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kandang kerbau women s and children s hospital is one of the major centers in singapore for the admission of sick children , including those showing respiratory illness . after obtaining prior approval from the hospital s ethics committee ( approval number ec/043/2004 )
, we collected nasopharyngeal swabs from 400 pediatric patients between october 2005 and january 2007 . when admitted to the hospital , these patients exhibited symptoms of acute lower respiratory tract infections ( lrti ) ( bronchiolitis , bronchitis , pneumonia , asthma , and wheezing ) and urti ( pharnygitis ) .
specimens were sent to the hospital s microbiologic laboratory for routine testing for influenza a and b viruses , rsv , adenovirus , and parainfluenza virus ( serotypes 13 ) by immunofluorescence assay ( lightdiagnostics , chemicon , tamacula , ca , usa ) .
the clinical specimens were stored at 80c until further analysis for hmpv was performed ( not longer than a week after collection ) . viral rna ( vrna )
was extracted from each of the thawed nasopharyngeal swabs with the qiaamp viral rna minikit ( qiagen inc . ,
of the total rna extracted from the clinical specimens , 5 l was subjected to real - time reverse transcription 
pcr ( rt - pcr ) testing by using the n gene specific primer set nl 
this was performed with the onestep rt  pcr kit ( qiagen ) on a corbett research rotorgene 3000 ( corbett life science , sydney , nsw , australia ) .
the pcr cycling conditions were 50c for 30 min , 95c for 15 min , and 45 cycles ( 95c for 20 s and 60c for 60 s ) .
specimens that tested positive by real - time rt - pcr analysis were confirmed by conventional rt  pcr by using the nl 
n primer set . the amplified products ( 163 bp ) were detected by using agarose gel electrophoresis , and their identity was confirmed by dna sequencing . of the 400 samples collected , 21 tested positive for hmpv infection , which suggests an incidence rate of 5.3% , compared with an 11.5% incidence rate for rsv ( table 1 ) .
previous reports have suggested that in some cases severe symptoms exhibited by rsv - infected patients are associated with dual infections involving hmpv ( 7 ) . although we detected the presence of hmpv and rsv in the patients screened , no evidence for co - infections was observed , which suggests a low occurrence for these viruses in singapore . in a recent study in australia ,
only 8 of 10,000 screened hospitalized patients showed evidence of co - infection with hmpv and rsv ( 8) . in contrast , several recent studies suggest that co - infections may account for a substantial number of instances in which hmpv has been detected .
for example , a recent study in brazil , which used a lower sample size than in our study , reported an 8% incidence rate for pediatric patients who had rsv and hmpv co - infections ( 9 )
. therefore , environmental factors may be a key feature in the development of co - infections .
the entire p gene sequences were amplified directly from the specimens by rt  pcr using the primers hmptpf 5-atgtcgttccctgaaggaaaagatattc-3 and hmptpr 5-ttaaactacataattaagtggtaaat-3. amplicons 884 bp in size were generated and corresponded to 1209 nt2093 nt of the hmpv genome ( strain jps03 - 240 , ay530095 ) .
touch - down  procedure ; conditions were 94c for 5 min followed by 30 cycles of 94c for 15 s , 62c ( reducing by 0.5c / cycle ) for 30 s , 72c for 1 min , and a final extension step of 72c for 7 min .
the sizes of the respective pcr - amplified products were examined by using agarose gel electrophoresis , gel - purified , and confirmed by dna sequencing .
the genetic relationship between the singapore hmpv isolates and those hmpv isolates described previously was analyzed by comparing the p gene sequences ( 10 ) .
alignments of nucleic acid sequences were created by using clustalx version 1.83 ( bips.u-strasbg.fr/fr/documentation/clustalx ) .
phylogenetic trees were constructed by using the neighbor - joining method ( 1,000 bootstrap replicates ) and edited with mega 3.1 ( 11 ) .
this analysis shows that although isolates representing both a and b genotypes were detected , the singapore isolates clustered more predominantly with representative hmpv strains in lineage a , in particular the sublineage a2 . in this study hmpv
was detected throughout the year , which suggests that in singapore , hpmv is present in the pediatric community throughout the year .
we also noted a slight increase in the incidence of b genotypes ( b1 and b2 ) during the last quarter of 2006 , but the implications of this finding are unclear .
phylogenetic analyses of nucleotide sequences of hmpv phosphoprotein showing comparisons with singapore - nanyang technological university ( sin06-ntu * ) sequences .
* the specimen number acquired during the course of the investigation ( e.g. , sin06-ntu14 ) was made with known strains ( highlighted  ) from canada [ can99 - 81 ( ay145294 , ay145249 ) , can97 - 83 ( ay297749 ) , can97 - 82 ( ay145295 , ay145250 ) , can98 - 75 ( ay297748 ) , can98 - 79 ( ay145293 , ay145248 ) ] , japan [ jps03 - 180 ( ay530092 ) , jps03 - 240 ( ay530095 ) , jps03 - 187 ( ay530093 ) , jps02 - 76 ( ay530089 ) ] , and the netherlands [ nl-1 - 00 ( af371337 ) , nl-17 - 00 ( ay304360 ) , nl-1 - 99 ( ay525843 ) , nl-1 - 94 ( ay304362 ) ] .
the age and clinical characteristics of the hmpv patients were next compared with the different hmpv lineages ( table 2 ) .
children with hmpv infection were 1 month to 12 years in age ; 67% were < 1 year of age compared with 63% of rsv - infected children .
of the hmpv - infected patients , 52% exhibited lrti ; of these , 82% were infected with the hmpv sublineage a2 .
in contrast , 43% of the patients exhibited urti caused by the sublineages a2 and b2 . in comparison
, 61% and 20% of the rsv patients had a clinical diagnosis of lrti or urti , respectively .
our data suggested an increased association of sublineage a2 with lrti in the hmpv - infected patients .
the implications of this are unclear , but several reports note a correlation between severity of infection and the presence of the a genotype ( 12,13 ) .
unfortunately , we were not able to make a strict comparison of our data with data from recent studies in southeast asia ( 14,15 ) ; these studies used significantly smaller sample sizes and a different selection criterion for the patients screened ( i.e. , lrti   and wheezing and asthma ) . *
l , lower respiratory infections including bronchiolitis , bronchitis , pneumonia , asthma , wheezing or chest infection ; u , upper respiratory infections including infantile pyrexia and pharyngitis ; o , febrile fit .
our study is the first , to our knowledge , that has attempted to assess the importance of hmpv among the pediatric population in singapore .
we analyzed 400 samples that were collected from pediatric patients who were admitted to a hospital over a 16-month period .
an infection rate of 5.3% was observed , which is consistent with the reported infection rates of several other industrialized countries .
we also noted that of the viruses detected , 67% were of the a subtype and 33% were of the b subtype , which suggests that the former was the predominant hmpv subtype causing illness in these patients
our findings suggest that hmpv is a substantial cause of illness among the pediatric population of singapore .
four hundred specimens were collected from pediatric patients hospitalized in singapore ; 21 of these specimens tested positive for human metapneumovirus ( hmpv ) , with the a2 genotype predominating . 
 a 5% infection rate was estimated , suggesting that hmpv is a significant cause of morbidity among the pediatric population of singapore .