Hematological and immunological abnormalities among children receiving highly active antiretroviral therapy at Hawassa University College of Medicine and Health Sciences, Southern Ethiopia

Background: The human immunodeciency virus (HIV) epidemic remains a serious challenge and continues to take its roll, on vulnerable populations such as children. Hematological and immunological abnormalities are common complications in children infected with human immunodeciency virus. They are associated with an increased risk of disease progression and death. Hence; specic diagnosis and determination of these parameters are required for monitoring of treatment to avert disease progression. Therefore, this study was aimed to determine hematological and immunological parameters among HAART-experienced children at Hawassa University comprehensive specialized Hospital. METHODS A hospital-based cross-sectional study was conducted among 273 HIV-infected children from July to December 2019. Data were collected using a structured questionnaire that included variables related to sociodemographic characteristics and clinical conditions of the study individuals. Blood samples for hematological and immunological parameters were collected and analyzed using SPSS version 20. P-Value < 0.05 considered statistically signicant. Treatment; AZT, Zidovudine; CBC, Complete Blood Count; CD4+, Cluster differentiation; FACS, Fluorescence-Activated Cell Sorting; Highly Active Antiretroviral Immunodeciency


Page 3/21
The human immunode ciency virus (HIV) epidemic remains a serious challenge and continues to take its roll, particularly on vulnerable populations such as children. At the end of 2016 UNAIDS reported that there were approximately 36.7 million people worldwide living with HIV/AIDS, of these, 2.1 million were children < 15 years old and 70% (1.5 million) of them were found in Sub-Saharan Africa (1). In the same year, one million people died from acquired immunode ciency syndrome (AIDS) related illnesses. The annual rate of new HIV infections globally was believed to be 1.8 million (2). Approximately; 12% of these infections (330,000 cases) occurred in children < 15 years of age annually In Ethiopia, there is a signi cant pediatric HIV burden with approximately 65,100 infected children, with an estimated 3200 AIDS-related child deaths occurring annually (3).  (5) HIV replicates not only in CD4 lymphocyte cells but also in macrophages and dendritic cells (6,7) such replication is followed by immune system depression, which can lead to life-threatening opportunistic infections.
Hematological complications such as mild-to-severe anemia are associated with HIV disease progression and subsequently reduced survival (8) Although numerous complications occur in HIV infected patients, (5,9) the most common hematological abnormalities are anemia and neutropenia (10). Anemia and neutropenia are generally caused by inadequate blood cell production because of bone marrow suppression by an HIV infection mediated by abnormal cytokine expression and alteration of the bone marrow microenvironment (8). Anemia in HIVinfected persons is associated with CD4 cell depletion and progression to AIDS (9) and is one of the strongest predictors of HIV mortality and poor responses to antiretroviral therapy (5) Neutropenia is frequently observed in advanced stages of HIV infection after the development of AIDS and has been associated with certain types of antiretroviral medications used to treat HIV infection (11).
The initiation of highly active antiretroviral therapy played a critical role in the clinical management of HIV infected individuals by restoring the immune function, preventing morbidity and mortality, improving quality of life, and preventing the transmission of the virus to other uninfected individuals (13). But a signi cant number of patients fail to achieve a sustained virological and immunological suppression to treatment among the HIV-infected patients receiving HAART (14). Even after 6-12 months of rst-line treatment, several children are failed to attain viral suppression and it could be because these children may have been infected with mutant resistant viruses from mothers that were already on treatment with rst-line HAART (15).
Hematological parameters are other important monitoring tools for assessing treatment and prognosis among HAART experienced HIV positive children. The use of antiretroviral drugs could positively or negatively affect these parameters, depending on the choice of combination of drugs used making hematological abnormalities common among HAART experienced HIV positive persons. For example, lamivudine and zidovudine combination may cause Neutropenia, anemia, thrombocytopenia, and transient rise in liver enzymes, while Nevirapine has been reported as eosinophilia, granulopenia, and jaundice. Anaemia, neutropenia, thrombocytopenia have also been reported as adverse effects of Stavudine (16). Those attributed to Zidovudine and Stavudine include neutropenia, anemia, and thrombocytopenia. Although many drugs used for the treatment of HIV-related disorders are myelosuppressive, severe cytopenia is most often related to the use of zidovudine (17).
Hematological complications have been documented to be the second most common cause of morbidity and mortality in HIV positive children on HAART and are generally marked with cytopenias such as anemia, neutropenia, lymphopenia, and thrombocytopenia (18). The incidence and severity of the cytopenia generally correlate to the stage of the disease with anemia being the most commonly encountered hematologic abnormality and a signi cant predictor of progression to AIDS or death.
There is limited data regarding the hematological complication, immunologic failure and associated factors among HAART experienced children in the southern region of the country. Therefore, this study was aimed to assess the prevalence of hematological and immunological abnormalities among children on HAART in HUCSH ART pediatrics clinic, Hawassa, southern Ethiopia.

Study design
A hospital-based cross-sectional study was conducted from July to December 2019 on 273 HIV-infected children receiving HAART.

Study area
The study was done from July to December 2019 at Hawassa University comprehensive specialized Hospital. It is one of the tertiary hospitals in the country with a catchment area of 15-22 million people and the largest academic institutions in Ethiopia. The university is located at the heart of Hawassa city, which is the capital city of the region and located 275 Km from Addis Ababa, the capital city of Ethiopia.
The altitude of the city is 1697meters above sea level with the mean annual temperature and rainfall of Participants.
All 273 HIV-infected children < 15 years of age who took HAART for > 6 months who had a follow-up on pediatrics ART clinic at Hawassa University Comprehensive and specialized hospital and who were voluntary to participate, in the study were included.
Children on treatment for the known hematological disorder and who had transfusion treatment within three months of data collection, those who had a traumatic injury or surgical interventions resulting in blood loss during the study period or within 3 months before the study period and children with their incomplete information, unreadable or their manual record is lost, and who do not have baseline CD4 count and those without a legal guardian or unaccompanied children were excluded from the study.

Sample size
To have a representative sample of our entire pediatrics ART follow-up clinic, all 273 children who were on follow up in ART clinic of Hawassa University College of Medicine and Health Science Comprehensive and Specialized Hospital were used as a sample size.
Data collection tools and procedure Data on the sociodemographic and clinical characteristics of the study participants were collected using a pretested structured questionnaire by interview and review of medical records these were composed of sociodemographic characteristics, and for baseline characteristics of the study subjects before initiation of HAART; these included mainly clinical, laboratory and immunological characteristics after initiation of HAART. Data were collected by two trained professional nurses and one pediatrics resident working at the pediatrics ART clinic.
The laboratory measurements were done by experienced laboratory technologists. The data collection process was supervised strictly by the investigator. All study subjects were approached during their respective appointment.

Laboratory Testing
Specimen Collection and Processing 5 ml of blood was collected by following standard operational procedure (SOP) for sample collection and transport to determine both CBC, CD4 tests, and viral load is drawn from each participant using vacutainer tube containing anticoagulant ethylene diamine tetra-acetic acid (EDTA). Hematological parameters were run on hematological auto-analyzer ruby Cell-Dyne 3000 USA (Abbott Laboratories Diagnostics Division, USA) whereas the immunological (CD4 + T cells) was run on BD FACSCOUNT system (Becton Dickenson and Company, California, USA). The performance of both analyzers was controlled by running quality control material alongside the study participant's sample. Besides, all agged specimen was subjected to the manual differential to con rm the results. Immunosuppression and anemia were de ned based on WHO criteria (Reference interval) as de ned operationally.

Quality Assurance
To ensure the quality of data, training was given to data collectors and supervisors, and a pre-test was done on adult patients. The necessary feedback was offered to data collectors in the next morning.
Besides, the collected data were checked for validity, completeness, and internal consistency by the principal investigator daily. Quality Control of both CBC and CD4 analyzer was run daily before patient sample analysis.

Data management and analysis
The data were cleaned, edited, checked for completeness, and entered and analyzed using SPSS version 20 statistical software. The results were reported as the mean and standard deviation for continuous variables and as percentages for categorical variables. An unpaired t-test was used to compare the means of all continuous variables. Categorical data were analyzed using the Fisher exact test. Logistic regression analysis was applied to determine the associations of established risk factors for hematologic abnormalities and to determine the determinant of immunologic failure. We used a CI of 95% and P < 0.05 to evaluate for any signi cant association.

Operational de nition
Clinical failure is de ned as the appearance or reappearance of WHO clinical stage 3 or stage 4 events after at least 24 weeks on ART in a treatment-adherent child (19).
Immunological failure and anemia: is de ned as developing or returning to the following age-related immunological thresholds after at least 24 weeks on ART, in a treatment-adherent child and anemia (19). CD4 count of < 200 cells/µL or percent CD4 cell count < 10% for a child ≥ 1 year to < 5 years of age and a CD4 count of < 100 cells/ µL for a child 5 years of age or older. Anemia was de ned based on WHO criteria (R) as mild anemia: hemoglobin level between 10 and 10.9 g/dL for under 5 and between 11 and 11.9 g/dL for under 18 years of age children; moderate anemia: hemoglobin level between 7.0 and 9.9 g/d for under 5 and between 8.0 and 10.9 g/dL for under 18 years of age children; severe anemia: hemoglobin level < 7.0 g/dL for both under ve and under 18 years of age children.
Virological failure is de ned as a persistent HIV viral load of ≥ 5,000 copies/ml, after at least 24 weeks on ART, in a treatment-adherent child (20).
Based on the de nitions of treatment failure above, ART treatment is deemed effective if none of the clinical, immunological, or virological criteria for failure are met after at least 24 weeks on ART. However, for this study, the de nition of effectiveness is based on the immunologic and virological criteria for failure. Therefore treatment effectiveness was de ned as HIV viral load < 5,000 copies/ml after at least 24 weeks on HAART.

Ethical Consideration
This study was reviewed and approved by the Institutional Review Board (IRB) of Hawassa University College of Medicine and Health Science. A support letter was obtained from HUCSH Chief clinical director o ce and permission was obtained from the pediatrics ART clinic to collect the necessary data. Informed written consent was taken from the caretakers and assent was obtained from older children (above 8 years old) before enrollment in the study. Then the objective of this research was explained to the study participants, and those willing to participate were included. To ensure con dentiality of participant's information, anonymous typing was applied. Each participant was interviewed alone to keep privacy. Test results were given to the clinicians who are working on the pediatric ART clinic of the hospital for further diagnosis and management.

Socio-Demographic and Clinical Characteristics of Study Participants
A total of 273 HAART-experienced HIV-positive children were enrolled in this study. Of whom: 139(50.9%) were females and 82.1% were urban residents. The majority, (80.6%) of the participants were above seven years old. The mean age and standard deviation (SD) of the study participant were 10.2 ± 3.2 years. Clinically, 49.5% of the participants were in WHO clinical stage I. From the total study participants, 89.4% were on AZT/3TC/EFV, 83.9% did not take other drug and 11.7% had CD4 cell count of less than or equal to 350 cells/mm 3 (Table 1).  The immunological progression of the participants was indicated by the increasing trend of the CD4 + T cell median percentage from 18.4% at baseline to 29.2% during the study period and similarly, the mean hemoglobin level also increased from 12 mg/dl at baseline to 13.1 mg/dl during the study period (Fig-1).

Prevalence of hematological abnormalities and its distribution by CD4 + T cell and viral load
Out of the total number of study participants, 18.3% had Neutropenia, 14.3% had Leucopenia, 4% had Thrombocytopenia, 2.9% had Pancytopenia and 11.4% had Anemia (Table 3).
In the current study, the association of the distribution of hematological abnormalities and different CD4 + cell percentage was observed and all forms of hematological abnormalities were highly prevalent among study participants with CD4 cell percentage of < 15%. However; it was statistically signi cant with leucopenia (P = 0.02), Leucocytosis (P = 0.02), and lymphopenia (P = 0.001) ( Table 3). While when we compare the hematological abnormalities and different strati ed (ND, < 150 and > 150) viral loads, all forms of hematological abnormalities were highly prevalent with study participants who had greater than 150 viral copies / mm3 and it was statistically signi cant with anemia (P = 0.002), Lymphopenia (P = < 0.001), and pancytopenia (P = 0.001) ( Table 3). Higher prevalence of anemia and Leucocytosis among children of age group < 5 years of age in the current study was indicated as (25%) and (18.8%) respectively. Leucopenia was highly prevalent among 5-10 years old children and Neutropenia and lymphopenia were higher among children within the age group of 11-14 years. Similarly higher prevalence of Neutropenia (21.6%), lymphopenia (12.3%), thrombocytopenia (5.3%), and pancytopenia (3.5%) was found among children of older than the age group of (11-14) years old.
The prevalence of hematological abnormalities such as anemia (11.5%), neutropenia (18.7%), and pancytopenia (3.6%) had higher distribution among female children than males, but; no statistically signi cant association was observed (Table 4). Based on WHO grading; Neutropenia grading was indicated in the current study as severe, moderate and mild as indicated in the gure below (Fig. 2)

Predictors of CD4 T + cell change among study participants
In linear univariate analysis, among the factors that predicted the CD4 T cell count change; only viral load (B. = -4.9; R2 = 0.050.09; p-value = < 0.001 was found to be a predictor of CD4 + T cell change among children receiving HAART in the current study.

Discussion
The baseline prevalence of anemia before the initiation of HAART in this study was 111(40.7%). This result was comparable with the studies reported from northwest Ethiopia (42.8%) (21) Uganda 47.8% (22) and Brazil (37.5%) (23). But; our result was lower than the Indian study 65.5% (24). It was also higher than the study done in Addis Abeba, Ethiopia (18.9%) (25). The difference could be due to the difference in the WHO clinical stage, presence of opportunistic infections, nutritional status, population, and geographic difference might some of the factors.
The prevalence of anemia after initiation of HAART was (11.4%) which was in line with the study conducted in Addis Abeba, Ethiopia(10.4%) (25) but; lower than the study done in northwest Ethiopia (18.9%) (26) and Nigeria (54.2%) (27). This variation might be attributed to the differences in ethnicity, study designs, and time of the study. Besides, variation in age of the study participants, HAART status and cutoff value in de ning anemia, local prevalence of parasitic infections such as malaria or hookworm, as well as local nutritional patterns might contribute to the variation in magnitude of anemia.
The prevalence of leukocytosis in our study was (4.8%) which is relatively comparable with the study done in North West Ethiopia (4.1%) (16) but; lower than the study done in Kenya (6.2%) (28). In this study, the prevalence of Neutropenia was (18.3%) which was higher than the study conducted in northwest Ethiopia (15.8%) (26), Bahir Dar, Ethiopia (9.8%) (29), Addis Abeba, Ethiopia (5.7%) (25) and Kenya (16%).The possible difference might be the difference in the immunological status of the study participants and sample size. Neutropenia has been widely reported to be associated with some HAART drugs, particularly combination drugs that included zidovudine. Prophylactic co-trimoxazole, which is often used in these patients to prevent opportunistic infections, is thought to be the cause of neutropenia through an unknown mechanism (30). Any in ltrative process involving the bone marrow (infection, malignancy) may produce granulocytopenia. In clinical practice, however, drug toxicity is responsible for most of the granulocytopenia seen in patients with HIV infection. AZT therapy is probably the most common cause of neutropenia in patients with HIV infection (31). The prevalence of leukopenia was 14.3% which is relatively comparable with the study done in northwest Ethiopia (13.5%) (26) but higher than the study done in Bahir dar, Ethiopia (4.5%).
The difference in the prevalence could be due to the difference in sample size, geographical location, and the cut off value used. The mechanism of thrombocytopenia in HIV infection is mainly due to ineffective platelet production and increased platelet destruction by the spleen. Other possible causes of thrombocytopenia in HIV patients are immune-mediated destruction, TTP, impaired hematopoiesis, and toxic effects of medications and infections (31).
The immunological and clinical failure among HAART-experienced children in the current study was 4% and 5.5% respectively. In which both of these parameters were lower than a retrospective cohort study conducted in the Oromia region, Ethiopia (6.69%), and (12.26%) respectively (40 D.)). An original copy of the approval, as well as the consent and assent form, is available upon request. A permission letter was obtained from Hawassa University hospital administrations.
The purpose and importance of the study were explained to each study participant, parents, and guardians. Finally, informed written consent and assent were obtained from each study participant, and any information obtained during the study was kept with utmost con dentiality, and those found infected with bacterial meningitis were treated in the hospital.

Consent for publication
Not applicable.
Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.  Neutropenia Grading among HAART-Experienced children at HUCSH ART clinic (n=273).

Figure 3
Clinical and Immunological characteristics of HAART-experienced children at HUCSH (n=273).

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. Table11.docx