Pyrosequencing Reveals Increase in Influenza Resistance to Adamantanes in USA; Canadian report confirms


On January 14 2006, the United States Centers for Disease Control and Prevention (CDC) released a Health Alert Notice informing of the high frequency of adamantane resistance found in currently circulating influenza strains in the USA. Out of 120 strains gathered from 23 states, 109 strains (91%) were found to contain mutations in the viral M2 gene which are known to confer resistance to the generic class of drugs the adamantanes (sold as Amantadine and Rimandatine). In an independent announcement on January 15, the Public Health Agency of Canada announced an identical prevalence of resistance among circulating strains in Canada, thus confirming the CDC findings with Pyrosequencing.

Consequently, CDC recommends against the use of the anti-viral drugs Amantadine and Rimantadine in the 2005/06 influenza season. For the full text of the CDC Health Alert, click here: http://www.cdc.gov/flu/han011406.htm

According to CDC officials, Pyrosequencing was useful to detect the resistance mutations in a timely and cost-efficient manner. The following links give information to health researchers on the use of Pyrosequencing for detection of the implicated M2 gene mutations.

 Pyrosequencing for rapid influenza A antiviral drug resistance surveillance

 Pyrosequencing system PyroMark ID

Pyrosequencing for rapid influenza A antiviral drug resistance surveillance

• The adamantane anti-viral drug class are inhibitors of the influenza M2 protein ion channel
  
  
• Resistance can develop against adamantanes through point mutations in the influenza M2 gene 
  
  
• Resistance was monitored in H3N2, H1N1 and H1N2 influenza A subtypes using Pyrosequencing^® Technology
  

Bright RA, Medina M, Xu X, Perez-Oronoz G, Wallis TR, Davis XM, Povinelli L, Cox NJ, Klimov AI, “Incidence of adamantane resistance among influenza A (H3N2) viruses isolated worldwide from 1994 to 2005: a cause for concern”. Lancet, 2005; 366: 1175–81.

Adamantane resistance in Influenza A (H3N2) - Pyrosequencing Assay Design

• Bright et al. sequenced 44-base region coding for amino acids 25-38
  
  
• 6,524 isolates of H3N2 were sequenced
  
  
• Pyrosequencing data for 165 randomly-chosen isolates confirmed by independent methods (Sanger sequencing and RFLP)
  

Bright RA et al., Lancet, 2005; 366: 1175–81.

Primer design

Method

Viral RNA was extracted and a 264-bp region of the Influenza A matrix gene was amplified using OneStep RT-PCR kit (Qiagen, Hilden, Germany) with 0.2 µM each of the primers M2F and MR. Twenty microlitres of the PCR product were prepared for sequencing essentially according to ‘Sample Preparation Guidelines for PSQ96 and PSQ 96MA Systems’ using M2FS as sequencing primer at a concentration of 0.45 µM in 40 µL Annealing Buffer. Annealing was performed by incubating at 80⁰C for 4 minutes and then slowly cooling by (i) placing the PSQ 96 Plate Low together with PSQ96 Sample Prep Thermoplate Low on the bench for 10 minutes, and (ii) placing the PSQ 96 Plate Low directly on the bench for at least 4 minutes. The 44-base region comprising nucleotides 784-827 of the matrix gene (amino acids 25-38 of the M2 protein) was sequenced on Pyrosequencing PSQ 96MA using PSQ SQA Reagent Kit and the dispensation order CATG.

Adamantane resistance in Influenza A (H3N2) - Workflow (96 samples)

Pyrosequencing is an appropriate technology to identify M2 mutations for larger numbers of isolates because it provides a simplified and faster preparative and analytical workflow compared to traditional sequencing. Pyrosequencing can sequence the specified M2 region of 96 samples within 1 hour.

Adamantane resistance in Influenza A (H3N2) - Assay Results

Bright RA et al., Lancet, 2005; 366: 1175–81.

The boxed region indicates amino acid 31, the position of one mutated codon that leads to resistance to the antiviral drug class (S31N substitution). Sequencing is appropriate for the detection of mutations spread out in a region, and even increases the chances of detecting new mutations that conventional, probe-based methods would miss (e.g. Q-PCR).

According to CDC, viral resistance to adamantanes can emerge rapidly during treatment because a single point mutation at amino acid positions 26, 27, 30, 31, or 34 of the M2 protein can confer cross-resistance to both amantadine and rimantadine. For the 2005–06 season, 120 influenza A (H3N2) viruses isolated from patients in 23 states have been tested at CDC through January 12, 2006; 109 of the isolates (91%) contain an amino acid change at position 31 of the M2 protein, which confers resistance to amantadine and rimandatine.

Adamantane resistance in Influenza A (H3N2) - Observed Growth Over Time

 

Bright RA et al., Lancet, 2005; 366: 1175–81

Bright RA, Shay D, Bresee J, Klimov A, Cox N, Ortiz J (2006). High levels of adamantane resistance among influenza A(H3N2) viruses and interim guidelines for use of antiviral agents – United States, 2005-06 influenza season. Morbidity and Mortality Weekly Report, January 17, 2006.

• Significant increase in resistance in Asia
  
  
• 91% resistance recently discovered in USA*
  
  
• Important to track emergence and spread of resistant influenza A viruses 
   
  

* Bright, RA et al., Morbidity and Mortality Weekly Report, January 17, 2006