Section 3 Testing for HIV

#### **Chapter 5**

### Streamlining Laboratory Tests for HIV Detection

*Ramakrishna Prakash and Mysore Krishnamurthy Yashaswini*

#### **Abstract**

HIV is a retrovirus that primarily infects CD4 presenting cells of the human immune system, such as macrophages and dendritic cells. People die of AIDS because the disease remains undetected for long periods of time. HIV diagnostic testing has come a long way since it was introduced in the early 1980s. Early diagnosis is key to successful treatment of HIV. Assay selection is based on initial screening results and clinical information provided by the physician, both of which are essential for the laboratory's ability to make accurate diagnoses. Detecting HIV with high specificity and sensitivity in the early stages of infection requires simple, accurate and economical methods. In this chapter we have described the indications & criteria's for HIV testing, HIV diagnosis by utilizing variety of immunological and molecular methods, like ELISA, rapid diagnostics, Western blotting, indirect immunoassays, and nucleic acid-based tests. Diagnostic laboratories must use testing algorithms to ensure the accuracy of results and the optimal use of lab resources. Participation in laboratory quality assurance programs are also essential to ensure that diagnostic laboratories provide accurate, timely and clinically relevant test results. HIV testing is the first step in maintaining a healthy life and preventing HIV transmission.

**Keywords:** generations, HIV antibody test, HIV diagnosis, HIV testing algorithm, quality assurance, Western Blot

#### **1. Introduction**

Early detection and diagnosis is key to ending the HIV/AIDs pandemic by 2030. The need for timely and quality programs to enhance rapid, timely, accurate and relevant tests as the initial step in reducing HIV and maintaining the quality of life is a fundamental process. This chapter describes the known tests for HIV Infection. The author bases his discussion on significant systematic review of literature of HIV diagnostic tests. The aim is to explain and provide rationale for the use of tests available. The chapter also describes, and discusses the various indications, criteria for HIV testing, detection and identifies phases and stages of laboratory detection. A variety of immunological and molecular methods are outlined and discussed. These include the simplest from point of care such as ELISA diagnostic tests, rapid tests and brands. It also extends the discussion to more advanced tests including indirect immunoassays, nucleic based assays and the Western blot confirmatory tests. The laboratory algorithms are discussed to promote quality assurance in practice. The

paper discusses the difficulties encountered during the early window period of infection and suggests appropriate detection tools. The staging and the dynamics of HIV viremia post infection and its implications for detection is discussed. The classifications of tests from first generation to forth generation is described and recommendations made on their appropriate usage for early and sustained quality in detection of infection. The challenges of detection during the acute and the window period post infection is discussed and suggestions made. Establishing several testing stages is discussed to support quality HIV detection and ideal screening and confirmatory tests for each stage are recommended. The review has a potential benefit to improve HIV [1, 2].

#### **2. Indications for HIV testing**

HIV testing should be considered in the following situations. The healthcare team should be aware of the screening recommendations [3].


#### **3. Criteria's for HIV testing**

There are ample of tests which can detect HIV starting from the point of care testing to confirmatory test. The test algorithm to be followed has been released by the Centre for Disease Control (CDC) and Association of Public Health Laboratories (APHL) as well as the national organizations in every country [4, 5].

#### **3.1 Clinical laboratory improvement amendments (CLIA)**

Centers for Medicare and Medicaid Services (CMS) has regulated for all the clinical laboratory testing to be done through the CLIA. As per this amendments, a three-level test complexity criteria has been established for HIV testing procedures. The criteria are as follows [4, 5]:

• **Waived:** These are the tests which are simple to perform with low-risk, it can be performed by any person with minimal training and the specimens do not require any centrifugation for testing.

*Streamlining Laboratory Tests for HIV Detection DOI: http://dx.doi.org/10.5772/intechopen.105096*


#### **3.2 Fiebig staging system**

The Fiebig staging system (2003) defines six stages on initial HIV infection. Stage I is defined as the emergence of HIV RNA and Stage VI is defined as full Western blot reactivity. The markers which appear as per timeline after HIV infection are HIV RNA after 10 to 11 days, p24 antigen after 4 to 10 days after emergence of HIV RNA, IgM antibodies after 3 to 5 days later, IgG antibodies after 2 to 6 weeks after HIV RNA emergence [6].

#### **4. Tests used for the diagnosis of HIV**

HIV tests were classified as first, second, third and fourth generation tests based on the substrate used for testing. First generation HIV antibody tests were developed using separate HTLV III and lymphadenopathy virus (LAV) isolates proteins isolated from virus-infected tissue cultures as antigenic targets. Initially window period was up to 12 weeks or more post-infection. These assays detected only IgG antibody of HIV-1. Second generation HIV test were based on the recombinant antigens for HIV-1 p24. The window period was up to 4 to 6 weeks post-infection. Third generation HIV test can detect IgM antibody in addition to second generation tests. Fourth generation tests is a test which can detect both HIV-specific antigen p24 and HIV antibodies. This test reduced window period to approximately 2 weeks [7]. Fifth generation HIV test detects both HIV-specific antigen p24 and HIV antibodies with increased sensitivity in detection of p24 and it also identifies the individual HIV1 and HIV2 markers. The different generations of HIV tests are shown in **Table 1**.


#### **Table 1.** *Different generations of HIV tests.*

With the invention of new HIV tests, the distinction between the different generations of ELISA test has been obscure. So the generation nomenclature is being modified as:


#### **4.1 Non-specific tests**

The non-specific tests for HIV diagnosis are [13]:


#### **4.2 Specific tests for HIV infection**

These are the tests which are specifically done for testing of HIV.

#### *4.2.1 Virus isolation*

This is a time-consuming procedure which is not routinely done. The viruses are present in the lymphocytes in the peripheral blood and also seen in lymphocytes in bone marrow, plasma and other body fluids. The procedure used to cultivate HIV virus is called as **Cocultivation.** In this both infected and noninfected mononuclear cells will be co-cultivated. The culture may become positive for HIV p24 antigen and HIV reverse transcriptase by 7–14 days or by 28 days. This test will be useful when the viral load is high especially in the initial stage of the disease [14].

#### *4.2.2 Serologic tests*

These tests include demonstration of antigens and antibodies in the serum. The tests have been classified as:

1.HIV antigen-antibody laboratory-based tests.

*Streamlining Laboratory Tests for HIV Detection DOI: http://dx.doi.org/10.5772/intechopen.105096*

2.HIV antigen-antibody point-of-care tests.

3.HIV antibody laboratory-based tests.

4.HIV antibody point-of-care tests.

5.HIV 1 and 2 differentiation tests.

6.HIV-1 Western Blot test.

7.HIV Nucleic acid diagnostic tests.

8. In-home HIV tests.

#### *4.2.2.1 HIV antigen-antibody laboratory-based tests*

These immunoassay tests are the preferred screening tests which detect HIV-1 p24 (capsid) antigen and antibodies (IgM and IgG) to HIV-1 and HIV-2. (**Figure 1A**–**C**) These antigen-antibody test detect HIV infection much earlier than the antibodybased tests. If found positive in these tests, then it may require a confirmatory test. Limitation of these tests are cross-reactivity to HIV-1 p24 antigen seen in HIV-2 infected persons. Examples are ADVIA Centaur HIV Ag/Ab Combo (CHIV) Assay, ARCHITECT HIV Ag/Ab Combo, BioPlex 2200 HIV Ag-Ab Assay, Elecsys HIV Combi PT, GS HIV Combo Ag/Ab EIA, & VITROS HIV Combo Test [10, 16].

#### *4.2.2.2 HIV antigen-antibody point-of-care tests*

This assay is a single use, rapid test which is a point-of-care test for the detection of HIV-1 p24 antigen, antibodies to HIV-1 (group 0), and antibodies to HIV-2. This test does not differentiate HIV-1 and HIV-2 antibodies. This test is less sensitive for acute or recent HIV infection when compared to laboratory-based HIV-1/2 antigenantibody tests. Example: Abbott Determine HIV-1/2 Ag/Ab Combo [17–19].

#### *4.2.2.3 HIV antibody laboratory based tests*

Laboratory-based HIV antibody tests were the first to be used for screening HIV since 20 years which has been replace by HIV antigen-antibody tests. These tests can detect the IgM/IgG-sensitive assays for HIV IgM antibodies in 23–25 days after HIV infection. Window period is around 90 days. The positive result in this tests would require an confirmatory test. Examples are: ADVIA Centaur HIV 1/O/2 Enhanced, Avioq HIV-1 Microelisa System, Genetic Systems (GS) HIV-1/HIV-2 Plus O EIA, VITROS Anti-HIV 1 + 2 Assay [5, 6, 10, 12].

#### *4.2.2.4 HIV antibody point-of-care tests*

Single-use, point-of-care tests can yield result in 40 min. These tests can detect antibodies to HIV-1 or HIV-2 or both but they will not be able to differentiate between HIV-1 and HIV-2. These tests are primarily used for testing (1) emergency situations (2) pregnant women whose HIV status in not known (3) occupational, and (4) in patients for whom follow-up for HIV result will not be possible. Examples are: Chembio DPP HIV 1/2 Assay, Chembio HIV 1/2 STAT-PAK Assay, Chembio SURE

#### **Figure 1.**

*(a) Components of HIV-1/2 antigen-antibody immunoassay. (b) Patient sample reacting with components in HIV-1/2 antigen-antibody immunoassay. (c) Reactive HIV-1/2 antigen-antibody immunoassay. Source: Illustration: David H. Spach, MD [15].*

CHECK HIV 1/2 Assay, INSTI HIV-1/HIV-2 Antibody Test, OraQuick ADVANCE Rapid HIV-1/2 Antibody Test, Reveal G4 Rapid HIV-1 Antibody Test (Reveal G4), Uni-Gold Recombigen HIV-1/2 [9, 20, 21].

#### *4.2.2.5 HIV 1 and 2 differentiation tests*

These tests will be able to differentiate between HIV-1 and HIV-2. These tests utilize multiple recombinant or synthetic peptides to detect HIV-1 antibodies and HIV-2

**Figure 2.** *Geenius HIV 1/2 supplemental assay. Source: modified from [15].*

antibodies. These immunochromatographic tests will contain 7 lines which consists of 6 HIV peptides and one control. A minimum of 2 envelope peptides (gp160 and gp41) or 1 envelope peptide plus either the p24 or the polymerase peptide p31 for HIV-1 reactive or HIV-2 envelope peptides gp36 and gp140 should be present for HIV-2 reactive test. (**Figure 2**) Example: Geenius HIV 1/2 Supplemental Assay [15, 22].

The Geenius HIV 1/2 Supplemental Assay is a single-use immunochromatographic test that utilizes multiple recombinant or synthetic peptides to detect HIV-1 antibodies (p31, gp160, p24, and gp41) and HIV-2 antibodies (gp36 and gp140). The test cassette as shown here contains seven test lines, including the six HIV peptides and one control.

#### *4.2.2.6 HIV-1 Western blot test*

Western blot test is used as supplemental tests for those tests which are reactive by rapid tests. It can detect the human antibodies for three HIV-1 gene regions: env (gp41, gp120/160), pol (p31, p51, p66), and gag (p15, p17, p24, p55) (**Figure 3A-D**).

This graphic shows the relationship of the HIV-1 genes and products with the corresponding band on the HIV-1 Western blot.

CDC and the Association of State and Territorial Public Health Laboratory Directors (ASTPHLD) have published the criteria for interpretation of Western blot tests [23].

Positive: A positive Western blot indicates the presence of at least two of the following bands: p24, gp41, and gp120/160.

Negative: A negative Western blot is defined by the absence of any bands.

Indeterminate: An indeterminate Western blot results from the presence of any bands, but not meeting positive criteria. Possible causes of an indeterminate Western blot include early HIV infection, HIV-2, pregnancy, or cross-reactivity with other antibodies, such as in persons who have recently received an influenza immunization or who have autoimmune disorder.

#### **Figure 3.**

*(a) Components used in the HIV-1 Western blot; (b) HIV-1 antibodies bound to HIV-1 antigens on Western blot test strip. (c) Addition of secondary anti-human antibody linked to enzyme signal. (d) HIV-1 Western blot genes. Source: Illustration: David H. Spach, MD [15].*

#### *4.2.2.7 HIV nucleic acid diagnostic tests*

HIV RNA nucleic acid test (NAT) is used in case of 1. Reactive HIV-1/2 antigenantibody immunoassay but a nonreactive or indeterminate HIV-1/HIV-2 differentiation test, 2. HIV-1/2 antigen-antibody immunoassay is negative but there is high suspicion of acute HIV, and 3. Confirmative test for chronic HIV-1 infection. The limitation of these tests are cost, time taken to perform the test is 3 hours and the expert is required to perform the test. Example: APTIMA HIV-1 RNA Qualitative Assay [9, 24–26].

#### *4.2.2.8 In-home HIV tests*

This test can be performed at home by the client itself within 40 minutes by simply collecting an oral sample and performing the test as per kit literature. A confirmatory test will be required if this test is reactive. Example OraQuick In-Home HIV test [27].

#### **5. HIV laboratory testing algorithms**

There are several algorithms published for HIV laboratory testing among which CDC, NACO (National AIDS Control Organization) and APHL are some of them (**Figure 4**) [28].

This graphic shows the HIV testing algorithm as recommended in 2014 and 2018 by the Centers for Disease Control and Prevention (CDC) and Association of Public Health Laboratories (APHL). Source: Centers for Disease Control and Prevention and Association of Public Health Laboratories [4, 5].

*Streamlining Laboratory Tests for HIV Detection DOI: http://dx.doi.org/10.5772/intechopen.105096*

**Figure 4.** *CDC and APHL Recommended Laboratory Testing for the Diagnosis of HIV Infection.*

#### **6. Interpretation of HIV test results**


### **7. Staging of HIV and tests recommended**

Days following HIV acquisition which of the HIV diagnostic tests can show positivity for infection are shown in **Figure 5** [14, 29].

This graphic shows the HIV testing algorithm as recommended in 2014 and 2018 by the Centers for Disease Control and Prevention (CDC) and Association of Public Health Laboratories (APHL).

The stages of HIV infection and the tests that are recommended are [5, 30].


#### **Figure 5.**

*Timing of positivity for HIV diagnostic tests.This figure shows estimates for the mean number of days for HIV diagnostic tests to become positive after acquisition of HIV. Abbreviation: POC = point-of-careSource: modified from Centers for Disease Control and Prevention and Association of Public Health Laboratories [5].*

window period for the HIV 1/2 antigen–antibody tests and 90 days for all HIV antibody tests and all HIV point-of-care tests.


#### **8. Performance of diagnostic tests**

#### **8.1 An ideal screening tests**

An ideal screening test should be able to accurately identify individuals with the HIV infection and rule out infection in individuals without HIV infection.

The characteristics that define a screening test are [31]

• The disease should be a health problem.

*Streamlining Laboratory Tests for HIV Detection DOI: http://dx.doi.org/10.5772/intechopen.105096*


#### **8.2 Sensitivity and specificity**

Sensitivity and specificity refers to the diagnostic ability of a given test. Sensitivity refers to the percentage of individual who are correctly identified as having disease if they are infected with HIV. A very high sensitivity is desirable for the initial screening test so that if we get a non-reactive result we can be 100% sure that the person is not having HIV infection [32]. Specificity refers to the percentage of individuals who are correctly identified as not having disease if the person does not have HIV infection. A very high specificity is desirable for the confirmation test as a reactive result means the person is suffering from HIV infection [33].

#### **8.3 Positive predictive value and negative predictive value**

The predictive value of a test refers to the accuracy of the test. Positive predictive value refers to the proportion of patients who are correctly diagnosed as reactive. Negative predictive value refers to the proportion of patients who are correctly diagnosed as non-reactive [32].

#### **8.4 False negative and false positive HIV test**

False negative HIV test result refers to the non-reactive report in a person who is possessing HIV infection.

A false negative HIV antigen–antibody test result can be seen in [34–46]:

	- acute HIV infection,
	- from error in laboratory reporting,
	- person on antiretroviral therapy,
	- Immunosuppression.
	- Hypogammaglobulinemia.
	- Immunosuppressant medications.
	- Chronic HIV.

A false negative p24 antigen test can be seen in the window period and in chronic HIV. A false negative HIV RNA tests can be seen in first one to two weeks after HIV infection and chronic HIV.

False positive HIV test result refers to the reactive report in a person who is not possessing HIV infection.

A false positive HIV test result can be seen in [47, 48]


A false positive HIV NATs can be seen in persons receiving chimeric antigen receptor (CAR) T-cell therapy.

#### **9. Special diagnostic situation**

#### **9.1 Diagnosis of Acute HIV-1**

HIV RNA is the most reliable test for diagnosis of acute HIV-1 infection as this test can detect HIV in about 17 days after HIV infection which is much earlier when compared to all other methods of testing [49, 50].

#### **9.2 Diagnosing HIV in persons receiving preexposure prophylaxis**

Diagnosis of HIV infection in persons receiving preexposure prophylaxis is difficult due to delayed seroconversion, indeterminate results in HIV differentiation tests, and low viraemia [51].

#### **9.3 Diagnosing HIV in HIV exposed infants and children**

Antibody tests or antigen-antibody immunoassays will not be useful in diagnosis of HIV in infants or children as they may have maternal HIV antibodies. Nucleic acid tests like HIV RNA, HIV DNA polymerase chain reaction or RNA qualitative or

quantitative tests will be better option for HIV diagnosis in infants. Qualitative HIV proviral DNA PCR assays detects cell-associated virus as they are less affected by the antiretroviral drugs [52].

#### **9.4 Diagnosis of HIV-2**

Diagnosis of HIV-2 should be done using a HIV-1/2 antigen-antibody immunoassay followed by HIV-1/HIV-2 differentiation test. Confirmation of HIV-2 can be done by HIV-2 DNA/RNA Qualitative and Quantitative assays. Western blot can give a negative, indeterminate or positive HIV-1 result in HIV-2 infected individuals. Western blot will be indeterminate with the presence of gag and pol bands but the env bands will be absent in HIV-2 infection [53–57].

#### **10. Laboratory quality assurance**

The laboratory should participate in quality assurance program to ensure the quality of reports. The quality control should be monitored in preanalytical, analytical and post-analytical stages with Internal QC (quality control), external QC as well as test kit controls [58].

#### **11. Conclusions**

Testing an individual having HIV infection is important using the appropriate test at the appropriate time. Differentiation of HIV-1 and HIV-2 can be done using the differentiation assays. Diagnosis of HIV-2 and infection in infants and children requires Nucleic acid tests. Quality assurance needs to be maintained in all the labs which do HIV testing as the entire process has to be done in an appropriate manner to get the perfect results.

#### **Acknowledgements**

I would like to acknowledge the head of the department of Microbiology, Dr. Lakshminarayana S A for his encouragement and support in writing this chapter.

#### **Conflict of interest**

"The authors declare no conflict of interest."

#### **Author details**

Ramakrishna Prakash\* and Mysore Krishnamurthy Yashaswini Rajarajeswari Medical College and Hospital, Bengaluru, India

\*Address all correspondence to: prakashssmc@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Streamlining Laboratory Tests for HIV Detection DOI: http://dx.doi.org/10.5772/intechopen.105096*

#### **References**

[1] Global AIDS Strategy 2021–2026. Available from: https://www.unaids.org/ en/Global-AIDS-Strategy-2021-2026. [Accessed: April 04, 2022]

[2] Li Z, Purcell DW, Sansom SL, Hayes D, Hall HI. Vital signs: HIV transmission along the continuum of care – United States, 2016. MMWR. Morbidity and Mortality Weekly Report. 2019;**68**(11):267-272

[3] Huynh K, Kahwaji CI. HIV Testing. Treasure Island (FL): StatPearls Publishing; 2021

[4] National Center for HIV/AIDS, Viral Hepatitis, and TB Prevention (U.S.). Division of HIV/AIDS Prevention; Association of Public Health Laboratories. 2018 Quick reference guide: Recommended laboratory HIV testing algorithm for serum or plasma specimens. Available from: https:// stacks.cdc.gov/view/cdc/50872

[5] Branson BM, Owen SM, Wesolowski LG, Bennett B, Werner BG, Wroblewski KE, Pentella MA. Laboratory testing for the diagnosis of HIV infection: Updated recommendations. Corporate Authors (s): Centers for Disease Control and Prevention (U.S.); Association of Public Health Laboratories; National Center for HIV/AIDS, Viral Hepatitis, and TB Prevention (U.S.). Division of HIV/AIDS Prevention, 2014. Available from: https://stacks.cdc.gov/view/cdc/23447

[6] Fiebig EW, Wright DJ, Rawal BD, Garrett PE, Schumacher RT, Peddada L, et al. Dynamics of HIV viremia and antibody seroconversion in plasma donors: Implications for diagnosis and staging of primary HIV infection. AIDS. 2003;**17**(13):1871-1879. DOI: 10.1097/ 00002030-200309050-00005

[7] Chappel RJ, Wilson KM, Dax EM. Immunoassays for the diagnosis of HIV: Meeting future needs by enhancing the quality of testing. Future Microbiology. 2009;**4**:963-982. DOI: 10.2217/fmb.09.77

[8] Alexander TS. Human immunodeficiency virus diagnostic testing: 30 years of evolution. Clinical and Vaccine Immunology. 2016;**23**(4):249-253

[9] Branson BM. State of the art for diagnosis of HIV infection. Clinical Infectious Diseases. 2007;**45**(Suppl. 4): S221-S225

[10] Hurt CB, Nelson JAE, Hightow-Weidman LB, Miller WC. Selecting an HIV test: A narrative review for clinicians and researchers. Sexually Transmitted Diseases. 2017;**44**:739-746

[11] Branson BM, Mermin J. Establishing the diagnosis of HIV infection: New tests and a new algorithm for the United States. Journal of Clinical Virology. 2011; **52**(Suppl. 1):S3-S4

[12] Delaney KP, Wesolowski LG, Owen SM. The evolution of HIV testing continues. Sexually Transmitted Diseases. 2017;**44**:747-749

[13] Baveja CP. Retroviruses: HIV in Textbook of Microbiology. 5th ed. New Delhi: Arya Publishers; 2017. pp. 509-522

[14] Masciotra S, McDougal JS, Feldman J, Sprinkle P, Wesolowski L, Owen SM. Evaluation of an alternative HIV diagnostic algorithm using specimens from seroconversion panels and persons with established HIV infections. Journal of Clinical Virology. 2011;**52**(Suppl. 1):S17-S22

[15] David H, Spach DH. HIV Diagnostic testing. In: Spach DH, Wood BR, Kalapila AG, Budak JZ, editors. National HIV Curriculum 2nd ed. University of Washington Infectious Diseases Education & Assessment Program. 31 Aug 2020. [Accessed: March 15, 2022]. Available from: https://www.hiv.uw.edu/go/ screening-diagnosis/diagnostic-testing/ core-concept/all

[16] Delaney KP, Hanson DL, Masciotra S, Ethridge SF, Wesolowski L, Owen SM. Time Until Emergence of HIV Test Reactivity Following Infection With HIV-1: Implications for Interpreting Test Results and Retesting After Exposure. Clinical Infectious Diseases. 2017;**64**: 53-59

[17] U.S. Food and Drug Administration. Alere Determine HIV-1/2 Ag/Ab Combo

[18] Masciotra S, Luo W, Youngpairoj AS, et al. Performance of the Alere Determine™ HIV-1/2 Ag/Ab Combo Rapid Test with specimens from HIV-1 seroconverters from the US and HIV-2 infected individuals from Ivory Coast. Journal of Clinical Virology. 2013; **58**(Suppl. 1):e54-e58

[19] Masciotra S, Luo W, Westheimer E, et al. Performance evaluation of the FDA-approved Determine™ HIV-1/2 Ag/Ab Combo assay using plasma and whole blood specimens. Journal of Clinical Virology. 2017;**91**:95-100

[20] Kuhar DT, Henderson DK, Struble KA, et al. Updated US Public Health Service guidelines for the management of occupational exposures to human immunodeficiency virus and recommendations for postexposure prophylaxis. Infection Control and Hospital Epidemiology. 2013;**34**:875-892

[21] Centers for Disease Control and Prevention. Advancing HIV prevention: New strategies for a changing epidemic– United States, 2003. MMWR. Morbidity

and Mortality Weekly Report. 2003;**52**: 329-332

[22] U.S. Food and Drug Administration. Geenius HIV 1/2 Supplemental Assay

[23] Centers for Disease Control (CDC). Interpretation and use of the Western blot assay for serodiagnosis of human immunodeficiency virus type 1 infections. MMWR. Morbidity and Mortality Weekly Report. 1989;**38**(Suppl. 7):1-7

[24] U.S. Food and Drug Administration. APTIMA HIV-1 RNA Qualitative Assay [U.S. FDA]

[25] Giachetti C, Linnen JM, Kolk DP, et al. Highly sensitive multiplex assay for detection of human immunodeficiency virus type 1 and hepatitis C virus RNA. Journal of Clinical Microbiology. 2002; **40**:2408-2419

[26] Pierce VM, Neide B, Hodinka RL. Evaluation of the Gen-Probe Aptima HIV-1 RNA qualitative assay as an alternative to Western blot analysis for confirmation of HIV infection. Journal of Clinical Microbiology. 2011;**49**: 1642-1645

[27] U.S. Food and Drug Administration. OraQuick In-Home HIV Test

[28] Branson BM, Stekler JD. Detection of acute HIV infection: We can't close the window. Journal of Infectious Diseases. 2012;**205**(4):521-524

[29] Owen SM, Yang C, Spira T, et al. Alternative algorithms for human immunodeficiency virus infection diagnosis using tests that are licensed in the United States. Journal of Clinical Microbiology. 2008;**46**:1588-1595

[30] Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the use of antiretroviral agents in

*Streamlining Laboratory Tests for HIV Detection DOI: http://dx.doi.org/10.5772/intechopen.105096*

adults and adolescents with HIV. Department of Health and Human Services. Considerations for antiretroviral use in special patient populations: Acute and recent (early) HIV infection. 2019

[31] WHO Recommendations on the Diagnosis of HIV Infection in Infants and Children. Geneva: World Health Organization; 2010. Annex 4, Characteristics of a screening test. Available from: https://www.ncbi.nlm. nih.gov/books/NBK138555/

[32] Altman DG, Bland JM. Diagnostic tests 2: Predictive values. BMJ. 1994;**309**:102

[33] Altman DG, Bland JM. Diagnostic tests. 1: Sensitivity and specificity. BMJ. 1994;**308**:1552

[34] Manak MM, Jagodzinski LL, Shutt A, et al. Decreased seroreactivity in individuals initiating antiretroviral therapy during acute HIV infection. Journal of Clinical Microbiology. 2019; **57**:e00757

[35] Kassutto S, Johnston MN, Rosenberg ES. Incomplete HIV type 1 antibody evolution and seroreversion in acutely infected individuals treated with early antiretroviral therapy. Clinical Infectious Diseases. 2005;**40**: 868-873

[36] Hare CB, Pappalardo BL, Busch MP, et al. Seroreversion in subjects receiving antiretroviral therapy during acute/early HIV infection. Clinical Infectious Diseases. 2006;**42**:700-708

[37] de Souza MS, Pinyakorn S, Akapirat S, et al. Initiation of antiretroviral therapy during acute HIV-1 infection leads to a high rate of nonreactive HIV serology. Clinical Infectious Diseases. 2016;**63**:555-561

[38] Sullivan PS, Schable C, Koch W, et al. Persistently negative HIV-1 antibody enzyme immunoassay screening results for patients with HIV-1 infection and AIDS: Serologic, clinical, and virologic results. Seronegative AIDS Clinical Study Group. AIDS. 1999; **13**:89-96

[39] Spivak AM, Brennan TP, O'Connell KA, et al. A case of seronegative HIV-1 infection. The Journal of Infectious Diseases. 2010;**201**: 341-345

[40] Spivak AM, Sydnor ER, Blankson JN, Gallant JE. Seronegative HIV-1 infection: A review of the literature. AIDS. 2010;**24**:1407-1414

[41] Ellenberger DL, Sullivan PS, Dorn J, et al. Viral and immunologic examination of human immunodeficiency virus type 1-infected, persistently seronegative persons. The Journal of Infectious Diseases. 1999;**180**: 1033-1042

[42] Donnell D, Ramos E, Celum C, et al. The effect of oral preexposure prophylaxis on the progression of HIV-1 seroconversion. AIDS. 2017;**31**: 2007-2016

[43] Smith DK, Switzer WM, Peters P, et al. A strategy for PrEP clinicians to manage ambiguous HIV test results during follow-up visits. Open Forum Infectious Diseases. 2018;**5**:180

[44] Padeh YC, Rubinstein A, Shliozberg J. Common variable immunodeficiency and testing for HIV-1. The New England Journal of Medicine. 2005;**353**:1074-1075

[45] Jurriaans S, Sankatsing SU, Prins JM, et al. HIV-1 seroreversion in an HIV-1 seropositive patient treated during acute infection with highly active

antiretroviral therapy and mycophenolate mofetil. AIDS. 2004;**18**: 1607-1608

[46] Roy MJ, Damato JJ, Burke DS. Absence of true seroreversion of HIV-1 antibody in seroreactive individuals. Journal of the American Medical Association. 1993;**269**:2876-2879

[47] Klarkowski D, O'Brien DP, Shanks L, Singh KP. Causes of false-positive HIV rapid diagnostic test results. Expert Review of Anti-Infective Therapy. 2014; **12**:49-62

[48] Theppote AS, Carmack AE, Riedel DJ. False positive HIV testing after T-cell receptor therapy. AIDS. 2020;**34**:1103-1105

[49] Cornett JK, Kirn TJ. Laboratory diagnosis of HIV in adults: A review of current methods. Clinical Infectious Diseases. 2013;**57**:712-718

[50] Cohen MS, Gay CL, Busch MP, Hecht FM. The detection of acute HIV infection. The Journal of Infectious Diseases. 2010;**202**(Suppl. 2):S270-S277

[51] Sivay MV, Li M, Piwowar-Manning E, et al. Characterization of HIV Seroconverters in a TDF/FTC PrEP Study: HPTN 067/ADAPT. Journal of Acquired Immune Deficiency Syndromes. 2017;**75**:271-279

[52] Management of Infants Born to People with HIV Infection. In: Recommendations for the Use of Antiretroviral Drugs During Pregnancy and Interventions to Reduce Perinatal HIV Transmission in the United States [Internet]. 2021. Available from: https://clinicalinfo.hiv.gov/en/guidelines/ perinatal/diagnosis-hiv-infection-infantsand-children#::text=Diagnosis%20of% 20HIV%20in%20Infants,age%204%20to %206%20months [Accessed: April 15, 2022]

[53] Peruski AH, Wesolowski LG, Delaney KP, et al. Trends in HIV-2 diagnoses and use of the HIV-1/HIV-2 differentiation test – United States, 2010-2017. MMWR. Morbidity and Mortality Weekly Report. 2020;**69**:63-66

[54] Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the use of antiretroviral agents in adults and adolescents with HIV. Department of Health and Human Services. Considerations for antiretroviral use in special patient populations: HIV-2 infection. 2019

[55] Centers for Disease Control and Prevention. HIV-2 Infection Surveillance–United States, 1987–2009. MMWR. Morbidity and Mortality Weekly Report. 2011;**60**:985-988

[56] O'Brien TR, George JR, Epstein JS, Holmberg SD, Schochetman G. Testing for antibodies to human immunodeficiency virus type 2 in the United States. MMWR - Recommendations and Reports. 1992; **41**:1-9

[57] Campbell-Yesufu OT, Gandhi RT. Update on human immunodeficiency virus (HIV)-2 Infection. Clinical Infectious Diseases. 2011;**52**:780-787

[58] Consolidated Guidelines on HIV Testing Services. 5Cs: Consent, Confidentiality, Counselling, Correct Results and Connection 2015. Geneva: World Health Organization; 2015, Quality assurance of HIV testing. Available from: https://www.ncbi.nlm. nih.gov/books/NBK316025/ [Accessed: April 15, 2022]

#### **Chapter 6**

## Challenges in Platelet Functions in HIV/AIDS Management

*Gordon Ogweno*

#### **Abstract**

The interest in platelet functions in HIV/AIDS is due to the high incidence of microvascular thrombosis in these individuals. A lot of laboratory data have been generated regarding platelet functions in this population. The tests demonstrate platelet hyperactivity but decreased aggregation, though results are inconsistent depending on the study design. Antiretroviral treatments currently in use display complex interactions. Many studies on platelet functions in these patients have been for research purposes, but none have found utility in guiding drug treatment of thrombosis.

**Keywords:** HIV, AIDS, platelet functions, light transmission aggregometry, flow cytometry, microparticles, combined antiretroviral, antiplatelets

#### **1. Introduction**

There is increasing focus on platelet functions in people living with HIV/AIDS. This is because of the high incidence of cardiovascular events in these individuals that is 10 times higher than general population [1] independent of traditional risk factors such as age, hyperlipidemia, and ethnic/racial differences. Acquired platelet dysfunctions are often observed in association with HIV/AIDS. Of the available tests for platelet functions [2, 3], none fully captures the complexity involved in this population group.

The results of the functional assays are modified by the viral count, CD4/CD8 ratio, and immunological response and whether or not on antiretroviral treatment. The effects of combined antiretroviral therapy (cART) on platelet functions are complex. Despite achieving viral suppression, these drugs have been demonstrated to have independent effects on platelet functions.

#### **2. Platelet count and indices in HIV/AIDS**

Complete blood count and microscopic examination of formed elements are often the first investigations in suspected hemostatic disorders in clinical situations. Platelet count and morphological changes have impact on bleeding or thrombosis.

#### **2.1 HIV-associated thrombocytopenia**

Globally, the prevalence of HIV-associated thrombocytopenia is 4–40% [4] though there are geographical, racial as well as ethnic differences from the same locality [5] and stage of disease. Indeed, thrombocytopenia has been considered as a marker of disease progression and improvement [6]. Whereas platelet counts improve with initiation of combined antiretroviral therapy (cART) viral suppression [7], beneficial effect does not apply to zidovudin (AZT) [8].

Despite thrombocytopenia, very low rates of clinical hemorrhage have been reported, estimated at only 3.2% among HIV thrombocytopenic patients [9] even with platelet count as low as 50 × 109 /L [10] casting doubt on the clinical relevance of the laboratory results. As a result of lack of clear correlation between HIV-associated thrombocytopenia and clinical significance, some authors have questioned benefit of treatments purely directed toward improvement of platelet count [11].

#### **2.2 HIV-associated thrombocytosis**

The prevalence of HIV-associated thrombocytosis, defined as platelet count of more than 400× 109 /L [12], is low but depends on the population studied and concurrent medications. Reported prevalence of thrombocytosis in pediatric group who were also HIV-positive cART naive was found at 6% [13], though could be higher at 14% (more than thrombocytopenia at 7% in same cohort) for children on co-trimoxazole prophylaxis [14]. Whether these findings were independent or dependent on co-administered drugs remains undetermined.

Thrombocytosis is an emerging toxic complication accounting for 9% on stable cART depending on the regimen [7] up from 5.8% in treatment-naïve individuals [7]. It remains undetermined the relationship between HIV-associated thrombocytosis and accelerated thrombosis.

#### **2.3 Platelet ultrastructure in HIV/AIDS**

Despite the thrombocytopenia being associated with HIV, peripheral blood film smears of platelets are either unremarkable or hypogranular, which are of different sizes appearing as fragments [15].

Ultrastructure of platelets from HIV individuals, apart from showing normal features of hyperactivated aggregates having membrane pseudopodia/filopodia formation, in addition have shriveled aggregates with irregular and torn membrane surfaces, membrane blebbing and shedding of vesicles [16, 17]. The most distinctive features are alteration of granular structure though data are limited.

#### **3. Tests based on platelet aggregation**

#### **3.1 Light transmission aggregometry (LTA)**

Most studies on platelet aggregation in HIV have used single or fewer than the recommended panel of agonists with conflicting results [18]. Application of escalating agonist concentrations has uncovered dose-response patterns [19]. In this study, while epinephrine demonstrated greater potency indicating hyperresponsiveness, responses with collagen, TRAP, and ADP showed lesser maximum aggregation indicating

*Challenges in Platelet Functions in HIV/AIDS Management DOI: http://dx.doi.org/10.5772/intechopen.105731*

lesser efficacy and hyporesponsiveness. The agonist dose-response curve is, however, modified by cART viral suppression, especially abacavir-containing regimens [20] depending on agonist [21]. It must be remembered that although cART is a commonly mentioned modifier, the effects of fever associated with HIV are neither reported nor analyzed in these studies. Hyperthermic conditions such as fever are associated with reduced platelet aggregation [22].

#### **3.2 Whole blood platelet aggregometry-multiple electrode aggregometry (MEA) and impedance aggregometry**

A study comparing whole blood platelet aggregation using MEA found hyporeactivity in both HIV-treated and untreated individuals [23], similar to findings by impedance aggregometry [24]. It is worth noting that co-infection with HBV (6 vs. 4%) and HCV (0 vs. 2%) and low CRP levels [23] could have obscured the overall response. Co-infection with other viruses modulates platelet responses in HIV [25].

#### **3.3 Thromboelastography (TEG)/ Thromboelastometry (ROTEM)**

Few studies have been performed using thromboelastography (TEG) in HIV individuals. Of the few studies done, MA amplitude was low despite higher normal fibrinogen levels in both cART-treated [21] and untreated HIV subjects [23]. These study results of hypocoagulability are not in keeping with other tests, probably reflecting lack of sensitivity of TEG as a platelet function assay.

#### **4. Platelet activation**

Activated platelets are characterized by surface expression of activation-specific molecules such as P-selectin or CD62P, active GPIIbIIIa (PAC-1), phosphatidyleserine (PS) externalization; platelet-leukocyte aggregates (PLA); platelet microparticle formation (PMP), in addition to granule secretion such as platelet factor 4(PF4), β-thromboglobulin, and intracellular calcium flux [26].

#### **4.1 Flow cytometry for membrane surface glycoprotein expression**

A number of studies have documented platelet hyperactivity in HIV characterized by increased plasma membrane surface expression of CD62P, PAC-1, PS, CD63, [27], but paradoxically decreased GPIbα [28]. The levels positively correlate with viral loads but not CD4 count [29].

Although activation markers are higher in HIV sero-positive individuals who are cART naïve compared to healthy controls [30], with cART treatment levels decrease but do not normalize to pre-treatment levels [20, 31]. The persistent levels are related to inflammatory markers in virally suppressed individuals [32].

#### **4.2 Intracellular signal transduction test—VASP**

There is evidence of altered signal transduction affecting protein synthesis, degranulation, and activation functioning in HIV platelets. Experimental data show that HIV platelets had upregulation of ABCC4 (ATP-binding cassette subfamily 4), increase in cAMP, decrease in vasodilator-stimulated phosphoprotein (VASP),

which correlated with increased membrane expression of CD62P and integrin αIIbβ3 (GPIIbIIIa) [33]. It must be noted that VASP is only sensitive to PY12 inhibitors, and not much data are available from HIV patients.

#### **5. Platelet secretion**

#### **5.1 Alpha granules**

People living with HIV have increased secretion of alpha granule contents such as RANTES, sP-selectin, and sCD40L [34], despite viral suppression [33]. The persistence of these chemokines, especially anomalous secretion of RANTES, despite cART treatment [28] remains unexplained to date.

#### **5.2 Dense granules**

HIV platelets have low basal dense granule content and diminished secretion response as evidenced by low mepacrine uptake and release [33]. Although platelet mepacrine uptake and release have been considered among dense granule assays, it is not as specific as serotonin and lummiaggregometry for ATP [35, 36]. Despite this knowledge, the measurements of platelet serotonin and ATP remain largely undescribed in people living with HIV.

#### **5.3 Concept of "platelet exhaustion" in HIV**

Although HIV-associated platelets display increased baseline expression of surface activation markers compared to healthy controls [32], there is evidence of refractoriness to further agonist stimulation. This behavior has been referred to as "platelet exhaustion" in many publications [25, 28, 32, 37, 38].

Platelet "exhaustion" as a concept was postulated in references to previous observations, before HIV era, where activated platelets continued to circulate [39, 40] and were shown to be activated [41] but with decreased aggregation [42, 43]. They were considered refractory to further agonist stimulation [44] owing to acquired storage pool granule depletion [45, 46].

In HIV, stimulation with increased agonist concentration leads to lesser response at each corresponding dose [21]. Specifically, decreased thrombin dose-response curve for granule content and secretions for P-selectin, PFA/CXCL4,TXA and RANTES in HIV platelets less than healthy controls [32]. The decreased P-selectin and PAC-1 secretory responses correspond to impaired c-AMP, ABCC4 and VASP signal transduction mechanisms [33]. Furthermore, HIV platelets display decreased mepacrine uptake and release [33], and wheat germ agglutinin staining (WGA) [32] indicating reduction of dense and alpha granule contents respectively.

Despite many studies mentioning "platelet exhaustion" in HIV, however the results in support are neither consistent for all agonists nor confirmed by other tests. In patients who are cART naïve, stimulation with AA, ADP or collagen, the doseresponse curves for CD62P are higher than the uninfected controls [30]. None of the LTA aggregation tests have been accompanied by corresponding Lummiaggregometry test which could have better characterized platelet ATP dense granule secretion [47, 48]. Platelet lumiaggregometry testing remains largely un-described in HIV.

Furthermore, the studies are on people who are already infected by HIV, but platelet responses prior to HIV infection remains unknown.

From the foregoing, evidence in support for "platelet exhaustion" in HIV is suggestive but inconclusive. Although decreased dose-response to thrombin has been described, however response to epinephrine was enhanced in some studies. The maintained response to epinephrine casts doubt on granule exhaustion, since true storage pool disorder do not respond to epinephrine [49] or variable [50]. Indeed HIV platelets maintain both alpha and dense granule secretions to collagen and ADP agonists stimulation [51]. Perhaps a better term to use could be "anergy," refractory or "tired" platelets.

#### **6. Platelet adhesion**

HIV platelets have enhanced adherence to fibrinogen-coated surfaces [32, 33]. However, testing by this method is technically difficult and not available in clinical situations.

Although platelet PFA-100/200 testing is always recorded as aggregation in most studies, in actual fact it is marker of adhesion [2, 52]. The few tests of PFA-100 in HIV compared those on cART treatment with untreated [31], or in addition to [53] all of which showed shorter closure time in treatment-naïve individuals. The short closure times were neither normalized with aspirin nor with cART. The results are strongly indicative of influence of vWF as a third dimension in platelet function testing [54, 55].

#### **7. vWF-ADAMTS-13 axis in HIV/AIDS**

People living with HIV (PLWHIV) despite having very low platelet counts do not have issues of bleeding [56–58]. Instead, HIV-associated thrombotic complications [59] are an emerging issue of concern [60]. Although congenital thrombotic thrombocytopenic purpura (TTP) is very rare, acquired TTP is on the increase and associated with HIV estimated to be 15–40 times than the HIV negative in the general population [61]. It has been reported that HIV is responsible for 80% of TTP cases [62].

TTP is characterized by reduced or absent ADAMTS-13 and elevated vWF antigen as well as activity [63] especially the Unusually Ultralarge vWF multimers [64]. Elevated vWF Ag and high-molecular-weight vWF multimers [65] with reduced ADAMTS-13 have been detected in acute and chronic HIV [66, 67] and those with confirmed thrombosis [68]. Unusually, ultralarge vWF multimers that have increased adhesion to platelet GPIbα-V-IX receptors [69] compensates for hemostasis in the presence of the low platelet count in HIV.

#### **8. Platelet microparticles**

It has been demonstrated that blood from HIV individuals have abundant circulating platelet microparticles [70], and this is despite viral suppression [71, 72]. The levels were associated with increased cellular ROS, caspases, eNOS [72], and mitochondrial membrane depolarization [73] indicative of apoptosis [74] . Further, co-existence of platelet microparticles with increased LPS and platelet P-selectin and TF [29] are strong indicators that they are products of platelet activation.

#### **9. Mechanisms of platelet activation in HIV**

#### **9.1 Direct effect of HIV**

Recently, in mice, HIV particles were shown to be endocytosed by platelets by binding to TLR-7&9 leading to increased secretion of alpha (PFA-4) and dense granules (serotonin), and membrane expression of P-selectin [75]. Additionally, HIV interacts directly with platelets CLEC-2 and DC-SIGN receptors [76] *via* its trans-activating factor (Tat) [77]. The consequence is increased intracellular calcium flux, translocation of P-selectin (CD62P) from the alpha granules to the membrane surface, secretion of chemokine CD154, and release of platelet microparticles [77]. The process is by enhancing platelet NOX-2 oxidative stress [78].

#### **9.2 Gut microbiol translocation**

HIV preferentially infects CD4-T lymphocytes present in the gut leading to reduction in number and function [79]. The consequence is loss of gut epithelial immune protection and disruption of gut epithelial barrier allowing luminal indigenous intestinal bacteria to translocate out of the mucosa and into circulation [80]. Once in circulation, bacterial products such as lipopolysaccharides (LPS) interact with platelet toll-like receptors 4 (TLR4) [81]. The microbial products induce signal transduction mechanisms that eventually lead to facilitating platelet membrane receptor expression [82, 83]. The phenomenon of gut microbial translocation has been used to explain enhanced platelet reactivity despite therapy with antiplatelets such as ticagrelor in myocardial infarction [84]. However, some studies have disputed the role of LPS in platelet activation instead of reporting attenuation of receptor expression and aggregation in the presence of agonists [85] contradicting earlier findings. The paradoxical result may be due to the absence or presence of other factors such as soluble CD14 that prime TLR4 sensing of LPS [86], extent of TLR expression [87] or the different LPS isoforms [88], and experimental conditions [89] as well as clinical condition [89].

#### **9.3 Immune complexes, cytokines, and inflammatory markers**

#### *9.3.1 Cytokines*

HIV infection is associated with elaboration of cytokines from inflammatory cells, and these have been shown to induce platelet activation [90, 91] The platelet activation is not limited to interleukins only, since tumor necrosis factor in blood leads to dose- and time-dependent increase in platelet expression of GPIIbIIIa, PS, and mitochondrial dysfunction [92]. The role of TNF-α in platelet activation and apoptosis are well supported by empirical evidence [93].

#### *9.3.2 Immune complexes*

Platelets express FcRIIA (CD32a) or simply FcR receptor that recognizes the constant region of IgG in immune complexes [94]. The consequence of platelet-immune *Challenges in Platelet Functions in HIV/AIDS Management DOI: http://dx.doi.org/10.5772/intechopen.105731*

complex binding leads to platelet activation [95], aggregation and release of contents from alpha and dense granules [94], and microparticle formation [96]. The platelet activation from immune complexes is dependent on membrane GP IIbIIIa [97]. However, the immune complex-induced platelet aggregation is dependent on dose and charge [98].

Cross-reactive antibodies between HIV epitopes and platelet receptors have been described [99, 100].

#### *9.3.3 Neutrophil extracellular traps (NETS)*

When neutrophils encounter viruses such as HIV, they respond by releasing reactive oxygen species and net-like structures called neutrophil extracellular traps [101, 102]. The NETs, composed of DNA, histones, myeloperoxidase, citrinulated histones, and elastases, are the potent inducers of platelet aggregation and activation [103–105].

#### *9.3.4 Platelet-leukocyte complexes*

There is often cross-talk between platelets and leukocytes associated with bidirectional priming and activation of each other [106, 107]. These two cells interact through platelets such as P-selecti-PSGL-1, GPIb-vWF-CD18, integrin IIaIIIb-fibrinogen-MAC-1 neutrophil linkages that lead to the formation of plateletleukocyte aggregates (PLA) [108] linked by P-selectin-PSGL. These PLA conjugates have been found in HIV patients involving T-cells associated with CD42b and CD62P [109]. Elevated PLA together with other immune markers is positively correlated with increased platelet CD36, CD62P, and platelet aggregation but inversely with CD4 count [110].

#### *9.3.5 vWF-GPIbα in platelet activation in HIV*

There is evidence of endothelial damage [111] and increased vWF levels in HIV patients [66–68, 112, 113]. Apart from the high vWF Ag levels, of significant is the persistently high functionally active Ultralarge vWF multimers (ULvWFM) in HIV individuals [65] that causes adhesion even at low platelet counts [114]. Correspondingly, as HIV disease progresses, platelet expression of the integrin GPIbα decreases paradoxically unlike the other surface receptors indicating consumption [28].

#### **9.4 Platelet apoptosis**

There are similarities in markers of platelet activation and apoptosis [115]. In both processes, there is phosphatidyleserine (PS) exposure on the membrane [116] and microparticles [117]. However, specific features of platelet apoptosis include mitochondrial membrane leakage characterized by changes in membrane depolarization (Δψm) and increase in cytosolic caspases 3&8, [118, 119]. Indeed, features of platelet apoptosis and activation have been demonstrated in HIV patients [25, 32, 38]. It should be noted that the few studies demonstrating occurrence of full spectra of apoptosis in HIV individuals were confounded by cART viral suppression [32] and dengue co-infection [25] and therefore, whether results were specific to HIV in itself largely remains undetermined.

Some of the consequences of platelet apoptosis include thrombocytopenia [120, 121]. This is because, apart from the fact that apoptotic platelet eventually disintegrates [74], the surface exposure of PS acts as "eat me" signal for engulfment by the macrophages thus removing the altered cells from circulation shortening survival [122–124].

#### **10. Antiretrovirals and platelet functions in HIV**

Despite the success attained by cART in viral suppression and recovery of platelet counts [125, 126], their effects on platelet function remain variable. In general, platelet surface markers such as CD62P, PAC-1 and CD40L, soluble sCD62P, sCD40L as well as platelet-secreted chemokines such as RANTES persist despite cART viral suppression [27] with some variations between the individual drugs and study designs.

Platelet signal transduction and secretory effects are enhanced by HIV, but these effects are accentuated by cART. This was demonstrated by Pastori et al.'s [78] study in which levels of sCD40L, platelet sNOX-dp, and 8-iso-PGF2-α were elevated, the effects of PIs greater than NNRTI. The mechanism appears to be induction of oxidative stress, ROS, and arachidonic pathways that synergistically augment AA platelet activation. cART causes mitochondrial toxicities [127] *via* ROS release and inner membrane depolarization that eventually lead to apoptosis that persists even with viral suppression [32].

Abacavir is unique among cART [51] since it is a guanosine analogue and induces platelet activation *via* its effects on NO-cGMP signal transduction pathway [20]. *In vitro*, incubation of platelets with abacavir inhibits cAMP pathway and dose-dependently increases surface expression of P-selectin, an observation that is augmented by ADP agonist [128]. Compared with TDF and TAF, abacavir enhances platelet aggregation and increases agonist-induced platelet activation *in vivo* (CD62P, PAC-1) [31]. It has been shown that it induces both alpha and dense platelet granule secretions thereby increasing membrane CD62P [128] levels as well as increasing PAC-1 [129]. It also alters metabolic enzymes that lead to increase in PAF from leukocytes [130]. This likely explains its association with cardiovascular events where enhanced platelet hyperactivity plays a central role [131, 132].

Despite other studies reporting levels of platelets MP remaining unchanged [29] or increased [71] after initiating antiretrovirals, one study found MP TF levels decreased with cART treatment [133]. The difference could be attributed to monocyte phenotypes [134] and level of activation and attendant TF expression with cART [135]. This is because platelets undergo decryption [136] and transfer TF to monocytes using microparticles as vehicles [137, 138].

The effects of cART on platelets are complicated by other factors such as TNF-α, a known platelet activator and apoptosis inducer. Although TNF levels are often elevated in HIV infection, levels persist despite cART [139] even if used over 24-month period [34]. Whereas cART treatment decreases circulating bacterial LPS levels in HIV patients, platelet reactivity is increased instead [23] suggesting intrinsic effects of the drugs independent of bacterial translocation.

#### **11. Antiplatelets in HIV/AIDS**

People living with HIV/AIDS are at increased risk of cardiovascular events [140, 141], especially coronary heart disease [142, 143] and ischemic stroke [144, 145], than the

general population. The increased risk is due to HIV infection alone and accentuated by cART [146, 147].

Although there is evidence of enhanced platelet activation in association with HIV [27], studies of antiplatelet therapy in these patients have yielded inconsistent results, perhaps owing to drug interactions [148]. It should be noted that the studies so far done were on patients concurrently taking cART.

In a study of HIV-1 infected patients who had been on 6-month cART, it was found that 325 mg of oral aspirin-attenuated platelet aggregation to agonists, activation markers [37]. In the same study, although levels of urinary thromboxane were decreased in both HIV-positive cART untreated and treated, it was least responsive to aspirin. Furthermore, despite aspirin administration, suppression of platelet hyperactivity did not decline to baseline levels indicating the contributory effects of cART. Apart from the small sample size and short duration of therapy, other limitations of this pilot study are that it evaluated only one antiplatelet drug, and it did not perform subgroup analysis among the different cART drugs (NNRTI, PI, Raltegravir, and abacavir) as well as the racial and ethnic differences.

Although aspirin and R406 (thromboxane analogue) but not ticagrelor inhibits platelet engulfment, they do not inhibit CD62P expression or PMA complex formation [149]. Other studies have confirmed the suboptimal effects of aspirin on platelets agonist (collagen and epinephrine)-induced aggregation, surface expression of CD62P, CD40L, and PAC-1 from individuals with HIV taking ABC [53]. This study identified subjects taking abacavir-containing cART as poor responders. While cART is currently standard of care in the treatment of HIV, there are no data on effects of antiplatelets in PLWH before adoption of practice.

Clopidogrel reduces thrombogenicity and platelet hyperreactivity better than aspirin in PLWH on cART [21]. The question whether dual antiplatelet therapy compared to single agent may have a better reduction in platelet hyperreactivity in HIV concurrently taking cART was evaluated in the EVERE2ST-HIV [18]. This study evaluated the extent of platelet inhibition patients with acute coronary patients on dual antiplatelet therapy undergoing PCI utilizing various platelet function assays [18]. The findings were that P2Y12 inhibitors (clopidogrel, prasugrel, and ticagralor) and aspirin were all associated with residual platelet reactivity on light transmission aggregometry (LTA), VerifyNow, and VASP assays. Furthermore, HIV infection was an independent risk factor for the high on antiplatelet reactivity that was increased by combined antiretroviral therapy (cART). Of the cART, protease inhibitors had greater effects than the NNRTIs. The residual platelet reactivity in PLWHIV despite viral suppression and dual antiplatelet therapy can probably be accounted by the active immune mechanisms and drug interactions [148].

Overall, few studies have evaluated the effects of antiplatelets in persons living with HIV. The available studies suffer from small sample sizes and have not been performed in populations not taking cART. Furthermore, the different classes of antiplatelets have not been evaluated. Of the studies done so far, the results do demonstrate neither efficacy nor improved outcomes with either aspirin or clopidogrel.

#### **12. Conclusion**

Infection with HIV is associated with reduced platelet count; extent of thrombocytopenia inversely correlates with viral load and disease progression. Despite thrombocytopenia, cardiovascular events are on the increase. There is associated

platelet hyperactivity, as evidenced by increased surface expression of CD62P, CD40L, platelet microparticles, and platelet leukocyte aggregates. There is enhanced secretion of chemokines such as RANTES. Combined antiretroviral drugs independently and synergistically with HIV enhance platelet hyperactivity that persists despite viral suppression. Data on the effects of antiplatelets in this population can at best be described as clinical equipoise.

### **Other declarations**

Autor's ORCID identifier: 0000-0001-6466-172X.

### **Author details**

Gordon Ogweno Department of Medical Physiology, School of Medicine, Kenyatta University, Nairobi, Kenya

\*Address all correspondence to: ogweno.gordon@ku.ac.ke

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Challenges in Platelet Functions in HIV/AIDS Management DOI: http://dx.doi.org/10.5772/intechopen.105731*

#### **References**

[1] Ahonkhai AA, Gebo KA, Steiff MB, Moore RD, Segal JB. Venous thromboembolism in patients with HIV/ AIDS. A case-control study. Journal of Acquired Immune Deficiency Syndromes. 2008;**48**(3):310-314. DOI: 10.1097/QAI.0b013e318163bd70

[2] Lordkipanidzé M. Platelet function tests. Seminars in Thrombosis and Hemostasis. 2016;**42**(3):258-267. DOI: 10.1055/s-0035-1564834

[3] Mansouritorghabeh H, De Laat B, Roest M. Current methods of measuring platelet activity : Pros and cons. Blood Coagulation & Fibrinolysis. 2020;**31**:426- 433. DOI: 10.1097/MBC.000000000 0000941

[4] Getawa S, Aynalem M, Bayleyegn B, Adane T. The global prevalence of thrombocytopenia among HIV-infected adults: A systematic review and meta-analysis. International Journal of Infectious Diseases. 2021;**105**:495-504. DOI: 10.1016/j.ijid.2021.02.118

[5] Sloand EM, Klein HG, Banks SM, Vareldzis B, Merritt S, Pierce P. Epidemiology of thrombocytopenia in HIV infection. European Journal of Haematology. 1992;**48**(3):168-172. DOI: 10.1111/j.1600- 0609.1992.tb00591.x

[6] Passos AM, Treitinger A, Spada C. An overview of the mechanisms of HIV-related thrombocytopenia. Acta Haematologica. 2010;**124**(1):13-18. DOI: 10.1159/000313782

[7] Li B et al. Manifestations and related risk factors of thrombocyte abnormalities in HIV-positive patients before and after the initiation of art. Infection and Drug Resistance.

2021;**14**:4809-4819. DOI: 10.2147/IDR. S334046

[8] Marchionatti A, Parisi MM. Anemia and thrombocytopenia in people living with HIV/AIDS: A narrative literature review. International Health. 2021;**13**(2):98-109. DOI: 10.1093/ inthealth/ihaa036

[9] Ongondi M, Amayo EO, Lule GN, Rajab JA. Thrombocytopenia in HAART NAIVE HIV infected patients attending the Comprehensive Care Clinic at Kenyatta National Hospital. East African Medical Journal. 2016;**93**(9):406-408

[10] Dominguez A, Gamallo G, Garcia R, Lopez-Pastor A, Peña JM, Vazquez JJ. Pathophysiology of HIV related thrombocytopenia: An analysis of 41 patients. Journal of Clinical Pathology. 1994;**47**(11):999-1003. DOI: 10.1136/ jcp.47.11.999

[11] Miguez-Burbano MJ, Jackson J, Hadrigan S. Thrombocytopenia in HIV disease: Clinical relevance, physiopathology and management. Current Medicinal Chemistry. Cardiovascular and Hematological Agents. 2005;**3**(4):365-376. DOI: 10.2174/156801605774322364

[12] Mathur A, Samaranayake S, Storrar NPF, Vickers MA. Investigating thrombocytosis. BMJ. 2019;**366**(July): 1-6. DOI: 10.1136/bmj.l4183

[13] Ellaurie M. Thrombocytosis in pediatric HIV infection. Clinical Pediatrics (Phila). 2004;**43**(7):627-629. DOI: 10.1177/000992280404300707

[14] Mateveke-Kuona P, Bwakura MF, Dzangare J, Pazvakavambwa I. Haematological features in children

less than 12 years on co-trimoxazole prophylaxis seen in opportunistic infection clinics at Harare and Parirenyatwa Teaching Hospitals. The Central African Journal of Medicine. 2010;**56**(9/12):51-56

[15] Bamberg R, Johnson J. Segmented neutrophil size and platelet morphology in HIV/AIDS patients. Clinical Laboratory Science. 2002;**15**(1):18-22

[16] Pretorius E, Smit E, Oberholzer HM, Steyn E, Briedenhann S, Franz RC. Investigating the ultrastructure of platelets of HIV patients treated with the immuno-regulator, Canova. Histology and Histopathology. 2009;**24**:399-405

[17] Jackson BS, Nunes Goncalves J, Pretorius E. Comparison of pathological clotting using haematological, functional and morphological investigations in HIVpositive and HIV-negative patients with deep vein thrombosis. Retrovirology. 2020;**17**(1):1-13. DOI: 10.1186/ s12977-020-00523-3

[18] Hauguel-Moreau M et al. Platelet reactivity in human immunodeficiency virus infected patients on dual antiplatelet therapy for an acute coronary syndrome: The EVERE2ST-HIV study. European Heart Journal. 2017;**38**(21):1676-1686. DOI: 10.1093/ eurheartj/ehw583

[19] Satchell CS et al. Platelet function and HIV : A case – Control study. AIDS. 2010;**24**:649-657. DOI: 10.1097/ QAD.0b013e328336098c

[20] Satchell CS et al. Increased platelet reactivity in HIV-1 – Infected patients receiving abacavircontaining antiretroviral therapy. JID. 2011;**204**:1202-1210. DOI: 10.1093/infdis/ jir509

[21] Brien MPO et al. Targeting thrombogenicity and inflammation in chronic HIV infection. Science Advances. 2019;**5**:eaav5463

[22] Etulain J et al. Hyperthermia inhibits platelet hemostatic functions and selectively regulates the release of alphagranule proteins. Journal of Thrombosis and Haemostasis. 2011;**9**(8):1562-1571. DOI: 10.1111/j.1538-7836.2011.04394.x

[23] Haugaard AK, Lund TT, Birch C, Trøseid M, Ullum H, Gerstoft J. Discrepant coagulation profile in HIV infection : Elevated D-dimer but impaired platelet aggregation and clot initiation. AIDS. 2013;**27**:2749-2758. DOI: 10.1097/01.aids.0000432462.21723.ed

[24] Muñoz RP et al. Whole blood platelet aggregometry in HIV-infected patients on treatment with abacavir \*. OJIM. 2012;**2012**:62-66. DOI: 10.4236/ ojim.2012.22013

[25] Hottz ED, Quirino-teixeira AC, Vallsde-souza R, Zimmerman GA, Bozza FA, Bozza PT. Platelet function in HIV plus dengue coinfection associates with reduced inflammation and milder dengue illness. Scientific Reports. 2019;**9**(1):1-13. DOI: 10.1038/s41598-019-43275-7

[26] Kannan M, Ahmad F, Saxena R. Platelet activation markers in evaluation of thrombotic risk factors in various clinical settings. Blood Reviews. 2019;**37**:100583. DOI: 10.1016/j. blre.2019.05.007

[27] Nkambule BB et al. Platelet activation in adult HIV-infected patients on antiretroviral therapy: A systematic review and meta-analysis. BMC Medicine. 2020;**18**:357. DOI: 10.1186/ s12916-020-01801-9

[28] Holme PA, Muller F, Solum NO, Brosstad F, Land Q, Aukrust PAL. Enhanced activation of platelets with abnormal release of RANTES

*Challenges in Platelet Functions in HIV/AIDS Management DOI: http://dx.doi.org/10.5772/intechopen.105731*

in human immunodeficiency virus type 1 infection. The FASEB Journal. 1998;**12**:79-89

[29] Mayne E et al. Increased platelet and microparticle activation in HIV infection : Upregulation of P-selectin and tissue factor expression. Journal of Acquired Immune Deficiency Syndromes. 2012;**59**(4):340-346

[30] Nkambule BB, Davison GM, Ipp H. The evaluation of platelet function in HIV infected, asymptomatic treatment-naïve individuals using flow cytometry. Thrombosis Research. 2015;**135**(6):1131-1139. DOI: 10.1016/j. thromres.2015.01.031

[31] Francisci D, Falcinelli E, Belfiori B, Petito E, Guglielmini G, Malincarne L. In vivo platelet activation and platelet hyperreactivity in abacavir- treated HIV-infected patients. Thrombosis and Haemostasis. 2013;**110**(8):349-357. DOI: 10.1160/TH12-07-0504

[32] Mesquita EC et al. Persistent platelet activation and apoptosis in virologically suppressed HIV-infected individuals. Scientific Reports. 2018;**8**(1):1-10. DOI: 10.1038/s41598-018-33403-0

[33] Marcantoni E et al. Platelet transcriptome profiling in as a mediator of platelet activity. JACC: Basic to Translational Science. 2018;**3**:9-22. DOI: 10.1016/j.jacbts.2017.10.005

[34] Landrø L, Ueland T, Otterdal K, Frøland SS, Aukrust P. Persistently raised plasma levels of plateletderived inflammatory mediators in HIV-infected patients during highly active anti-retroviral therapy. Journal of Thrombosis and Haemostasis. 2011;**9**(5):1075-1077. DOI: 10.1111/j. 1538-7836.2011.04242.x

[35] Mumford AD et al. A review of platelet secretion assays for the diagnosis of inherited platelet secretion disorders. Thrombosis and Haemostasis. 2015;**114**(1):14-25. DOI: 10.1160/ TH14-11-0999

[36] Pai M et al. Diagnostic usefulness of a lumi-aggregometer adenosine triphosphate release assay for the assessment of platelet function disorders. American Journal of Clinical Pathology. 2011;**136**(3):350-358. DOI: 10.1309/ AJCP9IPR1TFLUAGM

[37] O'Brien M et al. Aspirin attenuates platelet activation and immune activation in HIV-1-infected subjects on antiretroviral therapy: A pilot study. Journal of Acquired Immune Deficiency Syndromes. 2013;**63**(3):280-288. DOI: 10.1097/QAI.0b013e31828a292c

[38] Gama WM et al. Increased levels of reactive oxygen species in platelets and platelet-derived microparticles and the risk of respiratory failure in HIV/AIDS patients. Memórias do Instituto Oswaldo Cruz. 2020;**115**:e200082. DOI: 10.1590/ 0074-02760200082

[39] O'Brien JR. "Exhausted " platelets continue to circulate. The *Lancet*. 1978;**312**(8103):1316-1317. DOI: 10.1016/ s0140-6736(78)92087-1

[40] Pareti FI, Capitanio A, Mannucci L, Ponticelli C, Mannucci PM. Acquired dysfunction due to the circulation of 'exhausted' platelets. The American Journal of Medicine. 1980;**69**(2):235-240. DOI: 10.1016/0002-9343(80)90383-6

[41] Boneu B, Bugat R, Boneu A, Eche N, Sie P, Combes P-F. Exhausted platelets in patients with malignant solid tumors without evidence of active consumption coagulation. European Journal of Cancer & Clinical Oncology. 1984;**20**(7):890-903

[42] Evans RJ, Gordon JL. Refractoriness in blood platelets: Effect of prior

exposure to aggregating agents on subsequent aggregation responses. British Journal of Pharmacology. 1974;**51**(1):123

[43] Fong BJSC, Kaplan BS. Ipairment of platelet aggregation in Hemolytic uremic syndrome: Evidence for platelet 'exhaustion'. Blood. 1982;**60**(3):564-571

[44] O'Brien JR, Etherrington M, Jameson S. Refractory state of platelet aggregation with major operations. The *Lancet*. 1971;**2**(7727):741-743. DOI: 10.1016/s0140-6736(71)92107-6

[45] Pareti F, Capitanio A, Mannucci P. Acquired storage pool disease in platelets during disseminated intravascular coagulation. Blood. 1976;**48**(4):511-515. DOI: 10.1182/blood.v48.4.511.511

[46] Zahavi J, Marder VJ. Acquired 'storage pool disease' of platelets associated with circulating antiplatelet antibodies. The American Journal of Medicine. 1974;**56**(6):883-890. DOI: 10.1016/0002-9343(74)90819-5

[47] Hughes CE. How to perform aggregometry and lumi-aggregometry in mouse platelets. Platelets. 2018;**29**(7):638-643. DOI: 10.1080/ 09537104.2018.1478074

[48] Jurk K, Shiravand Y. Platelet phenotyping and function testing in thrombocytopenia. Journal of Clinical Medicine. 2021;**10**(5):1114. DOI: 10.3390/ jcm10051114

[49] Fritsma GA. Platelet function testing: Aggregometry and lumiaggregometry. Clinical Laboratory Science. 2007;**20**(1): 32-37. DOI: 10.29074/ascls.20.1.32

[50] Weiss HJ, Lages B. The response of platelets to epinephrine in storage pool deficiency - evidence pertaining to the role of adenosine diphosphate in mediating primary and secondary aggregation. Blood. 1988;**72**(5):1717- 1725. DOI: 10.1182/blood.v72.5.1717.1717

[51] Taylor KA et al. Pharmacological impact of antiretroviral therapy on platelet function to investigate human immunodeficiency virus - associated cardiovascular risk. British Journal of Pharmacology. 2019;**176**:879-889. DOI: 10.1111/bph.14589

[52] Paniccia R, Priora R, Liotta AA, Abbate R. Platelet function tests: A comparative review. Vascular Health and Risk Management. 2015;**11**:133-148. DOI: 10.2147/VHRM.S44469

[53] Falcinelli E et al. Effect of aspirin treatment on abacavir-associated platelet hyperreactivity in HIV-infected patients. International Journal of Cardiology. 2018;**263**:118-124. DOI: 10.1016/j. ijcard.2018.04.052

[54] Castaman G et al. The impact of bleeding history, von Willebrand factor and PFA – 100 â on the diagnosis of type 1 von Willebrand disease : Results from the European study MCMDM-1VWD. British Journal of Haematology. 2010;**151**:245-251. DOI: 10.1111/j. 1365-2141.2010.08333.x

[55] Gianetti J, Parri MS, Della Pina F, Marchi F, Koni E, De Caterina A, et al. Von willebrand factor antigen predicts response to double dose of aspirin and clopidogrel by PFA-100 in patients undergoing primary angioplasty for ST elevation myocardial infarction. The Scientific World Journal. 2013:313492. DOI: 10.1155/2013/313492

[56] Franzetti M et al. Changes in the incidence of severe thrombocytopenia and its predisposing conditions in HIVinfected patients since the introduction *Challenges in Platelet Functions in HIV/AIDS Management DOI: http://dx.doi.org/10.5772/intechopen.105731*

of highly active antiretroviral therapy. Journal of Acquired Immune Deficiency Syndromes. 2014;**67**(5):493-498. DOI: 10.1097/QAI.0000000000000347

[57] Nascimento FG, Tanaka PY. Thrombocytopenia in HIV-infected patients. Indian Journal of Hematology and Blood Transfusion. 2012;**28**(2):109- 111. DOI: 10.1007/s12288-011-0124-9

[58] Vannappagari V, Nkhoma ET, Atashili J, Laurent SS, Zhao H. Prevalence, severity, and duration of thrombocytopenia among HIV patients in the era of highly active antiretroviral therapy. Platelets. 2011;**22**(8):611-618. DOI: 10.3109/09537104.2011.582526

[59] Ahmed S, Siddiqui RK, Siddiqui AK, Zaidi SA, Cervia J. HIV associated thrombotic microangiopathy. Postgraduate Medical Journal. 2002;**78**:520-525

[60] Dentali F, Nicolini E, Ageno W. Venous and arterial thrombosis associated with HIV infection. Seminars in Thrombosis and Hemostasis. 2012;**38**(5):524-529. DOI: 10.1055/ s-0032-1306434

[61] Meiring M, Webb M, Goedhals D, Louw V. HIV-associated thrombotic thrombocytopenic purpura - what we know so far. European Oncology & Haematology. 2012;**8**(2):89-91. DOI: 10.17925/eoh.2012.08.02.89

[62] Gunther K, Garizio D, Dhlamini B. The pathogenesis of HIVrelated thrombotic thrombocytopaenic purpura – Is it different? ISBT Science Series. 2006;**1**(1):246-250. DOI: 10.1111/j.1751-2824.2006.00041.x

[63] De La, Rubia J, Contreras E, Del Río-Garma J. Thrombotic thrombocytopenic purpura. Medicina Clínica (Barcelona).

2011;**136**(12):534-540. DOI: 10.1016/j. medcli.2010.02.011

[64] Tsai H-M. Deficiency of ADAMTS13 causes thrombotic thrombocytopenic Purpura. Arteriosclerosis, Thrombosis, and Vascular Biology. 2003;**23**:388-397. DOI: 10.1161/01.ATV.0000058401. 34021.D4

[65] Aukrust P et al. Persistently elevated levels of von Willebrand factor antigen in HIV infection. Downregulation during highly active antiretroviral therapy. Thrombosis and Haemostasis. 2000;**84**:183-187

[66] Graham SM et al. Von willebrand factor adhesive activity and ADAMTS13 protease activity in HIV-1-infected men. International Journal of Medical Sciences. 2019;**16**(2):276-284. DOI: 10.7150/ijms.28110

[67] Graham SM et al. Elevated plasma von Willebrand factor levels are associated with subsequent ischemic stroke in persons with treated HIV infection. Open Forum Infectious Diseases. 2021;**1**:1-9. DOI: 10.1093/ofid/ ofab521

[68] van den, Dries LWJ et al. von Willebrand factor is elevated in HIV patients with a history of thrombosis. Frontiers in Microbiology. 2015;**6**:1-8. DOI: 10.3389/fmicb.2015.00180

[69] Varga-Szabo D, Pleines I, Nieswandt B. Cell adhesion mechanisms in platelets. Arteriosclerosis, Thrombosis, and Vascular Biology. 2008;**28**(3):403- 413. DOI: 10.1161/ATVBAHA.107.150474

[70] Kelly C et al. Circulating microparticles are increased amongst people presenting with HIV and advanced immune suppression in Malawi and correlate closely with arterial stiffness: A nested case control study.

Wellcome Open Research. 2021;**6**:264. DOI: 10.12688/wellcomeopenres.17044.1

[71] Corrales-Medina VF et al. Increased levels of platelet microparticles in HIVinfected patients with good response to highly active antiretroviral therapy. Journal of Acquired Immune Deficiency Syndromes. 2010;**54**(2):217-218

[72] Hijmans JG et al. Circulating microparticles are elevated in treated HIV-1 infection and are deleterious to endothelial cell function. Journal of the American Heart Association. 2019;**8**:e011134. DOI: 10.1161/ JAHA.118.011134

[73] van der, Heijden WA et al. Long-term treated HIV infection is associated with platelet mitochondrial dysfunction. Scientific Reports. 2021;**11**(1):1-12. DOI: 10.1038/s41598-021-85775-5

[74] Gyulkhandanyan AV, Mutlu A, Freedman J, Leytin V. Markers of platelet apoptosis: Methodology and applications. Journal of Thrombosis and Thrombolysis. 2012;**33**(4):397-411. DOI: 10.1007/s11239-012-0688-8

[75] Banerjee M et al. Platelets endocytose viral particles and are activated via TLR (toll-like receptor) signalling. Arteriosclerosis, Thrombosis, and Vascular Biology. 2020;**40**:1635-1650. DOI: 10.1161/ATVBAHA.120.314180

[76] Chaipan C et al. DC-SIGN and CLEC-2 mediate human immunodeficiency virus type 1 capture by platelets. Journal of Virology. 2006;**80**(18):8951-8960. DOI: 10.1128/ jvi.00136-06

[77] Wang J, Zhang W, Nardi MA, Li Z. HIV-1 Tat-induced platelet activation and release of CD154 contribute to HIV-1-associated autoimmune thrombocytopenia. Journal of

Thrombosis and Haemostasis. 2011;**9**(3):562-573. DOI: 10.1111/j. 1538-7836.2010.04168.x

[78] Pastori D et al. HIV-1 induces in vivo platelet activation by enhancing platelet NOX2 activity. The Journal of Infection. 2015;**70**(6):651-658. DOI: 10.1016/j. jinf.2015.01.005

[79] Hsue PY. Mechanisms of cardiovascular disease in the setting of HIV infection. The Canadian Journal of Cardiology. 2019;**35**(3):238-248. DOI: 10.1016/j.cjca.2018.12.024

[80] Shan L, Siliciano RF. Unraveling the relationship between microbial translocation and systemic immune activation in HIV infection. The Journal of Clinical Investigation. 2014;**124**(6):2368-2371. DOI: 10.1172/ JCI75799

[81] Lopes Pires ME, Clarke SR, Marcondes S, Gibbins JM. Lipopolysaccharide potentiates platelet responses via toll-like receptor 4-stimulated Akt-Erk-PLA2 signalling. PLoS One. 2017;**12**(11):1-22. DOI: 10.1371/journal.pone.0186981

[82] Nocella C et al. Lipopolysaccharide induces platelet activation in HIV patients: The role of different viral load patterns. HIV Medicine. 2021;**22**(6):434- 444. DOI: 10.1111/hiv.13059

[83] Zhang G et al. Lipopolysaccharide stimulates platelet secretion and potentiates platelet aggregation via TLR4/MyD88 and the cGMP-dependent protein kinase pathway. Journal of Immunology. 2009;**182**(12):7997-8004. DOI: 10.4049/jimmunol.0802884

[84] Zhang X et al. Gut microbiota induces high platelet response in patients with ST segment elevation myocardial infarction after ticagrelor treatment.

*Challenges in Platelet Functions in HIV/AIDS Management DOI: http://dx.doi.org/10.5772/intechopen.105731*

eLife. 2022;**11**:e70240. DOI: 10.7554/ eLife.70240

[85] Martyanov AA et al. Effects of bacterial lipopolysaccharides on platelet function: Inhibition of weak platelet activation. Scientific Reports. 2020;**10**(1):1-10. DOI: 10.1038/ s41598-020-69173-x

[86] Damien P et al. LPS stimulation of purified human platelets is partly dependent on plasma soluble CD14 to secrete their main secreted product, soluble-CD40-ligand. BMC Immunology. 2015;**16**(1):1-7. DOI: 10.1186/ s12865-015-0067-2

[87] Aslam R et al. Platelet toll-like receptor expression modulates lipopolysaccharide-induced thrombocytopenia and tumor necrosis factor-α production *in vivo*. Blood. 2006;**107**(2):637-641. DOI: 10.1182/ blood-2005-06-2202

[88] Berthet J et al. Human platelets can discriminate between various bacterial LPS isoforms via TLR4 signaling and differential cytokine secretion. Clinical Immunology. 2012;**145**(3):189-200. DOI: 10.1016/j.clim.2012.09.004

[89] Jonathan S, Rajendran V, Dash P, Ketan P, Darius W. Effect of ultrapure lipopolysaccharides derived from diverse bacterial species on the modulation of platelet activation. Scientific Reports. 2019;**9**:18258. DOI: 10.1038/ s41598-019-54617-w

[90] Lumadue JA, Lanzkron SM, Kennedy SD, Kuhl DT, Mt BS, Kickler TS. Cytokine induction of platelet activation. American Journal of Clinical Pathology. 1996;**106**:795-798

[91] Burstein SA et al. Cytokine-induced alteration of platelet and hemostatic

function. Stem Cells. 1996;**14**(Suppl 1): 154-162

[92] Davizon-Castillo P et al. TNF-a–driven inflammation and mitochondrial dysfunction define the platelet hyperreactivity of aging. Blood. 2019;**134**(9):727-740. DOI: 10.1182/ blood.2019000200

[93] Çevİk Ö, Adigüzel Z, Baykal AT, Şener A. Tumor necrosis factor-alpha induced caspase-3 activation-related iNOS gene expression in ADPactivated platelets. Turkish Journal of Biology. 2017;**41**:31-40. DOI: 10.3906/ biy-1509-64

[94] Arman M, Krauel K. Human platelet IgG Fc receptor Fc c RIIA in immunity and thrombosis. Journal of Thrombosis and Haemostasis. 2015;**13**:893-908. DOI: 10.1111/jth.12905

[95] Goette NP, Glembotsky AC, Lev PR, Grodzielski M. Platelet apoptosis in adult immune thrombocytopenia : Insights into the mechanism of damage triggered by auto- antibodies. PLoS One. 2016;**11**(8):e0160563. DOI: 10.1371/ journal.pone.0160563

[96] Larson A, Egberg N, Lindahl L. Platelet activation and binding of complement components to platelets induced by immune complexes. Platelets. 1994;**5**:149-155

[97] Zhi H, Dai J, Liu J, Zhu J, Newman DK, Gao C. Platelet activation and thrombus formation over IgG immune complexes requires integrin α IIb β 3 and Lyn kinase. PLoS One. 2015;**10**(8):e0135738. DOI: 10.1371/ journal.pone.0135738

[98] Schattner BM et al. Activation of human platelets by immune complexes prepared with cationized human IgG. Blood. 1993;**82**(10):3045-3051

[99] Li Z, Nardi MA, Karpatkin S. Role of molecular mimicry to HIV-1 peptides in HIV-1-related immunologic thrombocytopenia. Blood. 2005;**106**(2): 572-576. DOI: 10.1182/blood-2005- 01-0243

[100] Zhang W, Nardi MA, Borkowsky W, Li Z, Karpatkin S. Role of molecular mimicry of hepatitis C virus protein with platelet GPIIIa in hepatitis C-related immunologic thrombocytopenia. Blood. 2009;**113**(17):4086-4093. DOI: 10.1182/ blood-2008-09-181073

[101] Saitoh T et al. Neutrophil extracellular traps mediate a host defense response to human immunodeficiency virus-1. Cell Host & Microbe. 2012;**12**(1):109-116. DOI: 10.1016/j. chom.2012.05.015

[102] Barr FD, Ochsenbauer C, Wira CR, Rodriguez-Garcia M. Neutrophil extracellular traps prevent HIV infection in the female genital tract. Mucosal Immunology. 2018;**11**(5):1420-1428. DOI: 10.1038/s41385-018-0045-0

[103] Fuchs TA et al. Extracellular DNA traps promote thrombosis. Proceedings of the National Academy of Sciences of the United States of America. 2010;**107**(36):15880-15885. DOI: 10.1073/ pnas.1005743107

[104] Elaskalani O, Abdol Razak NB, Metharom P. Neutrophil extracellular traps induce aggregation of washed human platelets independently of extracellular DNA and histones. Cell Communication and Signalling. 2018;**16**(1):1-15. DOI: 10.1186/ s12964-018-0235-0

[105] Zhou P et al. Interactions between neutrophil extracellular traps and activated platelets enhance procoagulant activity in acute stroke patients with ICA occlusion. eBioMedicine. 2020;**53**:102671. DOI: 10.1016/j.ebiom.2020.102671

[106] Zarbock A, Polanowska-Grabowska RK, Ley K. Plateletneutrophil-interactions: Linking hemostasis and inflammation. Blood Reviews. 2007;**21**(2):99-111. DOI: 10.1016/j.blre.2006.06.001

[107] Stark K. Platelet-neutrophil crosstalk and netosis. HemaSphere. 2019;**3**(S2):89-91. DOI: 10.1097/ HS9.0000000000000231

[108] Carestia A, Kaufman T, Schattner M. Platelets: New bricks in the building of neutrophil extracellular traps. Frontiers in Immunology. 2016;**7**:271. DOI: 10.3389/fimmu.2016.00271

[109] Green SA et al. Activated platelet – T-cell conjugates in peripheral blood of patients with HIV infection : Coupling coagulation / inflammation and T cells. AIDS. 2015;**29**:1297-1308. DOI: 10.1097/ QAD.0000000000000701

[110] Nkambule BB, Davison G, Ipp H. Platelet leukocyte aggregates and markers of platelet aggregation, immune activation and disease progression in HIV infected treatment naive asymptomatic individuals. Journal of Thrombosis and Thrombolysis. 2015;**40**(4):458-467. DOI: 10.1007/s11239-015-1212-8

[111] Francisci D et al. HIV type 1 infection, and not short-term HAART , induces endothelial dysfunction. AIDS. 2009;**23**:589-596. DOI: 10.1097/ QAD.0b013e328325a87c

[112] Jong E, Louw S, Van Gorp ECM, Meijers JCM, Ten Cate H, Jacobson BF. The effect of initiating combined antiretroviral therapy on endothelial cell activation and coagulation markers in South African HIV-infected individuals. Thrombosis *Challenges in Platelet Functions in HIV/AIDS Management DOI: http://dx.doi.org/10.5772/intechopen.105731*

and Haemostasis. 2010;**104**(6):1228- 1234. DOI: 10.1160/TH10-04-0233

[113] Van Den Dries LWJ, Gruters RA, Van Der Borden SBCH. von Willebrand factor is elevated in HIV patients with a history of thrombosis. Frontiers in Microbiology. 2015;**6**:180. DOI: 10.3389/ fmicb.2015.00180

[114] Reininger AJ. The function of ultra-large von Willebrand factor multimers in high shear flow controlled by ADAMTS13. Hämostaseologie. 2015;**35**:225-233

[115] Mutlu A, Gyulkhandanyan AV, Freedman J, Leytin V. Concurrent and separate inside-out transition of platelet apoptosis and activation markers to the platelet surface. British Journal of Haematology. 2013;**163**(3):377-384. DOI: 10.1111/bjh.12529

[116] Reddy EC, Rand ML. Procoagulant phosphatidylserine-exposing platelets *in vitro* and *in vivo*. Frontiers in Cardiovascular Medicine. 2020;**7**:1-11. DOI: 10.3389/fcvm.2020.00015

[117] Böing AN, Hau CM, Sturk A, Nieuwland R. Platelet microparticles contain active caspase 3. Platelets. 2008;**19**(2):96-103. DOI: 10.1080/ 09537100701777295

[118] Gyulkhandanyan AV et al. Mitochondrial inner membrane depolarization as a marker of platelet apoptosis : Disclosure of nonapoptotic membrane depolarization. Clinical and Applied Thrombosis/Hemostasis. 2017;**23**(2):139-147. DOI: 10.1177/ 1076029616665924

[119] Leytin V, Allen DJ, Mutlu A, Gyulkhandanyan AV, Mykhaylov S, Freedman J. Mitochondrial control of platelet apoptosis: Effect of cyclosporin a, an inhibitor of the mitochondrial

permeability transition pore. Laboratory Investigation. 2009;**89**(4):374-384. DOI: 10.1038/labinvest.2009.13

[120] Thushara RM, Hemshekhar M, Kemparaju K, Rangappa KS, Girish KS. Biologicals, platelet apoptosis and human diseases : An outlook. Critical Reviews in Oncology/Hematology. 2015;**93**(3):149-158. DOI: 10.1016/j. critrevonc.2014.11.002

[121] Von Gunten S, Wehrli M, Simon H. Cell death in immune thrombocytopenia\_ novel insights and perspectives. Seminars in Hematology. 2013;**50**(1):S109-S115. DOI: 10.1053/j.seminhematol.2013.03.016

[122] Rand ML, Wang H, Bang KWA, Poon KSV, Packhams MA, Freedman J. Procoagulant surface exposure and apoptosis in rabbit platelets : Association with shortened survival and steady-state senescence. Journal of Thrombosis and Haemostasis. 2004;**2**:651-659

[123] Lebois M, Josefsson EC, Lebois M, Josefsson EC. Regulation of platelet lifespan by apoptosis. Platelets;**27**(6):497-504. DOI: 10.3109/ 09537104.2016.1161739

[124] Dasgupta SK et al. Platelet senescence and phosphatidylserine exposure. Transfusion. 2010;**50**(10):2167-2175. DOI: 10.1111/j.1537-2995.2010.02676.x.Platelet

[125] Carbonara S et al. Response of severe HIV-associated thrombocytopenia to highly active antiretroviral therapy including protease inhibitors. The Journal of Infection. 2001;**42**(4):251-256. DOI: 10.1053/jinf.2001.0833

[126] Servais J et al. HIV-associated hematologic disorders are correlated with plasma viral load and improve under

highly active antiretroviral therapy. JAIDSs. 2001;**28**:221-225

[127] Lewis W. Mitochondrial toxicity of antiviral drugs. Nature Medicine. 1995;**1**(5):417-422

[128] Baum PD, Sullam PM, Stoddart CA, McCune JM. Abacavir increases platelet reactivity via competitive inhibition of soluble guanylyl cyclase. AIDS. 2011;**25**(18):2243-2248. DOI: 10.1097/ QAD.0b013e32834d3cc3

[129] Khawaja AA et al. HIV antivirals affect endothelial activation and endothelial-platelet crosstalk. Circulation Research. 2020:1365-1380. DOI: 10.1161/ CIRCRESAHA.119.316477

[130] Chini M et al. Effects of highly active antiretroviral therapy on platelet activating factor metabolism in naïve HIVinfected patients: II study of the abacavir/ lamivudine/efavirenz haart regimen. International Journal of Immunopathology and Pharmacology. 2012;**25**(1):247-258. DOI: 10.1177/039463201202500127

[131] Jaschinski NJ, Greenberg L, Beesgaard B, et al. Recent abacavir use and incident cardiovascular disease in contemporary treated people living with HIV (PLWH) within the RESPOND cohort consortium. In: 18th European AIDS Conference. London, EACS 2021, October 27-30, 2021. Abstract BPD1/3

[132] Dorjee K, Baxi SM, Reingold AL, Hubbard A. Risk of cardiovascular events from current, recent, and cumulative exposure to abacavir among persons living with HIV who were receiving antiretroviral therapy in the United States: A cohort study. BMC Infectious Diseases. 2017;**17**(1):1-12. DOI: 10.1186/ s12879-017-2808-8

[133] Baker JV, Hullsiek KH, Bradford RL, Prosser R, Tracy RP, Key NS. Circulating levels of tissue factor microparticle

procoagulant activity are reduced with antiretroviral therapy and are associated with persistent inflammation and coagulation activation among HIVpositive patients. Journal of Acquired Immune Deficiency Syndromes. 2013;**63**(3):367-371. DOI: 10.1097/ QAI.0b013e3182910121

[134] Funderburg NT et al. Shared monocyte subset phenotypes in HIV-1 infection and in uninfected subjects with acute coronary syndrome. Blood. 2012;**120**(23):4599-4608. DOI: 10.1182/ blood-2012-05-433946

[135] Funderburg NT et al. Increased tissue factor expression on circulating monocytes in chronic HIV infection: Relationship to in vivo coagulation and immune activation. Blood. 2010;**115**(2):161-167. DOI: 10.1182/ blood-2009-03-210179

[136] Østerud B. The role of platelets in decrypting monocyte tissue factor. Seminars in Hematology. 2001;**38**(Suppl 12):2-5. DOI: 10.1053/shem.2001.29508

[137] Scholz T, Temmler U, Krause S, Heptinstall S, Lösche W. Transfer of tissue factor from platelets to monocytes: Role of platelet-derived microvesicles and CD62P. Thrombosis and Haemostasis. 2002;**88**(6):1033-1038. DOI: 10.1055/s-0037-1613351

[138] Lösche W, Scholz T, Temmler U, Oberle V, Claus RA. Platelet-derived microvesicles transfer tissue factor to monocytes but not to neutrophils. Platelets. 2004;**15**(2):109-115. DOI: 10.1080/09537100310001649885

[139] De Pablo-Bernal RS et al. TNF-α levels in HIV-infected patients after longterm suppressive cART persist as high as in elderly, HIV-uninfected subjects. The Journal of Antimicrobial Chemotherapy.

*Challenges in Platelet Functions in HIV/AIDS Management DOI: http://dx.doi.org/10.5772/intechopen.105731*

2014;**69**(11):3041-3046. DOI: 10.1093/ jac/dku263

[140] Laurence J, Elhadad S, Ahamed J. HIV-associated cardiovascular disease: Importance of platelet activation and cardiac fibrosis in the setting of specific antiretroviral therapies. Open Heart. 2018;**5**(2):1-13. DOI: 10.1136/ openhrt-2018-000823

[141] Feinstein MJ et al. Characteristics, prevention, and Management of Cardiovascular Disease in people living with HIV: A scientific statement from the American Heart Association. Circulation. 2019;**140**(2):e98-e124. DOI: 10.1161/ CIR.0000000000000695

[142] Currier JS et al. Coronary heart disease in HIV-infected individuals. JAIDS. 2003;**33**:506-512

[143] Boccara F et al. HIV and coronary heart disease: Time for a better understanding. Journal of the American College of Cardiology. 2013;**61**(5):511- 523. DOI: 10.1016/j.jacc.2012.06.063

[144] Benjamin LA et al. HIV infection and stroke : Current perspectives and future directions. Lancet Neurology. 2012;**11**(10):878-890. DOI: 10.1016/ S1474-4422(12)70205-3

[145] Lin HL, Muo CH, Lin CY, Chen HJ, Chen PC. Incidence of stroke in patients with HIV infection: A population-based study in Taiwan. PLoS One. 2019;**14**(5): 1-14. DOI: 10.1371/journal.pone.0217147

[146] Currier JS et al. Epidemiological evidence for cardiovascular disease in HIV-infected patients and relationship to highly active antiretroviral therapy. Circulation. 2008;**118**(2):e29-e35. DOI: 10.1161/CIRCULATIONAHA.107.189624

[147] Obel N et al. Ischemic heart disease in HIV-infected and HIV-uninfected

individuals: A population-based cohort study. Clinical Infectious Diseases. 2007;**44**(12):1625-1631. DOI: 10.1086/ 518285

[148] Archer O et al. Patients living with HIV and coronary disease: Are we using appropriate anti platelets as part of dual antiplatelet therapy? Cardiology & Vascular Research. 2021;**5**(1):1-5. DOI: 10.33425/2639-8486.1094

[149] Simpson SR, Singh MV, Dewhurst S, Schifitto G, Maggirwar SB. Platelets function as an acute viral reservoir during HIV-1 infection by harboring virus and T-cell complex formation. Blood Advances. 2020;**4**(18):4512-4521. DOI: 10.1182/bloodadvances.2020002420

Section 4
