![Red and white blood cells, Platelets
• Anemia - common in HIV positives. HIV itself and various
OIs such as Mycobacterium avium complex (MAC) can
affect red blood cells and their oxygen-carrying capacity
• Neutropenia - Various anti-HIV drugs, OI medications
[including ganciclovir (Cytovene), used to treat
cytomegalovirus], and cancer chemotherapies that
suppress the bone marrow may lead to low neutrophil
levels
• Thrombocytopenia - may be caused by certain drugs,
autoimmune reactions, accelerated destruction by the
spleen, or HIV disease itself.](https://image.slidesharecdn.com/labhivfinal-210411173339/85/Laboratory-monitoring-of-Progression-of-HIV-23-320.jpg)
Laboratory monitoring of Progression of HIV
- 1. Laboratory Monitoring of Progression of Human Immunodeficiency Virus infection By- Dr Ankita Mohanty Resident Department of Microbiology LTMMC , Sion
- 2. Contents A. Epidemiology – Global and India B. Overview of HIV infection & Immunity C. Laboratory Tests for Monitoring of HIV progression D. Treatment Monitoring E. Treatment Failure
- 5. Source: NACO
- 7. B. Overview of HIV life cycle • HIV life cycle: 1. Binding and Fusion 2. Entry 3. Reverse transcription 4. Integration 5. Viral RNA and protein expression 6. Assembly and budding 7. Maturation • HIV target cells: 1. CD4T cells, 2. Macrophages, 3. Dendritic cells
- 9. HIV disease progression –Acute infection
- 10. Stem Cell Mature T-Cells HIV Infected Cells HIV-Specific T Cell Expansion of HIV-Specific Cells Killing of HIV Infected Cells Incomplete Clearance of HIV Infected Cells and Exhaustion HIV Specific T Cell Responses Thymus Periphery R T R T R T R T R T R T R T R T R T R T R T R T R T R T
- 11. HIV disease progression -- Clinical Latency • During this period of the disease, the immune systems remains competent at handling most infections with opportunistic microbes • Few or no clinical manifestation. • Steady destruction of CD4+ T cells and steady decline of circulating blood CD4+ T cells
- 12. HIV disease progression -- AIDS • Acquired Immune Deficiency Syndrome: • Catastrophic breakdown of host defenses, marked increase in viremia and clinical disease. • CD4+ cell count less than or equal to 200 per microliter • Clinical Features: • Opportunistic infection • Neoplasms • CNS involvement
- 13. HIV disease progression Levels (Separate Scales) CD4+ T cell HIV viral load CD8+ T cell Neutralizing Antibodies Years AIDS and Death Acute Asymptomatic (clinical latency) 4 – 8 weeks Primary infection
- 14. C. Markers of Disease Progression Non-specific 1. CD4 Count 2. CBC 3. Serum Neopterin 4. Beta 2 microglobulin Specific • Viral Load
- 15. Non-specific - 1. CD4 Count • As HIV disease progresses, CD4 cell counts decline, typically by about 30-100 cells/μl per year (depending on viral load) • it provides information about how well anti-HIV therapy is working
- 16. Mechanism of CD4 T cell depletion in HIV infection
- 17. Methods for detection of CD4 counts Flow cytometric • Dual platform approach • Single Platform Non flow cytometric • Coulter manual CD4 count assay • Dynal T4-T8 quantitative assay • ELISA based counting systems (Capcellia ELISA and TRAx and Zymmune)
- 22. 2.Complete Blood count (CBC) • People with HIV may have low blood cell counts (cytopaenias) due to chronic HIV infection or as a side effect of medications, particularly drugs that damage the bone marrow, where all blood cells are produced. • Blood cell counts are typically reported as the number of cells per μl of blood (cells/μl) or as a percentage of all blood cells. • People with HIV infection should receive a CBC about every six months, and more often if they are experiencing symptoms or taking drugs associated with low blood cell counts
- 23. Red and white blood cells, Platelets • Anemia - common in HIV positives. HIV itself and various OIs such as Mycobacterium avium complex (MAC) can affect red blood cells and their oxygen-carrying capacity • Neutropenia - Various anti-HIV drugs, OI medications [including ganciclovir (Cytovene), used to treat cytomegalovirus], and cancer chemotherapies that suppress the bone marrow may lead to low neutrophil levels • Thrombocytopenia - may be caused by certain drugs, autoimmune reactions, accelerated destruction by the spleen, or HIV disease itself.
- 24. 3. Serum Neopterin • Neopterin (6-D- erythrohydroxy-propylpterin) is a low molecular weight compound derived from an intermediate product of the de novo biosynthesis of tetrahydrobiopterin from guanosine triphosphate (GTP) • It is an early marker of HIV infection • The levels rise further on progression from pre AIDS to clinical AIDS.
- 25. Application of Neopterin measurements
- 26. HPLC Method • Neopterin is measured by its native fluorescence (350nm excitation, 440nm emission).
- 27. Shimadzu LC-VP • HPLC System Available: Shimadzu LC-VP • Sensitivity: High sensitivity analysis possible – As little as 1 ppb of F-can be analyzed without any preconcentration procedures.
- 28. 4.Beta 2 microglobulin (β-2M) • 11kD protein which forms a heterodimer with class I MHC molecules present on the surface of most nucleated cells • Free β-2M can be measured in both serum and urine • It correlates with the degree of progression of HIV disease. It spikes in acute infection, declines and then rises during the infection.
- 29. Determination of β-2M levels • The β-2M levels can be determined by enzyme immunoassay (EIA) using the in vitro diagnostic test kit (Immunotech, France). • The EIA test used is a competition assay. The intensity of the colour developed and the measured optical density (OD) is inversely proportional to the concentration of β-2M in the samples
- 30. Specific Marker: Viral load • The number of copies of HIV-1 RNA per milliliter of plasma. • Viral load is an accurate reflection of the burden of infection and the magnitude of viral replication. • It is critical in monitoring virologic response to ART.
- 32. Viral Load - determined by PCR
- 34. Method COBAS AmpliPrep/ COBAS TaqMan HIV-1 Real Time HIV-1 NucliSENSEa sy Q HIV-1 v2.0 VERSANT HIV-1 RNA 1.5 (kPCR) Xpert HIV-1 Manufacturer Roche Abbott BioMerieux Siemens Cepheid Advantages •High throughput •Can be used for other diseases •Large range •High throughput •Compatible with dried blood spots as well as body fluids •Can be used for other diseases •Low risk of contaminatio n •Can be used for other diseases •Easy •Rapid •Flexible Disadvantages •High Cost •Requires trained personnel and support •High Cost •Requires trained personnel and support •High Cost •Prone to contamination •High Cost •Specific space and infrastructur e demand •High Cost •Specific space and infrastructure demand
- 35. Recommendations for Viral Load and CD4 Count Monitoring Clinical Scenario Viral Load Monitoring CD4 Monitoring Before initiating ART At entry into care If ART initiation is deferred, repeat before initiating ART At entry into care If ART is deferred, every 3- 6 months After initiating ART Within 2 to 4 weeks, after that every 4 to 8 weeks 6 months after initiation After ART modification( drug toxicity or regimen simplification with viral suppression) 4 to 8 weeks after modification According to prior CD4 count ART modification due to virological failure Within 2 to 4 weeks, after that every 4 to 8 weeks Till viral load is suppressed Every 3 to 6 months
- 36. E. Treatment Monitoring tests 1. Resistance testing a. Genotypic tests b. Phenotypic tests c. Virtual phenotype 2. Therapeutic drug monitoring
- 37. 1. Resistance Testing • HIV can develop drug resistance by mutating in such a way that the virus can continue to replicate despite the drug. • Usually occurs when there is inadequate treatment adherence or when drug is not completely effective, allowing HIV to multiply and mutate in the presence of the drug • Prevention of the emergence of HIV drug resistance (HIV DR), is the key for success of ART programme and prevention of transmission of resistant virus
- 38. a. Genotypic Tests • These tests examine the genetic sequence of HIV's reverse transcriptase and/or protease enzymes to look for mutations that are known to be associated with resistance to particular drugs. • These assays require a plasma viral load of at least 500 to 1,000 copies/mL.
- 39. Drug Resistance Testing- Panel recommendations • For Anti retroviral therapy- Naïve Persons - Entry into care - In persons with acute or recent(early) HIV Infection - In Pregnant people with HIV - In People who will initiate ART on day of or soon after HIV Diagnosis
- 40. Drug Resistance Testing- Panel recommendations • For Anti retroviral therapy- Experienced Persons - When changing ART regimen in following: 1) Virologic failure and RNA copies > 1000/ml 2) Virologic failure and RNA copies 500- 1000/ml 3) Sub optimal viral load reduction
- 41. Systems Available Manufacturer ViroLogic VIRCO Visible Genetics Applied Biosystems System Available GeneSeq HIV GENChec TruGene ViroSeq Principle RT-PCR RT-PCR RT-PCR RT-PCR Minimum Detectable Viral Load 500 copies/ml 500 copies/ml 500 copies per ml 1000 copies/ml TAT 10 days 7 to 14 days 7 to 14 days 7 to 14 days
- 42. • The highly sensitive parallel allele-specific sequencing (PASS) assay can simultaneously look at a large number of viral genomes and detect minor (0.1 to 0.01%) drug-resistant populations. • A dried serum spot method of HIV genotypic testing is also being developed. This method is relatively simple and it is a potential means of making resistance testing available throughout the world.
- 43. PASS • After isolation of the genetic material in blood samples, tiny fluorescent tags designed to stick to HIV genes in particular ways are added. Tags designed to stick to mutated gene locations known to produce drug resistance are labelled to appear green, while tags designed to stick to the same gene locations but where the genes had not mutated are labelled to appear red.
- 44. • A sophisticated computer program is used to count the number of molecules with green or red fluorescent tags in each sample. • The test proves sensitive enough to detect a single mutated virus out of 10,000 non mutated viruses in the patient samples • The test also can detect when a virus molecule has more than one mutation, a capability that no commercially available test has achieved.
- 45. b.Phenotypic tests • done by adding a medication to an HIV culture in the laboratory to determine how much drug is needed to inhibit viral replication • Resistance levels are usually reported in terms of how much drug is needed to inhibit viral replication by 50 or 90 per cent (the IC50 or IC90, respectively), compared with wild-type virus. • more direct measure of resistance, but are more difficult, time-consuming, and expensive.
- 46. Systems Available Manufacturer ViroLogic Viralliance Virgo System Available Phenosense Phenoscript Antivirogram Principle PCR and direct ligation PCR fully replication- competent recombinant virus Minimum detectable load 500 copies/ml 200-1000 copies/ml 500 copies/ml TAT 2-3 weeks 7 to 14 days 2 weeks
- 47. c.Virtual phenotype • Estimation of the viral phenotype using a large database of more than 18,000 pairs of genotypic and phenotypic data. • HIV with a similar genotype is identified in the database, and the corresponding phenotypic information is used to estimate resistance • The reliability of virtual phenotyping has been investigated in several studies
- 48. VIRA 3001 • The first was an analysis of VIRA 3001, where the actual phenotype established during the study was compared with a virtual phenotype derived from genotypic data and the Virco database. • There was an 86% agreement between actual and virtual phenotypes, with major discrepancies in only 3% of cases.
- 49. Virco TYPE HIV-1 • The Virco TYPE HIV-1 virtual phenotype assay uses one of the world's largest reference databases of viruses isolated from people with HIV. • As of January 2008, the database contained 314,000 genotype samples and 86,000 phenotype samples. • Samples of each assay for an individual were available for 53,000 and this is the basis of calculation for the correlative database.
- 51. 2. Therapeutic Drug Monitoring • used to help individualize anti-HIV therapy by measuring the amount of drug in an individual's blood. • different people absorb, process, and eliminate drugs at different rates, and blood levels vary . • Ideally, the lowest plasma drug concentration between doses (the trough level, or Cmin) should still be high enough to inhibit HIV, but the highest concentration (the peak level, or Cmax) should not cause intolerable side effects.
- 52. Normal Response to ART • Virological response to ART: The viral load done 6 months after initiation of ART should be less than 1000 copies/ml. • Immunological response to ART: CD4 count of the patients should increase after initiation of ART. (at least 50 CD4 cells/cubic mm at 6 months of the initiation of the ART in ARV naïve patients, who are adherent to their treatment)
- 53. Evidences of suspected treatment failure • New OIs/ recurrence/ clinical events after 6 months on ART (after ruling out IRIS) • Progressive decline in CD4 counts • Slow/ no clinical improvement over 6- 12 months, associated with stationary CD4 count, despite good adherence. • In cases of sub-optimal rise in CD4 count at 6 months, despite good adherence and absence of clinical symptoms, one may repeat CD4 count at 3 months.
- 54. Chronology of Treatment Failure
- 55. Definitions of ART Failure
- 56. SOP for Determining Failure • Treatment Adherence: A detailed assessment of reasons for non-adherence needs to be made. Unless these reasons are identified, a patient will find it difficult to adhere to the second-line regimen. • Drug- drug interactions: Assessing whether the patient is concomitantly taking medications that interfere with ARV activity. For example, many patients may not reveal that they take herbal treatments along with the prescribed ART regimen. • Certain Opportunistic infections may lead to decline in the CD4 count, which may revert after treating those infections.
- 58. Classification of Anti-HIV Drugs 1. Nucleoside reverse transcriptase inhibitors (NRTIs): Zidovudine, Stavudine, Lamivudine, Abacavir, Zalcitabine, Emtricitabine, Didanosine. 2. Non nucleoside reverse transcriptase inhibitors (NNRTIs): Efavirenz, Nevirapine, Delaviridine. 3. Nucleotide reverse transcriptase inhibitors (NTRTIs): Tenofovir 4. Protease inhibitors (PIs): Saquinavir, Indinavir, Nelfinavir, Amprenavir, Fosamprenavir, Ritonavir, Lopinavir, Atazanavir. 5. Entry/Fusion inhibitors: Enfuvirtide, Maraviroc
- 59. NACO guidelines on ART •First line anti- retroviral therapy •Second line ART , for HIV-1 patients failing to first line NNRTI based regimen •Second line ART , for HIV-1 patients failing to first line PI based regimen(not exposed to NNRTI) •Second line ART , for HIV-1 patients failing to first line PI based regimen(intolerant to NNRTI or where NNRTI cannot be used) •Second line ART , for HIV-2 patients failing to first line PI based regimen
- 60. First line anti- retroviral therapy
- 61. Steps in initiating Second-line ART
- 66. Summary Monitoring of HIV Disease progression must include CD4 count, CBC, Viral load Reaching an undetectable viral load is a key goal of ART. Treatment Monitoring : Resistance tests- Phenotypic Genotypic Virtual phenotype Therapeutic Drug Monitoring
- 67. References 1. Harrison’s Principles of Internal Medicine, 20th Edition 2. C. M. Tsoukas And N. F. Bernard ,Markers Predicting Progression of Human Immunodeficiency Virus- Related Disease, CLINICAL MICROBIOLOGY REVIEWS, Jan. 1994, p. 14-28 3. Madhu Vajpayee and Teena Mohan, Current practices in laboratory monitoring of HIV infection Indian J Med Res. 2011 Dec; 134(6): 801–822.
- 68. References-continued 4. www.naco.gov.in (Accessed on 1/6/2019) 5.https://www.unaids.org/en (Accessed on 1/6/2019) 6.https://www.who.int/ (Accessed on 1/6/2019)
Editor's Notes
- #13: Candidiasis of bronchi, trachea, esophagus, or lungs Invasive cervical cancer Coccidioidomycosis Cryptococcosis Cryptosporidiosis, chronic intestinal (greater than 1 month's duration) Cytomegalovirus disease (particularly CMV retinitis) Encephalopathy, HIV-related Herpes simplex: chronic ulcer(s) (greater than 1 month's duration); or bronchitis, pneumonitis, or esophagitis Histoplasmosis Isosporiasis, chronic intestinal (greater than 1 month's duration) Kaposi's sarcomav Lymphoma, multiple forms Mycobacterium avium complex Tuberculosis Pneumocystis carinii pneumonia Pneumonia, recurrent Progressive multifocal leukoencephalopathy Salmonella septicemia, recurrent Toxoplasmosis of brain Wasting syndrome due to HIV
- #31: Whereas the CD4 count is an indicator of immune system function, the HIV viral load (RNA level) gives prognostic information on how quickly the CD4 count is likely to decline and, consequently, the risk of disease progression. Patients with high HIV viral loads generally demonstrate a faster decline in CD4 count and progression to AIDS-related illnesses, whereas those with low viral loads will usually have higher CD4 counts and remain asymptomatic for prolonged periods. A small percentage of persons with HIV infection may have very low or undetectable viral loads for extended periods of time.