Slide1: HIV STUDIES Laboratory diagnosis
Slide3: Research in epidemiology
Central America collaborative project
-PASCA, M. of Health and the University of Washington
Sex workers and men that have sex with men from CA
SW: 2% MHSM: 11%
C. trachomatis: 15%
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 Slide8: Epidemiology
Immune markers of progression: Immune markers of progression Define less expensive markers of progression in HIV infected patients
75 HIV+ patients, 75 HIV- subjects
CD4, CD8, total lymphocytes
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. Slide10: 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 Slide11: 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
Slide12: p-24 in sera CD4 cells/ul p-24 (pg/ml) 52 40 18 <200 200-500 >500 HIV- p24 levels Slide13: IL-2 and TNF CD4 cells/ul IL-2 and TNF alpha (pg/ml) <200 200-500 >500 HIV- TNF IL-2 Slide14: 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 Slide16: Epidemiology
Co-receptors used by the HIV to invade cells: Co-receptors used by the HIV to invade cells CCR5
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
- 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. Slide26: Epidemiology
Slide27: HIV-1 SUBTYPES Slide28: 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 Slide29: 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.
Slide31: Methodology DNA extraction
gp41 Nested PCR Chunfu Y. et al. J. Clin. Microbiol, Vol. 38, 1999.
Sequencing 1058 pb 1516 pb 460bp Slide32: 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. Slide36: INSTITUTO CONMEMORATIVO GORGAS