we report results from an avian influenza quality assurance program ( qap ) that used an established , internet - based quality assurance reporting system ( www.rcpaqap.com.au/serology ) , allowing remote data entry , rapid result dissemination , and expert comment . the qap provided feedback to laboratories on nat characteristics ( pcr accuracy , sensitivity , and specificity ) , reporting optimization , and assessment of continuously updated laboratory - developed nat methods . during 2006 , three panels of specimens were distributed to 29 participating laboratories : 15 from australia ( including 4 veterinary laboratories ) ; 2 from hong kong special administrative region , people s republic of china ; 5 from singapore ; 1 from new caledonia ; 1 from malaysia ; and 5 from new zealand . the panels consisted of an indonesian and a vietnamese strain of avian influenza virus ( h5n1 ) , originally isolated from humans and grown in mdck cells . viral copy numbers were estimated by comparing real - time rt - pcr crossing - point values to a standard curve generated by using plasmid standards ; the amplicon was cloned into pgemt - easy ( promega , madison , wi , usa ) . plasmid standard concentrations were estimated as described previously ( 13 ) and as recommended by the lightcycler manufacturer ( roche , indianapolis , in , usa ) . sensitivity of nats was determined with a range of clinically relevant nucleic acid concentrations of both influenza ( h5n1 ) strains ( 10 to 10 copies/l ) to enable laboratories to assess limit of detection ( lod ) of their assays . specificity was assessed by inclusion of other influenza strains and a negative control ( table 1 ) . all strains and mdck cells were inactivated by exposure to 50 kgy of -irradiation , except for strain a ( h7n4 ) , which was inactivated by the addition of lysis buffer ( 14 ) . * these dilutions for panel 3 , the dilution for the influenza ( h5n1 ) vietnamese strain was changed to 1:1,000,000 , with a copy number of 5  10 l . 1 ) lod determinations , with a dilution series of all strains , were tested by using real - time rt - pcr ( 15 ) . 2 ) transport media were compared by using serial dilutions of inactivated influenza virus ( h5n1 ) in phosphate - buffered saline with gelatin ( with antimicrobial agents ) ( pbsg ) , te buffer , and buffer rlt ( lysis ) ( qiagen , valencia , ca , usa ) , placed at 80c , 20c , + 4c , + 25c , and + 37c for 10 days . each day , 1 tube at each temperature condition was removed , and viral dna was extracted by using the qiaamp viral rna minikit ( qiagen ) and tested with a real - time rt - pcr ( 15 ) . 3 ) for further stability testing , a test panel diluted in pbsg was sent by courier from sydney to hong kong , held by australian customs for 7 days , and returned unopened 15 days after dispatch . the temperature range the shipped panel was exposed to is unknown ; however , previous temperature loggers have recorded temperatures from 22c to 33c . the panel that traveled was tested against a panel that had been stored optimally ( 80c ) ( 15 ) . no difference was detected in the amount of virus in the specimens that traveled compared with optimally stored specimens , indicating that the specimens were stable under normal transport conditions ( results not shown ) . 4 ) homogeneity was established before distribution by having the panel tested and approved by 2 reference laboratories ( victorian infectious diseases laboratory , melbourne , victoria , australia ; western australian centre for pathology and medical research , perth , western australia , australia ) and 1 animal reference laboratory ( elizabeth macarthur agricultural institute , camden , new south wales , australia ) . for each panel the samples were diluted in pbsg and transported by courier at ambient temperature in 3 seasons ( autumn , winter , summer ) . participants were asked for information on methods used , including extraction and rt - pcr protocol and primer / probe sequences . a total of 780 results were analyzed , and a report was issued to participants within 3 weeks of each survey closing , well before the next panel shipment . this allowed participants to adjust their testing procedures if necessary before the next survey began . results were reported by participants as positive , negative , or equivocal . for simplicity , we report equivocal results as positive , given that participating laboratories retest an equivocal result and generally do not report such results as negative . on average for the 3 panels , 2.6% of results were reported as equivocal . panel 1 contained 8 specimens , which included 2 dilutions ( 10 and 10 copies/l ) of each subtype h5n1 strain ( table 1 ) . only 35% of participants correctly identified all samples ; 95% reported a correct result for the highest concentration of both subtype h5n1 strains ( 10 copies/l ) ; 70% could detect the lower concentration ( 10 copies/l ) . only 46% of participating laboratories used an influenza a matrix assay as well as a specific h5 assay ( table 2 ) . laboratories were advised to use both methods in tandem to reduce the chance of missing variant influenza ( h5n1 ) strains that might not be detected by their specific h5 assay . some false positives ( 1.3% ) were reported , and some confusion regarding terminology occurred : many laboratories reported results as for subtype h5n1 assays , despite most of these results being specific for the h5 gene only . * rt - pcr , reverse transcription  pcr . panels 2 and 3 had 2 very low dilutions of subtype h5n1 that were beyond the limit of detection for most laboratories . percentage of correct results reported for the indonesian and vietnamese strains , with these results included , is 59 and 59 for panel 2 and 56 and 66 for panel 3 , respectively . no participants correctly identified all samples because of the addition of 2 extremely dilute samples of influenza virus ( h5n1 ) ( 10 and 10 copies/l ) that were below the lod for most laboratories . eleven percent of participants detected 1 strain of hpai virus ( h5n1 ) by using primers specific for h5 or subtype h5n1 at 1 of the 2 highest dilutions , but not both . in our experience , dilute specimens are useful for assessing the lod of the testing system because they may highlight the most sensitive methods available . the number of laboratories using a generic influenza a test , in addition to a specific h5 test , increased to 73% ( table 2 ) . for panel 3 , sensitivity of detection improved compared with panel 2 : 25% of participants detected a strain of influenza virus ( h5n1 ) at the lowest concentrations . sensitivity of h5/h5n1 testing for the influenza ( h5n1 ) vietnamese strain increased over time , while sensitivity of testing decreased slightly over the 3 panels for the influenza ( h5n1 ) indonesian strain ( table 2 ) . laboratories had altered primer / probe sets to increase sensitivity for the vietnamese strain , which resulted in decreased sensitivity for the indonesian strain . sensitivities of other testing methods ( influenza a , b , h3 ) increased during subsequent testing of each panel ( data not shown ) ; the number of correct results reported by participants using influenza a matrix testing rose from 84% in panel 1 to 91% in panel 3 . most participants did not disclose their primer / probe sequence information , which made it difficult to recommend the most sensitive methods to other participants . however , during a prepandemic phase , having a range of primers / probes being used may be optimal , providing influenza a matrix detection is also conducted and qa is maintained , until who recommends a method to detect new pandemic strains . participants in the avian influenza qap made clear improvements in the sensitivity and specificity of their nat methods over time . it is important to provide continuing qa to expose inconsistencies in results or primers that may be skewed toward a particular strain .
an avian influenza quality assurance program was used to provide information for laboratories on the sensitivity and specificity of their avian influenza nucleic acid testing . 
 most laboratories were able to correctly detect clinically relevant amounts of influenza virus ( h5n1 ) , and results improved as each subsequent panel was tested .