28)	0.516
Marital Status
Married	199 (82.9)	41 (17.1)	1			1
Unmarried	184 (71.0)	75 (29.0)	1.69 (1.21–2.38)		0.002	1.46 (1.04–2.06)	0.028
WHO clinical stage
Stage I	243 (82.4)	52 (17.6)	1			1
Stage II	100 (69.0)	45 (31.0)	1.76 (1.25–2.50)		0.001	1.51 (1.08–2.10)	0.016
Stage III and IV	40 (67.8)	19 (32.2)	1.83 (1.17–2.85)		0.008	1.12 (0.69–1.79)	0.649
Regimen
ZDV based	178 (70.6)	74 (29.4)	1.73 (1.24–2.42)		0.001	1.25 (0.89–1.74)	0.187
Non ZDV based	205 (83.0)	42 (17.0)	1			1
Duration
<12 months	33 (71.7)	13 (28.3)	2.08 (1.09–3.90)		0.026	2.03 (1.01–4.07)	0.046
12–60 months	243 (73.9)	86 (26.1)	1.91 (1.18–3.07)		0.008	1.93 (1.24–3.02)	0.004
>60 months	107 (86.3)	17 (13.7)	1			1
Opportunistic infection
Yes	76 (58.9)	53 (41.1)	2.41 (1.78–3.28)		<0.001	1.69 (1.13–2.23)	0.007
No	307 (83.0)	63 (17.0)	1			1
Chronic disease
Yes	19 (63.3)	11 (36.7)	1.64 (0.99–2.70)		0.053	1.05 (0.59–1.88)	0.859
No	364 (77.6)	105 (22.4)	1			1
BMI category
18.5–24.9	258 (77.9)	73 (22.1)	1
<18	111 (74.5)	38 (25.5)	1.16 (0.82–1.63)	0.404
> = 25	14 (73.7)	5 (26.3)	1.19 (0.54–2.61)	0.657
CD4 count category
<200	47 (66.2)	24 (33.8)	1.76 (1.16–2.67)	0.007		1.53 (1.01–2.31)	0.045
200–499	138 (75.4)	45 (24.6)	1.28 (0.89–1.84)	0.178		0.91 (0.65–1.28)	0.594
> = 500	198 (80.8)	47 (19.2)	1			1

WHO = World Health Organization, ZDV = Zidovudine, PR = Prevalence ratio, CI = Confidence interval, 1.00 = Reference group.

In multivariable analysis, the prevalence ratio of having anemia in older age group (≥50 years) was 1.4 (PR = 1.39, 95% CI: 1.05–2.32, P = 0.027) compared to younger age group (18–29 years). The prevalence ratio of anemia was twice in rural residents (PR = 2.05, 95% CI: 1.48–2.84, P<0.001) compared to urban residents, 1.5 (PR = 1.46, 95% CI: 1.04–2.06, P = 0.028) in unmarried study participants compared to married participants, and 1.5 times higher in WHO clinical stage II (PR = 1.51, 95% CI: 1.08–2.10, P = 0.016) compare to participants in WHO clinical stage I. Anemia was also independently associated with 12–60 months duration of ART regimen (PR = 1.93, 95% CI: 1.24–3.02, P = 0.004) and opportunistic infection (PR = 1.69, 95%CI: 1.13–2.23, P = 0.007) as summarized in Table 5.

The prevalence of leucopenia among males and females were 13.4% and 14.2%, respectively but the difference were not statistically significant (P = 0.786). Leucopenia prevalence progressively increased with age: 8.0% for age 18–29 years, 10.4% for 30–39 years, 18.8% for 40–49 years and 26.5% for >50 years (P = 0.002). Leucopenia had decreased as CD4+ T-cell counts increased: CD4 count <200 cells/mm3 (38.0%), CD4 count from 200–499 cells/mm3 (15.8%), and CD4 count >500 cell/mm3 (5.3%) (P<0.001). Using a multivariate analysis, leucopenia was independently associated with age category 40–49 years (PR = 2.06, 95% CI: 1.12–3.79, P = 0.019), >50 years (PR = 2.40, 95% CI: 1.26–4.61, P = 0.008), ZDV based regimen (PR = 1.86, 95% CI: 1.15–3.00, P = 0.004), CD4 count less than 200 cells/mm3 (PR = 5.99, 95% CI: 3.18–11.29, P<0.001), and CD4 count from 200–499 cells/mm3 (PR = 2.91, 95% CI: 1.56–5.42, P = 0.001) (Table 6).

Table 6

Factors associated with leucopenia among HIV infected patients on HAART at Mahal-Meda Hospital, Northern Shewa Zone, Ethiopia, 2018 (n = 499).

Variable	Leucopenia		Bivariable	P value	Multivariable	P-value
	No, n (%)	Yes, n (%)	PR (95% CI)		PR (95% CI)
Age group (in years)
18–29	127 (92.0)	11 (8.0)	1		1
30–39	146 (89.6)	17 (10.4)	1.31 (.63–2.70)	0.467	1.61 (0.81–3.19)	0.172
40–49	121 (81.2)	28 (18.8)	2.36 (1.22–4.55)	0.011	2.06 (1.12–3.79)	0.019
> = 50	36 (73.5)	13 (26.5)	3.33 (1.59–6.94)	0.001	2.40 (1.26–4.61)	0.008
Sex
Male	207 (86.6)	32 (13.4)	1
Female	223 (85,8)	37 (14.2)	1.06 (0.68–1.65)	0.786
Residence
Urban	257 (85.4)	44 (14.6)	1
Rural	173 (87.4)	25 (12.6)	0.86 (0.55–1.36)	0.530
Educational status
Primary school and below	344 (87.3)	50 (12.7)	0.70 (0.43–1.14)	0.150	0.70 (0.44–1.12)	0.142
High school and above	86 (81.9)	19 (18.1)	1		1
Occupational status
Employed	138 (84.7)	25 (15.3)	1
Un employed	292 (86.9)	44 (13.1)	0.85 (0.54–1.35)	0.495
Marital status
Married	205 (85.4)	35 (14.6)	1
Unmarried	225 (86.9)	34 (13.1)	0.90 (0.58–1.40)	0.638
WHO clinical stage
stage I	254 (86.1)	41 (13.9)	1		1
Stage 2	133 (91.7)	12 (8.3)	0.59 (0.32–1.09)	0.097	0.42 (0.23–0.76)	0.004
Stage 3and 4	43 (72.9)	16 (27.1)	1.95 (1.18–3.23)	0.010	1.34 (0.78–2.29)	0.727
Regimen
ZDV based	206 (81.7)	46 (18.3)	1.96 (1.23–3.13)	0.005	1.86 (1.15–3.00)	0.011
Non ZDV based	224 (90.7)	23 (9.3)	1
Duration
<12 months	38 (82.6)	8 (17.4)	1
12–60 months	290 (88.1)	39 (11.9)	0.681 (0.34–1.37)	0.280
>60 months	102 (82.3)	22 (17.7)	1.02 (0.49–2.13)	0.958
Opportunistic infection
Yes	113 (87.6)	16 (12.4)	0.86 (0.51–1.46)	0.589
No	317 (85.7)	53 (14.3)	1
Chronic disease
Yes	26 (86.7)	4 (13.3)	0.96 (0.38–2.46)	0.936
NO	404 (86.1)	65 (13.9)	1
BMI category
18.5–24.9	281 (84.9)	50 (15.1)	1
<18	130 (87.2)	19 (12.8)	0.84 (0.52–1.38)	0.500
> = 25	19 (100)	0 (0)	0.00 (0.00)	0.985
CD4 count category
<200	44 (62.0)	27 (38.0)	7.16 (3.91–13.15)	<0.001	5.99 (3.18–11.29)	<0.001
200–499	154 (84.2)	29 (15.8)	2.99 (1.59–5.59)	0.001	2.91 (1.56–5.42)	<0.001
> = 500	232 (94.7)	13 (5.3)	1		1

WHO = World Health Organization, ZDV = Zidovudine, PR = Prevalence ratio, CI = Confidence interval, 1.00 = Reference group.

In this study, the overall prevalence of thrombocytopenia was 12.4%. The prevalence of thrombocytopenia among males (17.6%) were higher than females (7.7%) and the difference were statistically significant (P = .001). In bivariate analysis, thrombocytopenia was associated with age group, male gender, occupational status, marital status, WHO clinical stage III and IV, AZT based ART regimen, CD4 count less than 200 cell/mm3. In multivariate analysis, thrombocytopenia was independently associated with age group 40–49 years (PR = 0.33, 95% CI: 0.16–0.70, P = 0.004), male participants (PR = 2.36, 95% CI: 1.37–4.07, P = 0.002), unmarried (PR = 2.36, 95% CI: 1.33–4.20, P = 0.003), and CD4 count less than 200 cell/mm3 (PR = 2.91, 95% CI: 1.58–5.35, P = 0.001) (Table 7).

Table 7

Factors associated with thrombocytopenia among HIV infected patients on HAART at Mahal-Meda Hospitals, Northern Shewa Zone, Ethiopia, 2018 (n = 499).

Variable	Thrombocytopenia		Bi-variable	P value	Multivariable	P-value
	No, n (%)	Yes, n (%)	PR (95% CI)		PR (95% CI)
Age group (in years)
18–29	112 (81.2)	26 (18.8)	1		1
30–39	147 (90.2)	16 (9.8)	0.52 (0.29-.93)	0.028	0. 69 (0.38–1.23)	0.208
40–49	140 (94.0)	9 (6.0)	0.32 (0.16–0.66)	0.002	0.33 (0.16-.70)	0.004
> = 50	38 (77.6)	11 (22.4)	1.19 (0.64–2.23)	0.583	0.91 (0.45–1.84)	0.801
Sex
Male	197 (82.4)	42 (17.6)	2.28 (1.38–3.78)	0.001	2.36 (1.37–4.07)	.002
Female	240 (92.3)	20 (7.7)	1		1
Residence
Urban	260 (86.4)	41 (13.6)	1
Rural	177 (89.4)	21 (10.6)	0.78 (0.47–1.28)	0.322
Educational status
Primary school and below	352 (89.3)	42 (10.7)	0.56 (0.34–0.91)	0.020	0.82 (0.54–1.25)	0.356
High school and above	85 (81.0)	20 (19.0)	1		1
Occupational status
Employed	151 (92.6)	12 (7.4)	1		1
Un employed	286 (85.1)	50 (14.9)	2.02 (1.11–3.69)	0.022	1.57 (0.85–2.88)	0.147
Marital status
Married	226 (94.2)	14 (5.8)	1		1
Unmarried	211 (81.5)	48 (18.5)	3.18 (1.79–5.61)	<0.001	2.36 (1.33–4.21)	0.003
WHO clinical stage
Stage I	265 (89.8)	30 (10.2)	1		1
Stage 2	125 (86.2)	20 (13.8)	1.36 (0.79–2.31)	0.260	1.15 (0.68–1.96)	0.595
Stage 3and 4	47 (79.7)	12 (20.3)	2.00 (1.09–3.68)	0.026	1.17 (0.62–2.21)	0.624
Regimen
ZDV based	210 (83.3)	42 (16.7)	2.06 (1.24–3.40)	0.005	1.40 (0.82–2.33)	0.227
Non ZDV based	227 (91.9)	20 (8.1)	1		1
Duration
<12 months	43 (93.5)	3 (6.5)	1
12–60 months	285 (86.6)	44 (13.4)	2.05 (.67–6.34)	0.213
>60 months	109 (87.9)	15 (12.1)	1.86 (0.56–6.11)	0.310
Opportunistic infection
Yes	109 (84.5)	20 (15.5)	1.366 (0.83–2.24)	0.216
No	328 (88.6)	42 (11.4)	1
Chronic disease
Yes	23 (76.7)	7 (23.3)	1.99 (0.99–3.99)	0.052	1.28 (0.60–2.74)	0.519
NO	414 (88.3)	55 (11.7)	1		1
BMI category
18.5–24.9	291 (87.9)	40 (12.1)	1
<18	128 (85.9)	21 (14.1)	1.17 (0.71–1.91)	0.540
> = 25	18 (94.7)	1 (5.3)	0.43 (0.06–3.01)	0.399
CD4 count category
<200	55 (77.5)	16 (22.5)	2.40 (1.342–4.29)	0.003	2.91 (1.58–5.35)	0.001
200–499	160 (87.4)	23 (12.6)	1.34 (0.77–2.31)	0.295	1.12 (0.64–1.98)	0.684
> = 500	222 (90.6)	23 (9.4)	1		1

WHO = World Health Organization, ZDV = zidovudine, BMI = body mass index, PR = Prevalence ratio, CI = Confidence interval, 1.00 = reference group.

Discussion

This study assessed the prevalence and associated factors of cytopenias among adult HIV infected patients on HAART at Mehal Meda Hospital, North Shewa Zone, Ethiopia. The overall prevalence of at least one form of cytopenia (presence of anemia, thrombocytopenia or leukopenia) was 39.9% (95% CI;35.5% - 43.5%) and it was independently associated with older age groups, male gender, unmarried marital status, ZDV based ART regimen, taking ART regiment for 12–59 months, and CD4 count less than 200 cells/mm3. Prevalence of cytopenia in this study was higher than study done in Beijing Ditan Hospital, China [29]. These differences could be due to different cut-off values used to define the cytopenias, study population, socioeconomic status and dietary habits of study participants.

In this study, the most frequent type of cytopenia were anemia and its prevalence was 23.2% (95% CI; 19.4% - 26.8%) which was in agreement with other findings reported in Gondar (22.2%) [17], Northeastern Nigeria (24.3%) [30], and Kaduna State, Nigeria (23%) [31]. However, the prevalence was lower compared to studies conducted in Debre Tabor (29.9%) [16], Tikur Anbessa Specialized Hospital, Addis Ababa (34.6%) [18], Jimma University Specialized Hospital, Jimma (43.1) [19], South West Region of Cameroon (58.6%) [32], Benin City, Nigeria (51.15%) [33], and Brazil (37.5%) [34]. Our finding was higher than studies conducted in Gondar University Hospital (11.7%) [15], Black Lion Specialized Hospital, (11.4%) [12], and Zewditu Memorial Hospital (14.3%) [35]. The reasons for the observed differences in prevalence of anemia might be due to the difference in study population, socioeconomic status and dietary habits of study participants, sample size, and difference in the definition of anemia.

Of the anemic study subjects, about 9% had severe anemia, 54% had moderate anemia, and 37% had mild anemia. The predominance of moderate type of anemia in the current study is in line with a study conducted at Tikur Anbessa Specialized Hospital, Addis Ababa [18] but it deviates from the findings reported in Wolita Sodo University, Sodo [36], Black Lion Hospital, Addis Ababa [12], Debre Tabor [37], and rural China [13], which reported high rate of mild anemia.

In the present study, the prevalence of anemia was significantly higher in males than females which is consistent with other studies [13, 33, 35]; this might be due to the difference in the definition of anemia. However, many studies reported a high prevalence of anemia in females than male HIV infected patients [18, 37–40]. Similar to other studies findings [12, 13, 19, 39], we found that the prevalence of anemia was significantly higher as the CD4 T cell count of the study participants decreased and WHO clinical stage advanced. This might be due to high HIV infection that leads to compromised immune system and disrupts normal hematopoiesis by cytokine dysregulation [3, 4]. Anemia also independently associated with older age group (≥50 years) and opportunistic infections in this study, this might be associated with immunosuppression. The prevalence of anemia was decreased with longer ART regiment usage and this is attributed to the positive effect of ART on the differentiation and survival of erythrocytes. The finding is supported by other studies [12, 32].

Leukopenia was found in 13.8% (95% CI; 11.1% - 17.1%) of the study participants, this finding was supported by a study conducted in Jimma, Ethiopia (12.3%) [19]. However, the prevalence was lower than studies in Gonder, Ethiopia (35.9%) [15], South West Region of Cameroon (20%) [32], and Karnataka, India (35%) [41] and higher than studies in Ghana (6.5%) [42], Kaduna State, Nigeria (9%) [31], and Ranchi, India (3%) [43]. The observed difference in the prevalence might be due to variation in study populations, clinical conditions, study design methods, and leukopenia definition. In the present study, leukopenia were significantly increased as the CD4 T cell count decreased and age of HIV infected patients increased, which is in agreement with other studies [15, 19, 38]. This could be due to HIV mediated hematopoietic inhibition and direct infection of T cells [4].

Our study showed that the overall prevalence of thrombocytopenia was 12.4% (95% CI; 9.6% - 15.4%). It is consistent with other reports conducted in South West Region of Cameroon (14%) [32] and Southwestern Uganda (13%) [44]. The possible causes of thrombocytopenia could be immune-mediated platelet destruction, impaired platelet production by the infected magakaryocytes of the bone marrow, or myelosuppression effects of medications. On the other hand, the prevalence of this study was higher than studies done in northwest Ethiopia (4.1% and 6.3%) [15, 17], Jimma (6.9%) [19], Addis Ababa (5.7%) [45], and Yaoundé, Cameroon (6.9%) [46], and lower than studies in Ghana (18.5%) [42] and Kaduna State, Nigeria (24%) [26, 31]. The difference might be due to variation in the definition of thrombocytopenia, study design and size of the study population. Regarding the severity of thrombocytopenia, the predominant form was mild thrombocytopenia which is consistent with other studies [45, 46].

Similar to our finding, different studies reported thrombocytopenia were increased and independently associated with degree of immunosuppression [15, 31, 45]. However, our finding deviated from a study conducted in Jimma [19] that showed thrombocytopenia is not associated with neither the degree of immunosuppression nor with the clinical stage of HIV.

Pancytopenia in this study was 1% (95% CI: 0.4%-2%), which is nearly in agreement with a study conducted by Firnhaber C et al (0.3%) [47]. However, a study from Jimma reports no pancytopenia found [19] and studies conducted by Bukar A et al [31] and Santiago-Rodríguez EJ et al [48] reported 8% and 8.7% pancytopenia, respectively, which is higher than our finding. The difference might be due to variation in sample size and design of the studies.

The main limitation of this study is the cross-sectional nature of the study design which does not reveal causal links between independent variables and cytopenias, so a longitudinal study is recommended to generalize the related outcomes of this study. Despite the limitations, the study has determined the magnitude of cytopenias and identified important factors associated with cytopenias in HIV patients on HAART.

In conclusion, in this study prevalence of at least one form of cytopenia was 40% among HIV infected patients on HAART. The most frequent type of cytopenias was anemia followed by leukopenia and thrombocytopenia. Older age group (>50 years old), male gender, ZDV based ART regimen, and lower CD4 T cell count were identified as independent factors associated with having cytopenias (anemia, leucopenia or thrombocytopenia). Therefore, these warrant the need for monitoring hematological parameters of HIV infected patients on HAART to reduce morbidity and mortality.

(XLSX)

Click here for additional data file.

21 Apr 2020

PONE-D-20-04588

Prevalence of cytopenia and its associated factors among HIV infected adults on highly active antiretroviral therapy at Mehal Meda Hospital, North Shewa Zone, Ethiopia.

PLOS ONE

Dear Mr Gebreweld,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Jun 05 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Benn Sartorius, PhD

Academic Editor

PLOS ONE

Additional Editor Comments:

Please ensure that the revised manuscript is sent for a full, professional English edit.

Please include as a supplementary file for the revised manuscript a completed STROBE checklist.

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. 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: No

Reviewer #2: Partly

**********

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

Reviewer #1: No

Reviewer #2: No

**********

3. 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

Reviewer #2: Yes

**********

4. 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: No

Reviewer #2: No

**********

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 authors may need input from a native English speaker and the manuscript needs extensive editing for correction of grammatical and other errors for instance from the introduction alone lines 21, 35, 39, 40, 55,57,59,63,68,76-78, 83-85 have grammatical errors.

Introduction

Lines 64-65 China needs to be capitalized but also note that it is part of the industrialized countries. There is wrongful capitalization of words under "Operational definitions" from line 118-130.

Line 88-89: Please mention whether this hospital is located in an urban or rural settings as readers can not deduce this. Table 4 appears to imply that both contexts existed where the study took place and this makes interpretation of the findings difficult. It is sufficient to say "A cross-sectional study was conducted..." and drop the health facility based since the site being a hospital already implies a health facility.

Line 52: It is important for the authors to clearly point out at this point that the causes of cytopenia in HIV infection are multifactorial; HIV infection itself is just one of them but there are other causes of these abnormalities. This is relevant because effective HAART does not reverse all cytopenias

Line 104: No need to describe how BMI is calculated as this is general knowledge. Similarly, it's not relevant to this study to know the expertise of the phlebotomist.

Line 119: Should state clearly that severity of anemia was similarly graded as per WHO rather than state that anemia was further categorized. It is not simply categorizing but rather grading the severity because this has implications at the clinical as well as public health level.

Line 120 has a missing decimal point on 109 g/dl(This is important to correct!)

Line 132: Statistical Analysis

a)The use of the Chi square test in this context is erroneous. This test only provides us with information to the extent that categorical variables in the same population are independent of each other or have a statistical relationship. It is not a measure of the *strength* of an association.

b) The use of Odd Ratios (OR) and logistic regression in a cross-sectional study where the prevalence is high is problematic because it may lead to misinterpretation of the findings by over/underestimating the measure of association between the exposure and outcome. Secondly when OR is used in a cross-sectional study, the language used to report the findings should not use such wording as "higher or more at risk of" (e.g line 184) because the design is such that exposure and outcome are measured simultaneously and therefore can not evaluate risk. The Prevalence Ratio (PR) is recommended in cross-sectional studies with outcomes that have a high prevalence (generally >10%), together with the log-binomial regression model rather than the logistic regression model which may overestimate the measure of association but also give wider confidence intervals.

With that background it would require revising all the tables and reviewing the results and conclusions again. However, below are some additional general comments on the tables

1. Where possible it would be preferable to further combine the categories of the independent variables for instance Marital status: married or unmarried; employment status: employed versus unemployed, ART regimen:Zidovudine (AZT)-based versus non-AZT based and to specify that the duration is with respect to HAART use.

2. Table 3: Once you provide the n for "Yes "and the frequency(percentage) in each category, then the "No" becomes redundant. The "No" column is particularly difficult to interpret for the section of the table showing severity of anemia and thrombocytopenia for instance the row percentages for mild, moderate and severe anemia are difficult to interpret here.

Check typos as well here and throughout all tables.

Discussion:

1. This section should be revised once the issues with the statistical techniques are addressed however, line 283 defeats the purpose of the study. This is the golden opportunity to compare the findings of this study with studies done elsewhere-in Africa, Europe, Asia etc. It is the essence of research! Do my results compare with another study or are they different? What could explain the similarity or differences? Is my population older, more malnourished, more advanced in HIV, and so on. Do my results compare with other parts of Ethiopia or not? What is distinct about the population in Northern Shewa that could explain these findings?

2. Percentages reported need to be rounded of to the nearest whole with appropriate language such as "About 8% of the anemic subjects has severe anemia" instead of 8.62%

3. Line 286 contradicts line 291 with regard to the findings of this study and the one done in Gondar.

4. Lines 285-289: Herein lies the opportunity to give mileage to the importance of this study because you have findings from different parts of Ethiopia especially with regard to prevalence of anemia. Please explore this more and highlight it!

Conclusion: This section too should be revised once the statistical analysis is revised.

Line 343: How high is high considering that some studies have reported figures as high as 80%? What magnitude of abnormality is considered relevant at a public health or global health level? It would be more prudent to conclude within the context of your study for instance, "Anemia was the most common cytopenia (%) in HIV-infected adults receving HAART at the Mahal Meda hospital in Ethiopia. This finding however did not meet the WHO criteria for a severe public health problem"

Reviewer #2: This is an interesting paper describing the prevalence of cytopenias in an HIV population on HAART at a center in Ethiopia. However, substantial revision is necessary.

- This paper would benefit from thorough proof-reading for grammatical (e.g. subject-verb agreement, missing pronous, non-standard capitalization), typographical and spelling mistakes (e.g. "Privet" instead of "private" which are quite extensive throughout the manuscript

- In the tables presenting the main analyses (4,5 and 6), some of the data for the multivariate analyses is missing and it is not apparent why this is the case.

- In table 3, I would suggest using the words "prevalence" or "frequency" instead of "magnitude". Also, the definitions of "mild, moderate, severe" should be included either in the table or as a footnote to the table

-For Table 1, consider rearranging the table or selecting the appropriate presentation of the data, as some of the variables are summarized twice

-It was not clear how the sample size of 499 was selected. Was this based on potential precision of the prevalence estimates or the comparative univariate analyses?

- Some discussion of the limitations of the methods and statistical analysis is warranted. For example, most of the outcomes were common (>10%) and logistic regression has a tendency to be biased away from the null when the dependent variable is not rare. Additionally, please comment on whether any interactions between the variables were explored.

-For the result of a higher prevalence of anemia among males, it would be good to explain why that may be the case in this particular patient population given what has previously been observed from studies in the literature

**********

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.

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: No

Reviewer #2: No

[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 to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

5 Jun 2020

Response to Academic editor

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Response: We have followed PLOS ONE's formatting guidelines

2. Please ensure that the revised manuscript is sent for a full, professional English edit.

Response: The revised manuscript was edited by professional English editor.

3. Please include as a supplementary file for the revised manuscript a completed STROBE checklist.

Response: The completed STROBE checklist has included as a supplementary file

Response to reviewer #1

We appreciate that the reviewer’s comments. The followings are our point-by-point responses:

Comment: The authors may need input from a native English speaker and the manuscript needs extensive editing for correction of grammatical and other errors for instance from the introduction alone lines 21, 35, 39, 40, 55,57,59,63,68,76-78, 83-85 have grammatical errors.

Response: We tried to correct grammatical, typographical and spelling errors in the manuscript and the revised manuscript was edited by professional English editor.

Introduction

Lines 64-65 China needs to be capitalized but also note that it is part of the industrialized countries. There is wrongful capitalization of words under "Operational definitions" from line 118-130.

Response: We corrected it (see line 65 and from 118-133)

Line 88-89: Please mention whether this hospital is located in an urban or rural settings as readers cannot deduce this. Table 4 appears to imply that both contexts existed where the study took place and this makes interpretation of the findings difficult.

Response: As we stated in methodology section, the hospital is found in Mehal Meda town and provide service for both urban and rural residents (see line 91).

It is sufficient to say "A cross-sectional study was conducted..." and drop the health facility based since the site being a hospital already implies a health facility.

Response: We have already dropped it. (See line 88)

Line 52: It is important for the authors to clearly point out at this point that the causes of cytopenia in HIV infection are multifactorial; HIV infection itself is just one of them but there are other causes of these abnormalities. This is relevant because effective HAART does not reverse all cytopenias

Response: We have added the possible causes of cytopenia in HIV infection. (Line 52-55)

Line 104: No need to describe how BMI is calculated as this is general knowledge. Similarly, it's not relevant to this study to know the expertise of the phlebotomist.

Response: We have removed it (line 106).

Line 119: Should state clearly that severity of anemia was similarly graded as per WHO rather than state that anemia was further categorized. It is not simply categorizing but rather grading the severity because this has implications at the clinical as well as public health level.

Response: We have stated it clearly, anemia was categorized using the WHO classification criteria (see line 120).

Line 120 has a missing decimal point on 109 g/dl(This is important to correct!)

Response: We corrected it (see line 122).

Line 132: Statistical Analysis

a) The use of the Chi square test in this context is erroneous. This test only provides us with information to the extent that categorical variables in the same population are independent of each other or have a statistical relationship. It is not a measure of the *strength* of an association.

Response: We have corrected the statement (see line 138).

b) The use of Odd Ratios (OR) and logistic regression in a cross-sectional study where the prevalence is high is problematic because it may lead to misinterpretation of the findings by over/underestimating the measure of association between the exposure and outcome. Secondly when OR is used in a cross-sectional study, the language used to report the findings should not use such wording as "higher or more at risk of" (e.g line 184) because the design is such that exposure and outcome are measured simultaneously and therefore cannot evaluate risk. The Prevalence Ratio (PR) is recommended in cross-sectional studies with outcomes that have a high prevalence (generally >10%), together with the log-binomial regression model rather than the logistic regression model which may overestimate the measure of association but also give wider confidence intervals. With that background it would require revising all the tables and reviewing the results and conclusions again.

Response: Based on the recommendation, instead of odds ratio, we have estimated the Prevalence Ratio (PR) and its 95% confidence interval as a measure of association between the exposure and outcome using Poisson regression model with robust error variance (see from line 139-144). We couldn’t use log-binomial regression model because convergence problems. We revised all the tables, results, and conclusions.

Below are some additional general comments on the tables

1. Where possible it would be preferable to further combine the categories of the independent variables for instance Marital status: married or unmarried; employment status: employed versus unemployed, ART regimen: Zidovudine (AZT)-based versus non-AZT based and to specify that the duration is with respect to HAART use.

Response: We merged some categories of the independent variables for instance marital status: married or unmarried; employment status: employed versus unemployed, ART regimen: Zidovudine (AZT)-based versus non-AZT based (see tables).

2. Table 3: Once you provide the n for "Yes "and the frequency (percentage) in each category, then the "No" becomes redundant. The "No" column is particularly difficult to interpret for the section of the table showing severity of anemia and thrombocytopenia for instance the row percentages for mild, moderate and severe anemia are difficult to interpret here. Check typos as well here and throughout all tables.

Response: We omitted the ‘’No’’ column from table 3 to reduce redundancy. We have corrected the typographical errors.

Discussion:

1. This section should be revised once the issues with the statistical techniques are addressed however, line 283 defeats the purpose of the study. This is the golden opportunity to compare the findings of this study with studies done elsewhere-in Africa, Europe, Asia etc. It is the essence of research! Do my results compare with another study or are they different? What could explain the similarity or differences? Is my population older, more malnourished, more advanced in HIV, and so on. Do my results compare with other parts of Ethiopia or not? What is distinct about the population in Northern Shewa that could explain these findings?

Response: We have revised the discussion part and our finding is compared with other studies.

2. Percentages reported need to be rounded of to the nearest whole with appropriate language such as "About 8% of the anemic subjects has severe anemia" instead of 8.62%

Response: We rounded off the percentages to the nearest whole number (see line 289)

3. Line 286 contradicts line 291 with regard to the findings of this study and the one done in Gondar.

Response: line 286 not contradict with line 291 because they are two different studies conducted in Gondar by different authors.

4. Lines 285-289: Herein lies the opportunity to give mileage to the importance of this study because you have findings from different parts of Ethiopia especially with regard to prevalence of anemia. Please explore this more and highlight it!

Response:

Conclusion: This section too should be revised once the statistical analysis is revised.

Line 343: How high is high considering that some studies have reported figures as high as 80%? What magnitude of abnormality is considered relevant at a public health or global health level? It would be more prudent to conclude within the context of your study for instance, "Anemia was the most common cytopenia (%) in HIV-infected adults receiving HAART at the Mahal Meda hospital in Ethiopia. This finding however did not meet the WHO criteria for a severe public health problem"

Response: We have revised the conclusion section and substituted the word ‘’high’’ by the actual magnitude (see line from 341-347).

Response to reviewer #2

We appreciate that the reviewer’s comments. The followings are our point-by-point responses:

- This paper would benefit from thorough proof-reading for grammatical (e.g. subject-verb agreement, missing pronouns, non-standard capitalization), typographical and spelling mistakes (e.g. "Privet" instead of "private" which are quite extensive throughout the manuscript

Response: We tried to correct grammatical, typographical and spelling errors in the manuscript and the revised manuscript was edited by professional English editor.

- In the tables presenting the main analyses (4,5 and 6), some of the data for the multivariate analyses is missing and it is not apparent why this is the case..

Response: Variables with p < 0.20 at bivariate analysis were included in the multivariable analysis that is why some of the data for the multivariate analyses are missed. (see from line142-144)

- In table 3, I would suggest using the words "prevalence" or "frequency" instead of "magnitude". Also, the definitions of "mild, moderate, severe" should be included either in the table or as a footnote to the table

Response: based on the suggestion, we replaced the word "magnitude" by prevalence (see table 3). We have already stated the definitions of severity of anemia and thrombocytopenia (mild, moderate, severe) in method section under operational definitions. To reduce redundancy we haven’t included in the table or as a footnote to the table. (See from line 120-123 and line 127-129)

-It was not clear how the sample size of 499 was selected. Was this based on potential precision of the prevalence estimates or the comparative univariate analyses?

Response: We tried to make clear how the sample size of 499 was selected (line 96-100).

- Some discussion of the limitations of the methods and statistical analysis is warranted. For example, most of the outcomes were common (>10%) and logistic regression has a tendency to be biased away from the null when the dependent variable is not rare. Additionally, please comment on whether any interactions between the variables were explored.

Response: We have included some limitations of the study (see from line 336-340). However, we revised the statistical analysis we had used before. Instead of odds ratio, we have estimated the Prevalence Ratio (PR) and its 95% confidence interval as a measure of association between the exposure and outcome using Poisson regression model with robust error variance. So we didn’t included as limitation of the study (see line 139-144).

-For the result of a higher prevalence of anemia among males, it would be good to explain why that may be the case in this particular patient population given what has previously been observed from studies in the literature

Response: In our study, the prevalence of anemia was higher in males than females; this might be due to the difference in the definition of anemia (see line 294-296). However, it needs further study to know the exact reason in the study area.

-For Table 1, consider rearranging the table or selecting the appropriate presentation of the data, as some of the variables are summarized twice

Response: We have rearranged table 1 by merging some categories of the independent variables.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

6 Aug 2020

PONE-D-20-04588R1

Prevalence of cytopenia and its associated factors among HIV infected adults on highly active antiretroviral therapy at Mehal Meda Hospital, North Shewa Zone, Ethiopia.

PLOS ONE

Dear Dr. Gebreweld,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Sep 20 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Benn Sartorius, PhD

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

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 #2: (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 #2: Yes

**********

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

Reviewer #2: 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 #2: 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 #2: 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 #2: The authors have addressed most of my concerns. Although substantially improved from the first draft, the manuscript still requires additional thorough proof-reading and correction of grammatical/typographic errors such as consistency in verb tense, non-standard capitalization (e.g. "Male" instead of "male" in the middle of a sentence see line 256 as an example), missing words (e.g. see line 232 "Anemia *was* also...").

Additionally, the tables also require some revisions. One, each table should be able to stand alone and not rely on the text, hence the definitions of severity should be included in the footnote. Secondly, the standard number of decimal places for effect estimates (e.g. PR) is 2. In the tables the number of decimal places is inconsistent (sometimes 2 and sometimes 3). Additionally, there should be a space between the n and the %, e.g. 128 (66.0) instead of 128(66.0).

**********

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 #2: No

[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.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

18 Aug 2020

Response to reviewer

We appreciate the reviewer’s comments. The followings are our point-by-point responses:

Comment: The authors have addressed most of my concerns. Although substantially improved from the first draft, the manuscript still requires additional thorough proof-reading and correction of grammatical/typographic errors such as consistency in verb tense, non-standard capitalization (e.g. "Male" instead of "male" in the middle of a sentence see line 256 as an example), missing words (e.g. see line 232 "Anemia *was* also...").

Response: We have corrected the grammatical/typographic errors, non-standard capitalizations, and missing words in the manuscript.

Comment: Additionally, the tables also require some revisions. One, each table should be able to stand alone and not rely on the text, hence the definitions of severity should be included in the footnote. Secondly, the standard number of decimal places for effect estimates (e.g. PR) is 2. In the tables the number of decimal places is inconsistent (sometimes 2 and sometimes 3). Additionally, there should be a space between the n and the %, e.g. 128 (66.0) instead of 128(66.0).

.

Response: We have included the definitions of severity in the footnote of Table 3.We have corrected the number of decimal places for PR in the tables and texts and we have put a space between n and % in the tables.

Looking forward to hearing from you. Thank you again for your consideration!

Sincerely,

Angesom Gebreweld (BSc, MSc)

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

2 Sep 2020

Prevalence of cytopenia and its associated factors among HIV infected adults on highly active antiretroviral therapy at Mehal Meda Hospital, North Shewa Zone, Ethiopia.

PONE-D-20-04588R2

Dear Dr. Gebreweld,

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,

Benn Sartorius, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

7 Sep 2020

PONE-D-20-04588R2

Prevalence of cytopenia and its associated factors among HIV infected adults on highly active antiretroviral therapy at Mehal Meda Hospital, North Shewa Zone, Ethiopia.

Dear Dr. Gebreweld:

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,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Benn Sartorius

Academic Editor

PLOS ONE