Technological response

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hyperhomocysteinemia may add an additional risk among patients with other risk factors for venous clots.

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OPPORTUNISTIC INFECTION

In spite of the efficacy of HAART, HIV-positive individuals are at the greatest risk for developing opportunistic infections depending on their immunologic status. So, the concomitant presence of advanced HIV disease and opportunistic infections appears to be an additional risk factor for Thrombosis.20, 136,162,163

VTE is most commonly reported with Cytomegalovirus and Pneumocystis jiroveci pneumonia (PCP) and Mycobacterium avium-intracellulare.^135,164

HIV-ASSOCIATED MALIGNANCY

The risk of VTE in patients with cancer varies considerably between patients and even within an individual patient over time. Estimates ranging from 15 to 30 % have been reported.¹⁶⁵It is well-known how patients with no identifiable risk factors who develop DVT may have an underlying occult malignancy.¹⁶⁶

People with HIV infection and AIDS have an elevated cancer risk.¹⁶⁷ Kaposi's sarcoma and lymphomas are the only HIV-related tumours that have been proven to be procoagulant.^{109, 168}

The Swiss-Thai-Australia Treatment Interruption Trial (STACCATO), in which participants restarted ART when their CD4+ T-cell count fell below 350 cells/mm³ (rather than 250 cells/mm³ in SMART), also revealed a link between HIV viral load and inflammation biomarkers. A variety of markers, including D-dimer, VCAM-1, P-selectin, MCP-1, and leptin, decreased as HIV was suppressed on ARV therapy and rose during treatment

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interruption. In contrast, levels of anti-inflammatory biomarkers, including IL-10 and adiponectin, increased as viral load declined and fell during treatment breaks.¹⁶⁹

Neuhaus et al in a comparison of inflammation biomarkers in people with and without HIV looked at SMART participants and HIV-negative individuals in two large population-based cardiovascular studies. People with HIV had significantly higher levels of markers including IL-6, CRP, and D-dimer. Levels were higher in HIV-positive participants both on and off ARV therapy compared with HIV-negative people, and this link remained after adjusting for traditional cardiovascular risk factors.¹⁰⁴

The elevation of these biomarkers among HIV-infected persons on effective ARV therapy as well as those not on ARV therapies may reflect ongoing immune activation even with successful suppression of HIV replication.¹⁷⁰

Although many pathways have been investigated to determine the mechanism of thrombosis, the only strong evidence available appears to be a protein S deficiency.¹⁶¹Other factors appear to play at least some role, and it appears inevitable that the mechanisms underlying thrombosis associated with HIV infection are multimodal.

Reducing inflammation and thrombotic activity may represent an additional therapeutic goal in the clinical management of HIV infection in the current era.

Therapies that reduce the inflammatory response to HIV and decrease specific biomarkers such as hsCRP, D-dimer, IL-6, or TNF-α, should be studied as potential interventions.

D-dimer test may also be added as routine test for HIV patients to detect those at increased risk of thrombosis. So that thromboprophylaxis can be offered if necessary.

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LABORATORY TEST FOR D-DIMER

D-dimer is considered a marker of hypercoagulable state and of endogenous fibrinolysis, so increased D-dimer is detectable in patients affected by arterial and/or venous thrombosis.¹⁷¹ Clinical conditions, such as chronic inflammation, infectious disease (also as marker of disseminated intravascular coagulation if sepsis is associated) cancer, necrosis, in the elderly, pregnancy, immobility and sickle cell disease we may observe an increase of plasma D-dimer.89,172,173,174,175,176,177,178

Many dimer tests are available that use various methods. Laboratory testing for D-dimers may be qualitative, semiquantitative, or quantitative.¹⁷⁹

Qualitative D-dimer tests yield a positive or negative result depending on whether the level of D-dimer in the specimen exceeds the minimum detection level. Qualitative methods include whole blood agglutination (SimpliRed, D-dimer cassette test, simplify D-dimer test) and latex agglutination (Minutex D-dimer,Dimertest kit).¹⁷⁹

Semiquantitative dimer tests yield results in various ranges. Examples of this type of D-dimer test include the NycoCard D-D-dimer and INSTANT I.A.These tests are based on immunofiltration technology.¹⁷⁹

Quantitative D-dimer tests, considered the “gold standard” in D-dimer testing, directly measure the amount of D-dimer present. These tests have been labor intensive, but newer rapid tests are available with turnaround times of 1 hour.¹⁷⁹

Quantitative methods include the enzyme-linked immunosorbent assays (VIDAS D-dimer;

Asserachrom D-dimer, Enzygnost D-dimer Micro and FibrinostikaFbDP) and the newer

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latex-enhanced turbidimetric tests (Advanced D-dimer, LIATEST Ddimer, IL Test D-dimer and Tinaquant D-dimer).¹⁷⁹

ELISA assays are the reference standard for D-dimer quantitation. These assays utilize microtiter wells coated with an antibody with a high affinity for D-dimer. The classic enzyme-linked immunosorbent assay (ELISA) is highly sensitive but is also time consuming, labor intensive, and impractical for use as an emergency test.¹⁸⁰

The reporting standard for D-dimers varies with the test methodology and reagent manufacturer. Latex and whole blood agglutination results are usually reported as a D-dimer range (ng/mL). ELISA and immunoturbidimetric assays are usually reported in fibrinogen equivalent units (FEU). One FEU is the quantity of fibrinogen that was initially present before it was broken down. The actual quantity of D-dimer is approximately half of an FEU (when 1.0 of fibrinogen ug/mL is broken down, 0.5 ug/mL of D-Dimer remain).²⁴The normal reference range for D-dimer value is ≤ 500 ng/ml.

ELIZA assays have a sensitivity of 93-95% and a specificity of about 50% (typically between 40% and 60%) and the low specificity limits its usefulness, as this leads to a high rate of false-positive results.¹⁸¹

False positive may be seen with high levels of rheumatoid factor (a protein seen in patients with rheumatoid arthritis) high triglycerides, lipemia,bilirubin and hemolysis(caused by improper sample collection and handling).^{24, 182}

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LABORATORY TEST FOR CD4 COUNT

CD4(cluster of differentiation 4) is a glycoprotein expressed on the surface of T helper cells, monocytes, macrophages, and dendritic cells. The CD4 protein is encoded by the CD4gene located on chromosome 12.¹⁸³

CD4+ T-lymphocyte counts is essential in determining disease stage and progression. It is also crucial in determining when to start or change antiretroviral (ARV) therapy and in the monitoring of ARV therapy.¹³

Flow cytometry is the "gold standard" for accurate and automated measurement of CD4+

T- lymphocytes, but the technique is expensive and requires sophisticated equipment as well as trained personnel to perform it. Lack of ready access to technical support and quality assurance programs has also limited the use of flow cytometry techniques in resource-constrained countries.^{13, 14}

Current clinical flow cytometric methods use either dual (the product of flow cytometry derived lymphocyte subset percentages and the automated haematology instrument-derived absolute lymphocyte count) or single platforms (absolute counts instrument-derived directly from the flow cytometer) for the enumeration of absolute lymphocyte subpopulation counts.

Imade et al in a study carried out in Nigeria comparing a new, highly simplified, low-cost, true volumetric flow cytometer (Cyflow) with a manual magnetic bead (Dynabead) method produced a comparable and well-correlated CD4+ T-lymphocyte counts (P = 0.057, r

=0.93).The second important finding was that Cyflow produced more precise and reproducible CD4+ T-lymphocyte counts than the manual method and it was more cost

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effective.¹⁸⁴ These findings agree with earlier reports which involved the comparison of CD4+ T-lymphocyte counts by Dynabead and other flow cytometry techniques.^13,185

On the other hand, manual non-flow cytometric techniques such as the Dynabead and Coulter methods, which produce results comparable to those of flow cytometry, have their own limitations. They are labor-intensive, and only a few (10 to 15) CD4+ T-lymphocyte counts can be performed by an analyst per day.¹³

CHAPTER THREE

AIMS AND OBJECTIVES OF THE STUDY

General Objective

To study D-dimer level in HIV treatment-nạve and treatment-experienced patients in Port-Harcourt, Nigeria.

Specific Objective

1. To compare the D-dimer level between HIV-positive treatment-nạve and treatment- experienced patients.

2. To determine the correlation between D-dimer, CD4+ T-cell count and some other haematological parameters.

RATIONALE FOR THE STUDY

The main objective of this study was to document if HIV treatment-nạve patients have higher D-dimer level (which is a biomarker of thrombotic risk) compared to treatment- experienced patients in Port-Harcourt, Nigeria where there is paucity of data.

It will be interesting to know if HIV treatment-nạve patients in this region also have higher D-dimer level than treatment-experienced patients with its associated higher risk of thrombosis, leading to increased morbidity and mortality, which is potentially preventable.

The knowledge acquired will help us to manage our HIV positive patients better, by identifying individuals at high risk of VTE, and also help us to design a more rationale anticoagulant prophylaxis for varieties of surgical procedures and other conditions associated with increased predisposition to thrombosis, because of the high cost of treatment in a resource-constrained setting.

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