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Antiretroviral therapy has proven to be effective in the treatment of human immunodeficiency virus infection by suppressing replication of the virus within host cells (1). Viral replication within the host is followed by depression of the immune system. This predisposes the patient to life threatening opportunistic infections (2). Suppression of viral replication results in a significant improvement in immune status, quality of life and clinical outcomes (3-6). Although the success of ART is well established, some issues have remained of great concern. The drugs cannot eradicate the virus and like all drugs that are regularly administered for a long time, they have documented toxicities and adverse effects (7-11).
Haematological abnormalities are a common finding in HIV-patients and have been documented to be the second most common cause of morbidity and mortality in HIV- patients. The most common haematological abnormalities are cytopaenias such as anaemia, neutropaenia, lymphopaenia and thrombocytopenia (12-15). The origin of haematological abnormalities has been attributed to the direct effect of the HIV virus on the bone marrow, opportunistic infections, neoplasms and antiretroviral drugs (16-18). Findings from bone marrow studies showed that even in the absence of other disease processes, bone marrow morphology is invariably abnormal and cytopaenias are common during the course of the disease (19). Although ART has been shown to be effective in normalising haematopoiesis in HIV-patients, a number of haematological abnormalities are a direct effect of the administration of these drugs.
Macrocytosis is one of the haematological abnormalities that have been observed in association with the administration of antiretroviral drugs, especially zidovudine (20). Macrocytosis due to other nucleoside reverse transcriptase inhibitors (NRTIs) has also been reported (21, 22, 23). Although ARV-associated macrocytosis is well documented, the mechanisms of this macrocytosis have not been fully understood. No study in Zimbabwe has also looked specifically at evaluating this macrocytosis. The increased mean cell volume (MCV) in HIV-patients on antiretroviral therapy could be an early indicator of bone marrow toxicity or an indication of a good response to therapy by the bone marrow (24, 25, 26, 27). Some researchers argue that the high MCV is not an indicator of bone marrow toxicity but may actually help to define the subgroup of people resistant to the myelosuppressive effect of ARVs (28).
The term macrocytosis refers to a mean cell volume (MCV) greater than 97 femtolitres (fl). MCV is an automated red blood cell parameter that reflects the volume of the red blood cells and is important in the assessment of anaemia. Macrocytosis is commonly due to an increased number of reticulocytes in peripheral circulation. Other causes include vitamin B12 and folate deficiencies, medications, alcoholism, liver disease, malignancy, hypothyroidism, splenectomy, and chronic obstructive pulmonary disease (28).
A reticulocyte count is one of the initial evaluations in the investigation of possible causes and aetiology of macrocytosis in a patient presenting with a high MCV. This would allow a systematic consideration of the possible causes. Serum vitamin B12 and folate levels are then determined if the reticulocyte count is normal. Other studies may then be undertaken as appropriate. A reticulocyte count is a diagnostic haematological parameter that expresses the number of reticulocytes in peripheral blood as a percentage of total red blood cells. Apart from being used to investigate macrocytosis, a reticulocyte count is also useful in assessing the bone marrowâ€™s response to an anaemic condition, therapy and the production of new cells by a bone marrow transplant.
Reticulocytes are red blood cell precursors that precede the fully developed red blood cells during erythropoiesis. Under conditions of normal haematopoiesis, reticulocytes constitute about 0.5% to 2.0% of peripheral red blood cells. Reticulocytes are released into the circulation to replace aging and destroyed cells. A count less than 0.5% is termed reticulocytopaenia while a count greater than 2.0% is termed reticulocytosis. Normally the bone marrow is stimulated by erythropoietic stress associated with a rise in plasma erythropoietin levels to produce and release more reticulocytes into the circulation resulting in reticulocytosis and consequently macrocytosis. The bone marrow may fail to respond to stimulation because of an intrinsic problem within the bone marrow itself or due to suppression by therapy and inflammatory cytokines resulting in reticulocytopaenia (29, 30). The enumeration of reticulocytes in the peripheral blood is therefore a measure of the bone marrowâ€™s erythropoietic activity.
Macrocytosis in HIV-patients on ART could be an indication of two completely different processes involving the bone marrow. The high MCV after commencement of ART could mean a good response to therapy by the bone marrow. ARVs have been shown to be effective in improving the ability of precursor cells in the bone marrow to proliferate into clones. The ability of these cells to differentiate and proliferate is suppressed by HIV-infection (31). Increased formation and differentiation of BFU-E progenitor cells results in increased production of reticulocytes. The reticulocytes are then pushed into the circulation in increased numbers to meet increased demand for oxygen induced by impaired haematopoiesis. Reticulocytosis consequently result in macrocytosis. The high MCV could also be an indicator of bone marrow toxicity (24, 25). Determining the reticulocyte count would differentiate between the two possible mechanisms. Reticulocytosis (a count greater than 2%) indicates a good response to therapy while a low reticulocyte (a count less than 0.5%) could indicate suppression of the bone marrow by ARVs.
ARVs are known to cause bone marrow suppression resulting in macrocytosis with low reticulocyte counts (25). The myelosuppressive effects of nucleoside analogs (NRTIs) are thought to be due to their effect on the enzyme DNA polymerase in the erythroid precursors. These drugs compete with natural deoxynucleoside triphosphate for binding to reverse transcriptase of HIV and human DNA polymerase. As these drugs do not specifically the target the replicating virus binding to DNA polymerase in the rapidly dividing cells can result in impaired synthesis of erythrocyte precursors (31) .Other mechanisms have also been proposed. The lack of correlation between haematocrit and reticulocyte counts in HIV-patients with ARV-induced anaemia may be due to this suppression (32). Suppression of the bone marrow by ARVs is also characterised by ARV-induced cytopaenias. Treatment of HIV with the same ARVs has however been shown to improve haematopoiesis (28, 29, 33, 34). Improved haematopoiesis is characterised by improved reticulocyte counts.
Haematopoiesis in HIV-patients may also be suppressed by lymphoma cells, infections such as TB and inflammatory cytokines such as tumour necrosis factor, interleukin-1 and interferon-gamma (28, 29). Suppression of the bone marrow results in low reticulocyte counts despite stimulation of the bone marrow by decreased oxygen carrying capacity of the blood. However bone marrow suppression by opportunistic infections, lymphoma cells and inflammatory cytokines has not been associated with macrocytosis. Macrocytosis with low reticulocyte counts (counts < 0.5%) can also be attributed to bone marrow malignancies, pernicious anaemia, vitamins deficiencies (folate, vitamin B12). However in a study to determine causes of macrocytosis in HIV-patients on antiretroviral therapy, vitamin B12 and folate serum levels were not decreased in patients with macrocytosis (22).
A high reticulocyte count (count greater than 2%) is attributed to conditions that stimulate an increased red cell production to compensate peripheral blood cell destruction (haemolysis) or loss (haemorrhage). Such conditions may include autoimmune haemolytic anaemia (AIHA), microangiopathic haemolytic anaemia and gastrointestinal bleeding. These conditions have an increased incidence in HIV-patients. Reticulocytosis also occurs during active blood regeneration (stimulation of the bone marrow). Observations are that after the administration of highly active antiretroviral therapy (HAART) there was a significant increase in colony growth of all types of progenitor cells (CFU-E, BFU-E, CFU-GM and CFU-GEMM) in all patients who had decreased formation and differentiation of these progenitor cells due to HIV infection (29).
Another study demonstrated that cytopaenias due to HIV-infection were effectively reversed by administration of HAART. Reversal of the cytopaenias was characterised by a remarkable recovery of all cell lines (35).The increased formation and differentiation of the BFU-E progenitor cell results in increased production of reticulocytes. The reticulocytes are then pushed into the circulation in increased numbers to meet demand. The increased number of reticulocytes in peripheral blood can consequently result in macrocytosis as reticulocytes in the peripheral blood have a mean cell volume of 103 to 126 fl (27). This is the only mechanism of non megaloblastic macrocytosis that is fully understood.
An increase in reticulocyte count was observed after the discontinuation of zidovudine in a case report of zidovudine induced-pure red cell aplasia (PRCA). PRCA is one of the manifestations of the myelosuppressive effect of ARVs (25). The increase in reticulocyte count could consequently result in a high MCV. Only zidovudine was discontinued while the other ARV in the combination, lamivudine was not discontinued. Should the increase in reticulocyte count be attributed only to the discontinued use of zidovudine, which was suppressing the bone marrow or be also attributed to the use of lamivudine? Haematological reconstitution has been observed in HIV-patients on HAART because ARVs induce the ability of bone marrow precursors to proliferate into clones (29).Several explanations have been made for the mechanisms and indications of ARV-associated macrocytosis but no study has been done in Zimbabwe to evaluate this macrocytosis. This study intends to investigate the observed ARV-associated macrocytosis in Zimbabwe.
1.2 Statement of the problem
ARV-associated macrocytosis is well documented, however no research has been done in Zimbabwe to investigate the probable aetiology of the observed ARV-associated macrocytosis. This study aims to investigate the aetiology of ARV- associated macrocytosis.
H0: Reticulocyte counts in HIV-patients with macrocytosis are greater than 2%.
Ha: Reticulocyte counts in HIV-patients with macrocytosis are not greater than 2%.
1. To determine the reticulocyte counts of HIV patients who have been on ARVs for more than six months showing increased MCV values.
2. To determine drug regimens associated with macrocytosis.
CHAPTER TWO: MATERIALS AND METHODS
Listed in appendix 1
2.2.1 Study site
Parirenyatwa Opportunistic Infections Clinic.
2.2.2 Study design
Laboratory based cross sectional analytical study.
2.2.3 Study subjects
188.8.131.52 Inclusion criteria
HIV- patients eligible for inclusion in this study were those with high MCV values after being on ARVs for more than six months.
184.108.40.206 Exclusion criteria
HIV-patients excluded in this study were those on ARVs for less than six months and those on ARVs for six months but without increased MCV values. Patients with conditions other than HIV or AIDS that may cause bone marrow suppression or cytopaenia were also excluded. These include patients with: (1) patients with any malignancy such as lymphoma or patients who had received chemotherapeutic agents within six months prior to enrolment, (2) patients with medication history, for example antibiotics, ganciclovir, trimethoprim-sulfarmethoxazole, anti-TB medications known to induce cytopaenia within two weeks prior to enrolment, (3) patients with HBV or HCV co-infection or liver cirrhosis(11).
2.2.4 Ethics statement
Permission to carry out the proposed project was sought from the Joint Parirenyatwa and College of Health Sciences Research Ethics Committee. Informed consent was obtained from participants on their visit to the clinic. Access to the blood samples and patient notes of consenting patients was sought from authorities in charge of OIC patient samples and PGH Laboratory. Whole blood samples were used in this study.
2.2.5 Sample size
Reticulocyte counts of 259 participants were determined.
2.2.6 Sample size calculation
See appendix 2
2.2.7 Laboratory methods
220.127.116.11 Sample identification and patient confidentiality
In order to maintain patient confidentiality no patient names were used. Samples were each allocated a unique research number, for example S1, S2etc. Any other required information was recorded against corresponding research numbers.
18.104.22.168 Sample analysis
Samples were well mixed and checked for clots before analysis. Reticulocyte counts were determined using a Sysmex XT 4000i haematology blood analyser.
22.214.171.124 Principle of method
Reticulocytes can be distinguished from mature macrocytes by their polychromatophilic or diffusely basophilic staining. The polychromatophilic staining is due to remnant ribonucleic acid (RNA) which can be stained supravitally with new methylene blue or brilliant cresyl blue. This property is the basis of the manual reticulocyte count. Fully automated blood analysing machines such as a Sysmex XT 2000i or X T4000i employ the principle of flow cytometry using RNA-specific fluorescent dyes and a semiconductor laser. Mature red blood cells, reticulocytes and white blood cells are differentiated based on the difference in nucleic acid content. Platelets and nucleated red blood cells are excluded from consideration by gating (forward scatter versus side scatter)