The burden and types of anaemia among HIV infected, ART-naive injection substance users in Kenya

Introduction Illicit substance use and HIV infection cause haematological derangements. Anaemia characterized by a reduction in the quality and quantity erythrocytes is the most common disorder in both HIV-positive persons and illicit substance users. Objective To describe anaemia burden, types, and its association with HIV in injectable substance users in Mombasa, Kenya. Methods This descriptive case-control study evaluated red cell indices and morphology in 494 adults. The primary outcome was anaemia. The association of anaemia with HIV in injection substance users was determined using the chi-square test. Results The participants included 275 injection substance users (ISU), (HIV-positive, n=62 and HIV-negative, n=213); and 219 non-injection substance users (nonISU), (HIV-positive, n=33 and HIV-negative, n=186). Overall, 49% were anaemic with anaemia burden significantly differing across the groups, X2(3, N=494) =12.1, p=0.0070. Anaemia burden was higher in HIV-positive ISU compared to HIV-negative ISU (odds ratio (OR) = 1.59, 95% confidence interval (CI): 0.85, 2.96); and HIV-positive nonISU compared to HIV-negative nonISU (OR = 0.37, 95% confidence interval (CI): 0.17, 0.79). Most of the anaemia was dimorphic in both HIV-positive (ISU, 67% and nonISU, 52%) and HIV-negative (ISU, 43% and nonISU, 55%) participants. Conclusion Infection with HIV is associated with increased risk of anaemia in injectable and non-injectable substance users. Majority of the anaemia was dimorphic suggestive of multiple aetiologies. Establishing the related aetiologies is essential for the effective treatment of anaemia. The accurate evaluation of thin blood films remains an essential tool in diagnosing an array of haematologic disorders and as a reference for further tests and patient management.


Introduction
The use of illicit substances and the non-medical use of prescription medicines is an increasing global health problem 1 . Reports by the United Nations Office on Drugs and Crime (UNODC) indicate that 5.6% of ciated with life-threatening medical conditions such as anaemia, substance dependence and blood-borne infections such as the human immunodeficiency virus (HIV), the hep-atitis B virus (HBV) and the hepatitis C virus (HCV) 4,29,30 In 2018, the UNODC reported that 450,000 deaths in 2015 were directly attributable to substance use 5 . Illic-it substance use is also implicated in the soaring HIV burden and HIV disease progression 6 . Statistics from the UNODC in 2018 showed that 12.5% of illicit in-jection substance users were positive with HIV by the end of 2017 7 . In addition, HIV transmission rates are higher in illicit substance users compared to the gener-al population 8 . Furthermore, people who inject drugs (PWID) constitute 30% of new HIV infections in the world 9,10 . The underlying factors influencing the HIV burden include sharing hypodermic needles, flashing blood and high-risk sexual practices amongst substance users1 11 .
Illicit substance use and HIV infection are implicated in hematologic derangements 12 . Anaemia is the most frequent hematologic manifestation in both the HIV positive 13 and illicit substance users 14 . Whereas studies implicate anaemia as the prevalent hematologic disorder, little is known about the burden and types of anaemia in injectable substance users. Anaemia in HIV-positive persons is life threatening as it is associated with enhanced HIV disease progression and increased mortality and morbidity 28 . Substance use is also associated with haematological derangements including anaemia. Some of the interventions undertaken to manage and treat anaemia have yielded little or no results even after prolonged management/treatment 31,32 . This necessitated the need to understand the types of anaemia for effective management and treatment. The World Health Organization (WHO) laboratory guidelines recommend the establishment of haemoglobin concentration and the evaluation of use of complete blood count (CBC) in diagnosing anaemia 15 . This method is prone to bias as the blood parameters are altered with variations in plasma volume without changes in the erythrocyte mass 16 , as well as the body's ability to compensate for blood loss during the onset of anaemia 17 . Moreover, CBC does not amply reveal the morphological changes in blood cells, which are important in elucidating primary and secondary blood disorders and their pathophysiology. To overcome these drawbacks, we combined the CBC and the microscopic examination of thin blood films to adequately diagnose and characterize anaemia in HIV infected injection and non-injection substance users.

Materials and methods Study design and study site
This descriptive clinical case-control study was conducted as part of a wider study investigating the socio-demographic, nutritional and microbiologic determinants of HIV infections among injection substance users in Mombasa County 11 . Further information about the study area has been previously published 18 .

Study population
The study population comprised of 494 adults (males, n=329 and females, n=165) aged between 18 and 65 years. The study subjects were grouped as follows: 1) HIV-positive injection substance users (HIV+ISU, n=62); 2) HIV-negative injection substance users (HIV-ISU, n=213); 3) HIV-positive non-injection substance users (HIV + nonISU, n=33) and 4) HIV-negative non-injection substance users (HIV-nonISU). Injection substance users were individuals having visible needle scars and having reported using an injection substance at least once in the previous month. Participants were classified as either HIV-seropositive (cases) or HIV-seronegative (controls) using the DetermineTM HIV test kit. All the HIV-positive participants recruited into this study had not been previously initiated to antiretroviral therapy. A detailed description of the sampling techniques and study population have been published 11 .

Ethical considerations
Ethical approvals for the study were obtained from the Kenyatta University (Protocol KU/R/COMM/51/32-4) and the Masinde Muliro University of Science and Technology (Protocol MMU/COR: 403012-vol2 [8]) institutional review board (IRB). All the respondents were exhaustively educated as per the internationally recommended guidelines 19 and written informed consent obtained prior to enrolment.

Collection of blood samples
Venous blood samples were collected from the freely consenting participants by a certified phlebotomist using a Vacutainer assembly into 5ml EDTA vacutain-erTM tubes (BD, Franklin Lakes, USA). Blood was collected between 8.00am and 10.00am to obtain strictly comparable values and control for diurnal variations in blood parameters 20 . All laboratory tests were performed within the hour of sample collection to maintain sample integrity.

Preparation and examination of thin blood film (BF) for red cell morphology
Duplicate thin blood films were made on new microscope slides (Thermo-ScientificTM) to prevent cell aggregation and stain precipitation. The prepared films were Leishman stained 22 . Briefly, the freshly prepared blood films were thoroughly air-dried and fixed in absolute methanol for 2 minutes. Afterwards, the blood films were flooded with undiluted Leishman stain for a few seconds. The stain was then diluted with buffered water (pH. 6.8) and left to stand for 10 minutes for differentiation. The stained blood films were then washed off under a gentle stream of running tap water to remove excess stain. The back of the slide was wiped, and the blood films placed standing on a draining rack to dry. Once dried, two independent blinded hematologists assessed the blood films for erythrocyte morphology and recorded their findings on a standard RBC morphology form. Slides with extreme variations in the results of the two haematologists were re-read by a third independent haematologist. Ten per cent (n=50) of the read slides were randomly selected and the results confirmed by a pathologist.

Assessing anaemia Anaemia burden
Anaemia was defined as haemoglobin (Hb) concentration less than 13.0g/dL and 12.0g/dL in males and females, respectively 15 . The burden of anaemia was defined as the ratio of the occurrences of anaemia to the number of sampled individuals within each study group.
Haemolytic anaemia: Prescence of schistocytes, basophilic stippling, nucleated erythrocytes, spherocytosis, reticulocytosis, polychromasia with half-ghost cells African Health Sciences, Vol 22 Issue 1, March, 2022 and erythrocytes with "bitten out" margins, and sickle cells in the blood smear 31 . Additionally, we assessed the full blood count for reduced MCV (<80fl), normal MCV (80-100fl), raised MCH (>33pg) and MCHC (>36g/dL) as seen in haemolytic anaemia 32 . Extensive spherocytosis with reduced MCV and elevated MCH was used as a marker of autoimmune haemolytic anaemia while extensive schistocytosis with normal MCV was used as a marker of microangiopathic haemolytic anaemia.

Body mass index (BMI)
Anthropometric measures were obtained from each study participants at enrolment as per the Centres of Disease Control guidelines 33 . Height (m) was measured to the nearest 0.1 cm using the Health-o-meter PORTROD wall mounted height rod (Health O me-ter®, McCook, USA). Study participants were weighed in kilograms (kg) using a portable digital weight scale (Rich forth Electronics Co., Fuzhou, China). The BMI was calculated using the height and weight measurements of the study participants using the formula:

Statistical analysis
Statistical analyses were conducted in GraphPad Prism Version 6.01 (©2012GraphPad Software, Inc.). We used Chi-square test to determine the association between anaemia and HIV infection in injectable and non-injectable substance users. The continuous variables such as weight and height were compared across the groups using a one-way ANOVA test. Erythrocyte measures were compared using non-parametric ANO-VA (Kruskal-Wallis Test) followed by Dunn's post-hoc corrections for multiple comparisons to control for the overall type-I error. All tests were two-tailed and p values <0.05 was considered statistically significant.

Baseline characteristics
The baseline characteristics of the study participants are presented in Table 1. A total of 494 adults (males, n=329 and females, n=165) were recruited into the study. The number of participants that were substance users significantly differed by gender, X2 (3, N = 494)=120.4, p <0.0001. One-way analysis of variance showed that the effect of age was not significant across the study groups F (3, 490) =2.551, p=0.055.
Approximately 73% of HIV+ISU reported to have used injectable substances for more than a year compared to 67% HIV-ISU X2 (1, N = 275) =0.7,p=0.4172. Amongst the non-injectable substance users, 21% HIV + non ISU and 22% HIV-non ISU reported to have used substances for more than year X2 (1, N = 58)=1.4, p =0.2404. Assessment of the frequency of substance use showed that 79% of HIV+ISU reported to use the substances more than once daily compared to 94% of HIV-ISU X2 (1, N = 275) =13.7, p =0.0002. In contrast, 21% of HIV+nonISU and 26% of HIV-nonISU reported to have used the substances more than one daily X2 (1, N = 70) =0.5, p =0.4755.

Erythrocyte measures in substance users
Erythrocyte measurements are summarized in Table 2. The median RBC counts significantly differed across the groups H (3,490) =4.9, p=0.0029. Post hoc analyses using the Dunn's multiple comparisons test showed that the medial RBC count was higher amongst the HIV-ISU compared to the HIV+ISU (p=0.4062). Similarly, HIV-nonISU exhibited elevated RBC counts compared to the HIV+nonISU (p=0.1793). Significantly high RBC counts was observed amongst HIV-ISU compared to HIV+nonISU (p=0.0047). The medial haemoglobin concentrations were differing across the study groups H (3,490) =8.6, p=0.0002. Dunn's multiple comparisons test showed that haemoglobin concentration was higher in HIV-nonISU compared to HIV+nonISU (p=0.0001). Additionally, HIV+nonISU exhibited significantly lower haemoglobin concentration compared to the HIV-ISU (p=0.0035). However, no significant changes in the medial haemoglobin concentration were observed amongst the HIV-ISU and HIV+ISU (p>0.9999).
The MCV and MCH did not significantly vary across the study groups. The MCHC however, differed across the study groups (p<0.0001) being lower in HIV-ISU comparative to HIV-nonISU (p<0.0001). The RDW varied across the study groups (p=0.0024) with increased anisocytosis observed in HIV-nonISU compared to HIV-ISU (p=0.0020).

Discussion
This study describes the burden and types of anaemia in injection and non-injection substance users from Mombasa Kenya. In Addition, a detailed characterization of erythrocyte morphology and the association of HIV with anaemia in injectable and non-injectable substance users are reported.
Our results show that the burden of anaemia was higher in the HIV-positive substance users compared to the HIV-negative substance users. HIV-positive injection and non-injection substance users were more likely to suffer anaemia compared to their HIV-negative counterparts. Moreover, anaemia severity was marked in injectable substance users compared to non-injectable substance users. Significantly low erythrocyte counts were observed in HIV-positive non-injection substance users compared to the HIV-negative non-injection substance users. Therefore, HIV infection coupled with substance use exacerbates anaemia. This is likely due to the synergistic effect of excessive oxidative stress on cells associated with substance use and the direct induction of apoptosis on haematopoietic cells by HIV. Also, HIV associated auto-immune reactions combined with the impeded iron absorption in the duodenum by some illicit substances such as alcohol and khat could be a factor. Other studies show that HIV anti-viral antibodies against the Gag fragment cross-react with erythropoietin 1 (EP1) resulting in impaired erythropoiesis and the consequent manifestation of anaemia 34 . A study in West Africa revealed that HIV is associated with abnormal erythrocyte quantities and qualities 35 . HIV has been shown to disturb the division and survival of hematopoietic progenitor cells 36,37 . Moreover, HIV infection of the bone marrow stromal cells has been documented to negatively influence the production of erythropoietin, a key hormone for erythropoiesis 34,38 . This is consistent with our laboratory findings where erythrocytes were significantly reduced amongst the HIV-positive participants. Likewise, haemoglobin levels were lower in HIV+nonISU compared to HIV-nonISU and the HIV-ISU. This outcome is coherent with a Ghanaian study that reported a decline in haemoglobin concentration as a marker of HIV-disease progression 39 , and folic acid deficiency which is concomitant with jejunal pathology in HIV-positive patients 38 .
Dimorphic anaemia was prevalent across all the groups indicative of multiple aetiologies of anaemia at different stages of progression. Most participants reported to have suffered from chronic recurrent and unresolved anaemia likely due to the manifold causes of anaemia. Erythrocyte morphology amongst these individuals were predominantly consistent with those seen in iron deficiency anaemia coupled with either 1) haemolytic anaema, 2) anaemia of inflammation and 3) vitamin B12/folate deficiency. A small proportion of this population had a combined erythrocyte morphology consistent with haemolytic anaemia and anaemia of inflammation. Furthermore, anaemia of vitamin/ mineral deficiency was the third most common type of anaemia. Substance addicts avoid meals and fast to prolong the psychedelic effects of substances 40 . The participants in this study were from resource-limited backgrounds. They primarily spend whatever money they must to sustain their substance use habit. Consequently, they have poor intake of fruits, vegetables and animal products resulting in several vitamin deficiencies. Vitamins are necessary for haemoglobin synthesis (for example vitamin B12, folate) and the absorption of iron from the intestines (for example vitamin C) 41, 42 .
Moreover, nutritional deficiency could be because of substance-induced damage of the gastrointestinal mucosa. Mal-absorption states in these groups need to be investigated including the production and inhibition of the intrinsic factor, and examination bone marrow smears whch are important in differentiating real nutritional deficiencies from impaired nutrient absorption/ utilization.
Anaemia of inflammation was the second most prevalent type of anaemia. Substance use has been shown to trigger the inflammatory responses. For example, khat and alcohol use are associated with intestinal lesions that promote gastritis [43][44][45] . This intestinal inflammation is likely to cause the liver to secrete more of the hormone hepcidin which inhibits the body from utilizing stored iron (ferritin) and reduces iron absorption in the duodenum 46 .
Haemolytic anaemia was the least frequent type of anaemia. Laboratory analyses showed that 13% of the participants possibly suffered either autoimmune haemolytic anaemia (observable spherocytosis) or microangiopathic haemolysis (observable schistocytosis). It is likely that the observed intravascular haemolysis was attributable to the damping effect where drug metabolites are adsorbed on erythrocytes which become antigenic resulting in their untimely splenic clearance by drug dependent/independent antibodies and macrophages 47 . Normochromic anaemia is also associated with the accelerated red blood cell turnover and suppression of red blood cell production even when there Over 50% of the anaemia was hypochromic. Normocytic-hypochromic and microcytic-hypochromic anaemia were the predominant hypochromic anaemias. A study by Dancheck et al.,49 demonstrated that normocytic hypochromic anaemia in HIV-positive and HIV-negative women was associated with the use of illicit injection substances resulting in iron deficiency 49 . We speculate that injection substance users suffer chronic inflammation due to frequent skin abscesses and vasculitis resulting in increased circulating inflammatory cytokines (e.g., Tumour necrosis factor alpha (TNFα)) that affect erythropoiesis and could contribute to microcytic hypochromic anaemia 50,51 . Normocytic normochromic anaemia is associated with chronic inflammation, erythrocyte annihilation and the desertion of erythrocyte precursors in the bone marrow 52 .
HIV disease progression has been associated with the desertion of erythrocyte precursors in the bone marrow 53,54 . Substance use has been shown to activate the inflammatory response. For example, khat and alcohol use have been documented to cause intestinal lesions leading to gastritis [43][44][45] . This intestinal inflammation is likely to cause the liver to secrete more of the hormone hepcidin which inhibits the body from utilizing stored iron (ferritin) and reducing iron absorption in the duodenum 46 . It would be of interest to evaluate markers of inflammation such as the C-reactive protein (CRP) and procalcitonin (PCT) in these groups.
One potential limitation of this study is in its design. A longitudinal survey would be more informative in assessing the association and development of anaemia with other factors such as the duration, frequency and type of substance used. However, this limitation does not significantly impact our findings as the outcome would possibly be similar. Second, invitro cultures for erythropoiesis with physiological concentrations of the substances used would be informative in evaluating the specific erythropoietic pathways affected. Third, conducting reticulocyte counts would have given a more detailed evaluation of anaemia in the study population.
However, we opted to analyse RBC histograms which are faster and as informative as the manual reticulocyte count. Toxicological analyses would be of value in correlating the concentration of substance metabolites in body fluids to the severity of anaemia. Finally, biochemical investigations such as iron, vitamin B12 and folate studies would be critical in distinguishing nutritional deficiencies due to intake and/or bioavailability. Investigating indirect bilirubin levels and haemoglobinuria would have added important information in characterizing haemolysis.
This study provides a detailed description of erythrocyte changes associated with anaemia in HIV-positive and HIV-negative injectable substance users. This information is valuable for the effectual clinical treatment and management of anaemia, more so in these groups who suffer from unresolved and recurrent episodes of anaemia despite of commencing treatment. The early and accurate diagnosis of dimorphic anaemia is important such that treatment may be effective amongst injection substance users. Thus, it is desirable that the clinico-haematological diagnosis of anaemia be accompanied by the critical evaluation of erythrocyte morphology on thin blood smears. Current practices in the diagnosis of anaemia rely majorly on the estimation of haemoglobin concentration which alone is inadequate to accurately identify dimorphic and other types of anaemia with adverse consequences on the choice of method for anaemia treatment and management. Therefore, regardless of the technological advancement in medical diagnostics, the accurate evaluation of thin blood films remains an essential tool in diagnosing an array of haematologic disorders and a reference for further tests and patient management.

Conclusion and Recommendations
Infection with HIV is associated with increased risk of anaemia in injectable and non-injectable substance users. Majority of the anaemia was dimorphic suggestive of multiple aetiologies. Establishing the respective aetiologies is essential for the effective management of anaemia in illicit substance users. The accurate evaluation of thin blood films remains an essential tool in diagnosing an array of haematologic disorders and as a reference for further tests and patient management.

Funding
This study was supported, in part, by the Kenya Nation- Authors Contribution "TW and VB designed the study", "EMK, TW and VB conducted data collection and laboratory studies", "TW and EMK, data analysis, interpretation and co-drafted the manuscript"; "DHM critically revised the manuscript for important intellectual content".

Declarations of interest
None.