title: 'Sentinel' mutations in standard population sequencing can predict the presence of HIV-1 reverse transcriptase major mutations detectable only by ultra-deep pyrosequencing.
abstract: OBJECTIVE
  This proof-of-concept study aimed to identify whether mutations considered not yet relevant for drug resistance (but located at key drug-resistance positions) can act as 'sentinels' of minority resistant variants in HIV-1 drug-naive patients.
  
  
  METHODS
  We focused our attention on three reverse transcriptase (RT) mutations (T69S, L210M and K103R) easily detected by standard population sequencing [i.e. the genotypic resistance test (GRT)]. Ultra-deep pyrosequencing (UDPS) of HIV-1 RT was performed using GS-FLX Roche, on plasma RNA from 40 drug-naive patients infected with HIV-1 subtype B without primary resistance detected by GRT. Only RT drug resistance mutations detected at >0.1% in both forward and reverse directions were considered. Associations between GRT sentinel mutations and UDPS drug resistance were assessed using Fisher's exact test.
  
  
  RESULTS
  UDPS detected drug resistance mutations in 18/40 drug-naive patients. Patients carrying HIV-1 strains with T69S and L210M by GRT showed a trend to greater infection by minority drug-resistant variants than control patients infected by HIV-1 without these mutations (5/10 and 7/10 versus 3/10; P = not significant). No association was found for K103R by GRT. Notably, T69S and L210M (but not K103R or control viruses) were associated with GRT minority drug-resistant variants with a prevalence >1% (3/10 and 4/10 versus 0/20 in K103R and controls; P = 0.03 and P = 0.008, respectively). Moreover, the presence of L210M or T69S viruses by GRT significantly correlated with that of minority thymidine analogue mutations by UDPS (6/20 patients carrying HIV-1 strains with T69S/L210M versus 0/20 patients carrying HIV-1 having K103R or none of these mutations; P = 0.03).
  
  
  CONCLUSIONS
  This proof-of-concept study suggests the existence of genetic markers, detectable by routine testing, potentially acting as sentinel mutations of minority drug resistance. Their identification may help in the selection of patients at high risk of resistance in reservoirs without the necessity of using UDPS.
authors: Department of Experimental Medicine and Biochemical Sciences,  University of Rome 'Tor Vergata', Rome,  Italy.
authors: Claudia
authors: C
authors: Alteri
authors: Maria Mercedes
authors: MM
authors: Santoro
authors: Isabella
authors: I
authors: Abbate
authors: Gabriella
authors: G
authors: Rozera
authors: Alessandro
authors: A
authors: Bruselles
authors: Barbara
authors: B
authors: Bartolini
authors: Caterina
authors: C
authors: Gori
authors: Federica
authors: F
authors: Forbici
authors: Nicoletta
authors: N
authors: Orchi
authors: Valerio
authors: V
authors: Tozzi
authors: Guido
authors: G
authors: Palamara
authors: Andrea
authors: A
authors: Antinori
authors: Pasquale
authors: P
authors: Narciso
authors: Enrico
authors: E
authors: Girardi
authors: Valentina
authors: V
authors: Svicher
authors: Francesca
authors: F
authors: Ceccherini-Silberstein
authors: Maria Rosaria
authors: MR
authors: Capobianchi
authors: Carlo Federico
authors: CF
authors: Perno
mesh_terms: D000328:Adult; D023241:Antiretroviral Therapy, Highly Active; D001483:Base Sequence; D024882:Drug Resistance, Viral; D005260:Female; D005819:Genetic Markers; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology; D054303:HIV Reverse Transcriptase / Q000235:genetics*; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D018571:Sentinel Surveillance; D017423:Sequence Analysis, RNA
chemical_list: D005819:Genetic Markers; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase