A prospective longitudinal observational study, the objective is to observe and describe the prevalence of INRT, INNRT, Protease and INIS mutations detected by proviral DNA in patients with virologic failure and low level viremia.
Inclusion/Exclusion: Our Study will include only patients with HIV that had been confirmed by "Instituto de Salud Pública de Chile" (ISP-Chile). Children less than 18 years are excluded.
Number of Patients: A total of 1,200 patients will be included in a period of three years (Including statistical justification, if appropriate).
1. \- Only patients who have signed informed consent will be admitted to our study.
2. \- The admitted patients will have a blood sample taken for an HIV genotypic resistance test.
3. \- from the clinical record of each patient admitted, some relevant clinical data will be taken to interpret the results such as CD4 T lymphocyte count, Viral Load, date of HIV diagnosis, date of initiation of therapy in addition to number and type of treatments antiretrovirals
4. \- By way of genotype control, a second blood sample will be taken every year.
Detailed Description
Detection of Resistance Mutations in Proviral DNA in HIV-1 Infection
Background The genetic test HIV proviral DNA (HPD) for resistance to antiretroviral drugs is very useful to detect mutations in patients under antiretroviral therapy who has failed to achieve undetectable viral loads levels or with low level viremia (LLV).
HPD allows the detection of circulating resistant variants and archived which could not be detected in the plasma.
HPD is independent of the viral load of patients. This makes it very useful for studies of primary resistance as it can detect resistance mutations in recently diagnosed patients with low baseline viral loads (\<1,000 copies/mL).
HPD is recommended by the European AIDS Clinical Society EACS for cases where there is not HIV RNA for analysis (Barcelona, October 2015).
HPD is able to analyze resistance to all antiretroviral drugs currently available in the market. Therefore, with the HPD innovation we can carry out the following genetic resistance tests: HIV Tropism (Maraviroc), resistance to Integrase Inhibitors (Dolutegravir) and resistance to Transcriptase reversa/Proteasa (Abacavir).
Size Sample and Methodology
During a period of two years, we will study a population of 1,200 patients. These include all patients we have analyzed in our HIV center until December 2012. The methodology includes nucleic acid extraction, amplification of HIV-1 genes that are targets of therapeutic interest and sequencing by traditional and new generation sequencing methods.
Patient Recruitment: a Total of 1.200 (400 by year)
Obtaining blood samples: two samples: sample to the start and one sample to the end after one year
DNA and viral RNA extraction:
RT-PCR amplification:
Electrophoresis visualization in agarose gels
Sending samples to Macrogen for sequencing services
Automatic sequencing by Sanger method in Macrogen USA
Sequencing by deep sequencing (NGS) in Chile or Macrogen USA
Sequence Analysis
Quality Analysis and assemble of raw sequences in software ReCall
Registry of mutations of clinical interest
Report on resistance to antiretroviral drugs
Biostatistical analysis
Results Report and Publications
Preliminary results Preliminary analysis of 100 patients with viral load \<1,000 copies/mL, detected 10% of patients with mutations of resistance to drugs that inhibit reverse transcriptase and viral protease. This valuable information enabled earlier therapeutic intervention by changing the drug scheme before they reach higher levels of viral load with consequent improvement in the clinical management.
Projections Project Due to the success and immediate application of this preliminary study, we believe it become very important to validate this technique as a daily routine genetic testing for HIV clinic patients in the public health system. Permanent financing for this genetic HIV test for HIV proviral DNA should be given by the Ministry of Health, Government of Chile.
In conclusion, at present HPD is very useful tool to study virologic failure and LLV in order to an early change of the antiretroviral therapy.
