Computational protocol: Recent HIV-1 Infection Contributes to the Viral Diffusion over the French Territory with a Recent Increasing Frequency

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Protocol publication

[…] The Ethics Committee of Cochin Hospital approved the study, and all the patients gave their written informed consent. The next of kin, carers or guardians gave their written informed consent on the behalf of the enrolled minors/children.Acute infection was defined as the period between exposure to the virus and completion of the initial immune responses, i.e. by detectable HIV RNA in plasma in the setting of a negative or indeterminate HIV antibody test. Primary and recent infections were defined as the period between 6 and 24 months following the exposure to the virus, respectively. Chronic infection was defined as evolving for more than 24 months after the viral exposure.Transmission cluster was defined as a clade of patients infected with strains whose phylogenetic analysis revealed a strong homology, suggesting a reduced (or direct) transmission chain of the virus.This study involved patients with primary HIV-1 infection (PHI) enrolled in the French ANRS PRIMO CO6 cohort between January 1999 and September 2010.PHI was defined by a western blot profile compatible with ongoing seroconversion (incomplete western blot with absence of antibodies to pol proteins) in most patients (94%), detectable plasma HIV RNA with a negative or weakly reactive ELISA (2%), or an interval of less than 6 months between a negative and a positive ELISA result (4%), as previously described . The date of infection was estimated as the date of symptom onset minus 15 days or, in asymptomatic patients, the date of incomplete western blot minus 1 month, or the midpoint between a negative and a positive ELISA result. Patients were enrolled if HIV infection was estimated to have occurred less than 6 months previously. At enrolment blood samples were collected for immunological and virological studies. All patients were antiretroviral (ART)-naïve at enrolment in the cohort. The present analysis included clinical epidemiological data and results of the serological screening for syphilis (Treponema palladium haemagglutination assay (TPHA) and Venereal Diseases Research Laboratory test (VDRL)), hepatitis B and hepatitis C viruses performed at enrolment. Participants completed standardized questionnaires describing HIV acquisition risk group, frequency of previous HIV screening tests and sexual behaviour (including number and characteristics of sexual intercourses before diagnosis of PHI and history of sexually transmitted infections (STI)).HIV-RNA was quantified with the Cobas Amplicor HIV-1 Monitor 1.5 assay, the Cobas Taqman HIV-1 v1.0 and v1.5 assay (Roche Diagnostics, Meylan, France) or the Versant HIV-1-RNA 3.0 assay (Bayer Diagnostics, Emeryville, CA, USA), as recommended by the manufacturers. Both methods have a detection limit of 20 or 50 HIV-RNA copies/mL.Drug resistance was evaluated by amplifying and sequencing the HIV-1 reverse transcriptase (RT) and protease genes in plasma HIV-RNA samples obtained at enrolment, as described elsewhere , . Resistance to nucleoside RT inhibitors (NRTI), non-nucleoside RT inhibitors (NNRTI) and protease inhibitors (PI) was defined according to the 2010 ANRS HIV-1 genotypic resistance interpretation algorithm ( RT nucleotide sequences were aligned with previously reported representatives of group M subtypes and circulating recombinant forms (CRFs) for which sequences are available in the HIV database (, using Clustal W (v1.7) with minor manual adjustments . Phylogenetic trees were constructed with the neighbor-joining method , and reliability of the branching orders was implemented by Clustal W using the bootstrap approach . Neighbor-joining plot was used to draw trees for illustrations.Phylogenetic interrelationships among viral sequences were estimated using neighbor-joining trees and maximum likelihood methods with BioEdit and MEGA4 integrated molecular evolutionary genetic analysis software , . The existence of clusters was ascertained using the statistical robustness of the maximum likelihood topologies assessed by high bootstrap values (>98%) with 1000 resamplings and short branch lengths (genetic distances <0.015%) . Infections in clusters were validated for congruent polymorphisms and mutational motifs.Comparisons between clustered and unique PHI, and between patients involved into small versus large clusters were made by using the Chi-square or the Fisher's exact test for categorical variables, and the t-test or the Wilcoxon test for continuous variables. Logistic regression was performed to study the factors independently associated with belonging to a transmission cluster.All RT nucleotide sequences were submitted to GenBank (accession numbers [JQ291804–JQ292793]). […]

Pipeline specifications

Software tools Clustal W, BioEdit, MEGA
Application Phylogenetics
Organisms Human immunodeficiency virus 1, Homo sapiens, Human immunodeficiency virus 2
Diseases Virus Diseases, HIV Infections