Investigación de VIH ICGES

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Slide1: 

HIV STUDIES Laboratory diagnosis Serology Viral load Genotyping CD4/CD8 Research Epidemiology Immune response Co-receptors Virus subtypes

Slide2: 

Epidemiology Immune response Genetic factors Virus subtypes

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Research in epidemiology Central America collaborative project -PASCA, M. of Health and the University of Washington -Study population: Sex workers and men that have sex with men from CA -Preliminary results HIV prevalence: SW: 2% MHSM: 11% Other STDs C. trachomatis: 15% NG: 4% NG+CT: 2%

Prevalence of HIV, Sifilis y Trichomonas and any other STD in CSW : 

Prevalence of HIV, Sifilis y Trichomonas and any other STD in CSW

Prevalence of HIV, Sifilis y Trichomonas and any other STD in CSW by place of work : 

Prevalence of HIV, Sifilis y Trichomonas and any other STD in CSW by place of work

Prevalence of HIV, Sifilis y Trichomonas and any other STD in CSW by type of activity : 

Prevalence of HIV, Sifilis y Trichomonas and any other STD in CSW by type of activity

Prevalence of N. gonorrhoeae and C. trachomatis in CSW in Panama, N=434: 

Prevalence of N. gonorrhoeae and C. trachomatis in CSW in Panama, N=434

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Epidemiology Immune response Genetic factors Virus subtypes

Immune markers of progression: 

Immune markers of progression Define less expensive markers of progression in HIV infected patients Study population: 75 HIV+ patients, 75 HIV- subjects Markers: CD4, CD8, total lymphocytes Beta 2-microglobulin P-24 Immunoglobulins Pascale, J. M. et al. Immunological markers of disease progression in patients infected with the Human Immunodeficiency Virus. Clin Diag Lab Immunol Vol.4, No. 4, 1997.

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T lymphocytes in HIV+ and HIV- subjects CELLS/MICROLITER HIV- SUBJECTS 766 1120 2596 1112 784 2393 708 238 1451 CD4/CD8 1.46 0.70 0.34 HIV+ PATIENTS ASYMPTOMATIC AIDS

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B-2 MICROGLOBULIN CONCENTRATION (mg/l) 2.2 + 0.7 3.4 + 2.1 5.7 + 3.6 HIV- SUBJECTS ASYMPTOMATIC AIDS HIV + PATIENTS B-2 Microglobulin Beta 2 Microglobulin in plasma of HIV+ and HIV- subjects

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p-24 in sera CD4 cells/ul p-24 (pg/ml) 52 40 18 <200 200-500 >500 HIV- p24 levels

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IL-2 and TNF CD4 cells/ul IL-2 and TNF alpha (pg/ml) <200 200-500 >500 HIV- TNF IL-2

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IgG, IgA and IgM levels in HIV+ and HIV- subjects CD4 cells/ul Ig concentration mg/dl <200 200-500 >500 HIV - Total IgG Total IgA Total IgM

Percentages of HIV+ patients showing specific levels of immune markers of progression: 

Percentages of HIV+ patients showing specific levels of immune markers of progression Immune marker Asymptomatic AIDS (%) (%) CD4 <200 cells/ml 4 96 TL <1750 cells/ ml 15 89 B2-M >4 mg/l 25 81 IgA >450 mg/dl 34 72 TL+B2M 11 91

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Epidemiology Immune response Genetic factors Virus subtypes

Co-receptors used by the HIV to invade cells: 

Co-receptors used by the HIV to invade cells CCR5 Macrophages APC CXCR4 T cells

CCR5 mutations: 

CCR5 mutations CCR5 is coded by the CMKBR5 gen. At least 20 mutations of the CCR5 receptors have been identified. The 32 deletion protects homozygous subjects from HIV infection, and delays progression of the disease in heterozygous patients.

Receptor del CCR5-wt: 

Receptor del CCR5-wt

Receptor de CCR5-32: 

Receptor de CCR5-32

Methodology: 

Methodology DNA extraction: - Buffy coat Amplification of DNA: - One tube PCR Electrophoresis: - 2% Agarose-TBE

Results: 

Results 188 bp Wt/Wt 156 bp Wt/32

32 Deletion : 

32 Deletion

Results: 

Results 1- From the 83 patients 8 were (wt/32). 2- 75 patients were wt/wt. 3- All (wt/32) survived more than 11 years without treatment (slow progressors). 4-None of the (wt/32) children were infected.

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Epidemiology Immune response Genetic factors Virus subtypes

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HIV-1 SUBTYPES

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Determine HIV-1 subtypes circulating in Panama. Evaluate the genetic diversity of the HIV subtypes identified. Correlate HIV subtypes variability with transmission patterns, immune response, and progression. OBJECTIVES

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Why gp41? Small fragment Highly conserved region between M,N and O (Agwale et al (2001) J. Clin. Microbiol. 39:2110-2114). Can be used for phylogenetic analyses

Study Samples: 

Study Samples Subjects: 25 HIV positive patients from Saint Thomas Hospital that came for viral load evaluation. Method: Gp41 Sequencing

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Methodology DNA extraction gp41 Nested PCR Chunfu Y. et al. J. Clin. Microbiol, Vol. 38, 1999. Sequencing 1058 pb 1516 pb 460bp

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SEQUENCING 1-Genotyping: drug resistance 2-HIV subtypes

Panama vs other Subtypes: 

Panama vs other Subtypes Subtype B Other Subtypes

Future Research: 

Future Research 1-To determine the importance of other mutations in progression of the infection. 2-Immune mediators of resistance and/or progression. 3-Circulation of different HIV subtypes and recompbinant forms in Panama.

Where we are??: 

Where we are?? 1-What do we have? More than 300 samples (plasma and buffy coat) of HIV infected patients. 2-What facilities do we have? Flow cytometer, ABI-310 sequencer, viral load and genotying equipments. 3-What do we need? Improved softwares for subtyping and genetic analysis of recombinant forms.

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INSTITUTO CONMEMORATIVO GORGAS

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