Anemia Burden, Types and Associated Risk Factors among Kenyan Human Immunodeficiency Virus-1 and Mycobacterium Tuberculosis Co-infected Injection Substance Users

Background Although injection substance users and individuals co-infected with Human Immunodeficiency Virus-1 and Mycobacterium tuberculosis suffer marked hematologic derangements, the rates, levels, morphologic types and associated risk factors of anemia among Human immunodeficiency virus and Mycobacterium tuberculosis coinfected injection substance users has not been reported in Kenya. Methods This cross-sectional study determined anemia rates, levels and morphologic types. Anemia was associated with clinical markers of disease- underweight, immunosuppression and viral load. Complete blood count, CD4 T-cell enumeration and viral load were determined via standard laboratory methods. Results All injection substance users had higher rates of anaemia (HIV+TB+ ISUs, 79.3%; HIV-TB+ISUs, 70.0%; HIV+TB- ISUs, 56.6% and HIV-TB- ISUs, 56.2%) relative to non-ISUs (16.6%; P<0.05). A significant proportion of HIV+TB+ISUs (47.8%) developed severe anemia than other clinical groups. The commonest morphologic type of anemia in HIV+TB+ISUs was microcytic hypochromic (43.5%) followed by normocytic hypochromic (17.4%) relative to the other clinical groups. HIV+TB+ ISUs with CD4 T-cells <200/uL (OR: 2.94, 95% CI: 1.41–6.13, P=0.004) and CD4 Tcells of 200–349/uL (OR: 3.24, 95% CI: 1.66–6.31, P=0.001) associated with higher odds of developing anemia. Conclusion This study revealed that severe anemia and microcytic hypochromic anemia are the most common erythrocytic sequelae among Human Immunodeficiency Virus-1 and Mycobacterium tuberculosis co-infected ISUs. Those with CD4 T-cells < 350/uL are utmost expected to develop anemia.


INTRODUCTION
Injection substance use is an emerging public health problem which exacerbates the rate of HIV infection (1). It also raises the risk of HIV infected injection substance users (ISUs) developing or acquiring other comorbidities (2,3). Approximately, 13 million people inject illicit substances globally; of these, about 1.7 million are HIV positive (4). HIV infected ISUs suffer other co-infections like hepatitis B virus, hepatitis C virus, oral candidiasis, herpes zoster and tuberculosis (TB) (3). Tuberculosis is among the most prevalent co-infections in HIV-1 infected ISUs (2).
In Africa, close to 0.6 million people are injection substance users (ISUs) of whom 13.6% are living with HIV (5). In Kenya, over 50,000 ISUs have been identified, and the number is on the increase predominantly in coastal, urban sites, peri-urban areas and cities (6). The prevalence of HIV infection in Kenyan ISUs is ~18.3% with higher incidence of 18.7% mainly at the coast linked to high rates of injection substance use (7). Previous studies have reported TB to be the common co-infection in Kenyan HIV infected ISUs (8).
HIV infection is associated with marked hematological derangements including leucopenia, thrombocytopenia, immunosuppression and anemia (9,10). Anemia is the most frequent hematological abnormality among HIV infected individuals more so the immunocompromised and those with higher viral load (11). Anemia can be classified as mild, moderate and severe based on the hemoglobin levels (12). Moreover, anemia can be categorized morphologically on the basis of mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC), all derived from peripheral blood films microscopically (13,14). Determination of anemia types in patients is vital since it informs patient prognosis and management (15).
Anemia in HIV is further complicated by other comorbidities including injection substance use and TB (16). For instance, chronic use of methylamphetamine leads to severe aplastic anemia (17). In addition, cocaine and heroin use have been linked with iron deficiency anemia in HIV-1 infected females (18,19). Furthermore, TB has been shown to cause marked hematologic derangements especially anemia in TB mono-infected patients (20). Taken together, injection substance use, HIV and TB seem to have adverse hematologic changes with severe immunosuppression and abnormal iron metabolism which can possibly lead to anemia. However, anemia rates, levels, morphologic types and related risk factors have not been characterized in Kenyan HIV-1 and TB co-infected ISUs. Therefore, we established anemia burden, levels, morphologic types and the impacting factors among HIV and TB coinfected injection substance users in Kenya.

Study site and population:
This cross-sectional study was conducted at Bomu Hospital, Mombasa County, Kenya. Upon obtaining written informed consent, 443 adults (males, n = 309 and females, n = 134) were recruited, categorized into HIV+TB+ISUs (n = 29); HIV-TB+ISUs (n = 20); HIV+TB-ISUs (n = 129); HIV-TB-ISUs (n = 201) and non-ISUs (n = 64) with no evidence or history of injection substance use. ISUs were defined as individuals exhibiting needle scars and reporting use of any illicit injection substance from the United Nations Office on Drugs and Crime (UNODC) report (21).
Sample collection: From every non-ISU and ISU, 10 ml of venous blood was collected. The sample was divided into EDTA and plain BD vacutainer tubes (BD, Franklin Lakes, USA). Complete blood count was conducted immediately using the fresh EDTA samples. Clotted samples in Plain BD vacutainer tubes were centrifuged at 1500 r.p.m for 10 minutes. The serum samples were obtained and used for HIV sero-testing. Sputum samples were collected in dry, clean, leak proof sterile container from ISUs with the history of prolonged coughing, fever and chest pain for more than two weeks for TB diagnosis.
HIV-1 testing: HIV-1 was tested using rapid immunochromatographic tests, Determine TM (Abbot Laboratories, Tokyo, Japan) and Unigold TM (Trinity Biotech Plc, Bray, Ireland). Based on the Kenyan national HIV testing algorithm (22), ISUs who tested positive for both Determine and Unigold were confirmed infected with HIV.
TB testing: Standard sputum smears were prepared under level-3 Biosafety cabinet, heatfixed, and then covered with carbol fuchsin stain. Carbol fuchsin covered smears were then heated until vapor just begun to rise at about 60 • C ensuring no overheating. The heated smears were allowed to remain on the slide for 5 minutes then the stain washed off with clean water. The smears were then covered with 3% v/v acid alcohol for 5 minutes until were sufficiently decolorized (pale pink), washed well with clean water and covered with malachite green stain for 2 minutes. The stains were washed off with clean water. The back of the slides were wiped clean, and placed in a draining rack to air-dry. The stained smears were then examined microscopically, using the 100 oil immersion objective to detect the presence of mycobacterium tuberculin.

Measurement of WBC, Platelets and Red Cell Indices:
BC-3200 Mindray automated Hematology analyzer (Mindray Inc, New Jersey, USA) was controlled using the normal, low and high blood controls for quality outcomes. Hematological profiles comprising total and differential WBC, RBC, Hb, HCT, MCV, MCH, MCHC, RDW, PLT, PCT and MPV were determined and assessed for derangements across, between and within study groups. Viral load determination and enumeration of CD4 Tcells were conducted as described elsewhere (23). Demographic and anthropometric characteristics (age, gender, weight, height, BMI), red blood cell counts and its indices, CD4 T-cell counts and viral load, all presented as medians and interquartile ranges (IQR), were compared across groups using Kruskal Wallis test, followed by Dunn's posthoc test for multiple comparison. Differences in the distribution of anemia, its severity and morphologic types were compared using Chisquare test. Multiple logistic regression analyses were conducted to determine associations between anemia development and BMI, CD4 Tcells and viral load in HIV+TB+ISUs. P ≤ 0.05 was considered statistically significant.
Clinically, erythrocytic size and intra-RBC hemoglobin are used as diagnostic markers for determining morphological types of anemia (27). As a result, we established morphological types of anemia across the clinical groups.

DISCUSSION
This study focused on the rates, levels, morphologic types and related risk factors of anemia in ISUs. We compared distribution in HIV+ TB+ ISUs, HIV-TB+ ISUs, HIV+ TB-ISUs, HIV-TB-ISUs and non-ISUs with the main outcome group being HIV+ TB+ ISUs. RBCs, in their normal quantities and morphology, transport and deliver oxygen to all human body tissues (28). Derangements in RBC indices impair the body's physiological and homeostatic roles (29,30). This further leads to tissue hypoxia (31), disease progression, poor prognosis hence shortened life span (32). While studies have described levels, rates, morphologic types and associated risk factors of anemia in HIV-1 infected individuals, TB and HIV-TB coinfected individuals (26,33,34), little is known about anemia in the context of HIV-1 and TB co-infected Kenyan injection substance users.
We found high anemia rates and levels in all diseased clinical groups. These findings imply that injection substance use, HIV-1 and TB appear to modulate anemia. Similarly, other studies have reported anemia independently in the context of injection substance use, HIV-1 and TB. For instance, injection substance use has been shown as an independent risk factor associated with iron deficiency anemia (35). Also, HIV-1 infection is associated with low hemoglobin levels (36). Likewise, several recent studies have reported anemia in TB infected patients (34). However, a combination of all these possible anemia etiologies has not been studied in the milieu of Kenyan ISUs.
Consequently, the current study found very high rates and levels of anemia in HIV and TB co-infected ISUs with a majority presenting with severe anemia. A case study in a heroin addicted injection drug user suggested that hitting the groinal femoral artery with a syringe instead of the femoral vein could be causing iron deficiency anemia in the patient (37). Additionally, Cocaine use has been linked with iron build up in the brain hence depleting iron supply for hemoglobin synthesis (38). Like other studies, we found ISUs to have low BMI suggesting malnutrition among injection substance users which is associated with iron deficiency anemia. HIV has been suggested to directly infect erythroid progenitor cells causing anemia (39,40). Moreover, HIV derived proteins and cytokines have been proposed as inhibitors of hematopoietic cells in the bone marrow leading to anemia (41,42). The most plausible mechanism implicated in anemia in pulmonary TB is chronic inflammation that leads to malabsorption of iron (34). Consequently, there is a possibility of an amalgamation of these mechanisms as well as malnutrition resulting in high anemia rates and levels among HIV+ TB+ ISUs.
Normocytic normochromic and microcytic hypochromic anemia were the most common morphologic types in ISUs mainly, HIV-TB-, HIV+ TB -, HIV-TB+. This suggests that TB infection, HIV disease, cocaine and heroin use play key roles in pathophysiology of these anemia types. This finding is corroborated with previous studies which have reported the majority of TB and HIV patients with normocytic normochromic and microcytic hypochromic anemias (11,43,44). Furthermore, heroin and cocaine have been shown to disrupt iron regulation and depress developmental potentials of hematopoietic stem cells (17,45,46). Contrarily, we found dominance of microcytic hypochromic anemia in HIV+ TB+ ISUs (43.8 %). Consistent with lower BMI suggesting malnutrition, altered iron metabolism, reduced production of erythropoietin and suppressed bone marrow activity could be the probable explanations for this phenomenon.
Nonetheless, injection substance users with CD4 <350 cells/uL were highly associated with anaemia, a finding in agreement with previous studies which have reported CD4 <350uL as one of the major predictors of anaemia among injection substance users (47). This could suggest suppressed erythropoietic activity by heroin or cocaine, viral progression and aggressive invasion by opportunistic tuberculosis. Thus, HIV, its virulent proteins, TB, heroin, cocaine and proinflammatory cytokines including IL-1, IL-6, TNF-α altogether could be responsible for anemia severity among HIV+ TB+ ISUs (48).
In conclusion, these results indicate that weight loss, severe anemia and microcytic hypochromic anemia are the most common anthropometric and erythrocytic deviations among HIV and TB co-infected ISUs with CD4 T-cells < 350/uL in the Kenyan Coast. This is probably due to micronutrient deficiencies, chronic inflammation and direct or indirect effects of HIV, TB and illicit substance injection. The management of HIV and TB coinfected ISUs in our view should incorporate iron, folate and vitamin B12 levels determination. In addition, cases with insufficiencient iron or vitamins be given supplements to improve haematologic recovery.
Definition of ISUs was based on selfreported definition. Also, no toxicological analysis was performed on urine or serum samples to identify and quantify substances used including their metabolites. Moreover, ferritin levels and reticulocyte counts were not done to reinforce diagnosis of iron deficiency anemia suggested by microcytic hypochromic cells. Likewise, bone marrow aspirations and biopsies were not examined to give more information on marrow hematopoietic potentials among ISUs.