Rapid communications Increased detection of Mycoplasma pneumoniae infection in children in England and Wales, October 2011 to January 2012

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1 Rpid communictions Incresed detection of Mycoplsm pneumonie infection in children in Englnd nd Wles, October 2011 to Jnury 2012 V J Chlker (vicki.chlker@hp.org.uk) 1, T Stocki 1, D Litt 1, A Berminghm 1, J Wtson 1, D M Fleming 2, T G Hrrison 1 1. Helth Protection Agency, Colindle, London, United Kingdom 2. Birminghm Reserch Unit of the Royl College of Generl Prctitioners, Birminghm, United Kingdom Cittion style for this rticle: Chlker VJ, Stocki T, Litt D, Berminghm A, Wtson J, Fleming DM, Hrrison TG. Incresed detection of Mycoplsm pneumonie infection in children in Englnd nd Wles, October 2011 to Jnury Euro Surveill. 2012;17(6):pii= Avilble online: Article published on 9 Februry 2012 Community surveillnce dt, bsed on quntittive rel-time polymerse chin rection nlysis, showed tht one in seven children ged 5 14 yers with respirtory signs tested positive for Mycoplsm pneumonie in Englnd nd Wles from October 2011 to Jnury 2012 higher proportion thn tht seen in previous yers. Multilocus vrible number tndem repet nlysis indictes tht t lest seven known nd two novel strin types were circulting in Englnd nd Wles during this period. Recent reports indicte tht n incresed number of Mycoplsm pneumonie infections hve been detected in seven Europen countries including Denmrk, Norwy nd Finlnd [1-4]. To determine the number of ptients infected with M. pneumonie in Englnd nd Wles nd to see if the number hd incresed, compred with previous winters, community surveillnce dt nd lbortory reports submitted to the Helth Protection Agency (HPA) from 10th October (week 42) 2011 to 20th Jnury (week 3) 2012 were reviewed. Our study shows n increse in the number of children with M. pneumonie infection by PCR-bsed surveillnce in the community during the study period. Further nlysis ws crried out to determine which strins of M. pneumonie were present in this period in the community surveillnce smples, in ddition to nlysis of genetic mrkers for mcrolide resistnce. Bckground M. pneumonie is respirtory pthogen tht is common cuse of pneumoni nd my cuse other serious sequele such s encephlitis. The pthogen is found in ll ge groups, with higher prevlence in children ged 5 14 yers [2,5]. In Englnd nd Wles, epidemic periods lsting on verge 18 months hve occurred t pproximtely four-yerly intervls [6]. In ddition, low-level spordic infection occurs with sesonl peks from December to Februry [5,6]. Since 2005, community surveillnce scheme for M. pneumonie using quntittive rel-time polymerse chin rection (qpcr) nlysis hs been used to monitor M. pneumonie infection in Englnd nd Wles [7]. Until 2010, this scheme ws used for monitoring ptients of ll ges nd from 2010 to dte, for children ged under 16 yers [7]. It is n extension of the virologicl community surveillnce tht is undertken nnully in Englnd nd Wles for rnge of respirtory viruses including influenz virus, respirtory syncitil virus nd humn metpneumovirus [8]. Combined nsl nd throt swbs were tken during the winter months (from October to Mrch, 2005 to 2012, nd throughout the recent influenz A(H1N1) pdm09 pndemic) from ptients with respirtory symptoms including influenz-like illness, upper respirtory trct infection, lower respirtory trct infection, or fever or mylgi who ttended generl prctitioner clinics [5]. Additionl voluntrily submitted reports from regionl lbortories nd hospitls in Englnd nd Wles were collted by the Helth Protection Agency (HPA) ccording to ge nd region to give n indiction of the number of ptients testing positive for M. pneumonie by serologicl, moleculr or culture tests ech week. Detection nd nlysis of M. pneumonie in clinicl smples Lbortory reports The number of M. pneumonie-positive lbortory reports submitted to the HPA during the study period (week to week ) vried from 11 to 36 per week, s shown in the four-weekly moving verges in [9]. From week to week , totl of 353 reports were received, higher thn the number in the sme period in 2010 (week to week ), when 290 were received. Reports were received from ll res of Englnd nd Wles during this period (Tble 1). The ptients were of ll ges, with the youngest 1

2 being less thn one week old nd the oldest 92 yers of ge (Tble 2). This ge profile of submitted M. pneumonie-positive reports ws very similr to tht for ll such reports received from week to week Community surveillnce We crried out qpcr nlysis on 144 nonymised combined nose nd throt swbs tken s prt of community surveillnce from ptients ged under 15 yers with respirtory symptoms during October 2011 to Jnury 2012 ( totl of 144 swbs were tken during tht time). Nucleic cid ws extrcted nd stored s previously described before qpcr testing for the presence of the M. pneumonie P1 gene [5,10]. A totl of 13 of the smples (9.0%; 95% CI: ) were M. pneumonie positive. One in seven of the children ged 5 14 yers (12/84) hd detectble Tble 1 Percentge of Mycoplsm pneumonie-positive smples from lbortory reports by region, Englnd nd Wles, 10 October (week 42) Jnury (week 3) 2012 (n=353) Percentge of smples positive for M. pneumonie Region positive smples Est Midlnds 5.7 ( ) 20 Est 8.5 ( ) 30 London 24.4 ( ) 86 North Est 6.0 ( ) 21 North West 13.9 ( ) 49 South Est 4.0 ( ) 14 South West 7.1 ( ) 25 West Midlnds 5.4 ( ) 19 Wles 14.5 ( ) 51 Yorkshire nd Humberside 10.8 ( ) 38 Tble 2 Percentge of Mycoplsm pneumonie-positive smples from lbortory reports by ge, Englnd nd Wles, 10 October (week 42) Jnury (week 3) 2012 (n=353) nd 1 Jnury 1975 (week 1) 20 Jnury (week 3) 2012 (n=38,221) Age in yers Percentge of smples positive for M. pneumonie Week week Week week positive smples positive smples < ( ) ( ) 3, ( ) ( ) 9, ( ) ( ) 16, ( ) ( ) 4,806 > ( ) ( ) 1,957 Informtion bout ge ws not vilble for ll reports. M. pneumonie, wheres only one of the 60 children ged under 5 yers ws positive (Fisher s exct test p=0.008) (Figure 1). The percentge of positive cses per week (from week 42 to week 3 of the following yer) for children ged under 15 yers is shown for 2005 to 2012 in Tble 3. This shows n increse from November 2011 to Jnury 2012 (week to week ). Smples were more likely to be positive during this period in 2011/12 (13/91; 12.5%; 95% CI: ) thn in the previous four weeks (weeks ) nd the following two weeks (weeks ) (0/40; 0%; 95% CI: ; Fisher s exct test p=0.02). In November 2011 (week 46), December 2011 (weeks 50 nd 51) nd Jnury 2012 (week 1), the number of M. pneumonie infections significntly incresed in comprison with ll previous weeks of smpling since 2005 (binomil probbility test p= , , 0.05 nd 0.01, respectively). The men ge of the 144 ptients ws 6.5 yers (stndrd devition (SD)±4.4; rnge: 0 14) with the mjority of M. pneumonie-positive ptients being over 5 yersold (n=12 of 84). The men ge of the positive ptients ws 8.7 yers (SD±2.6). Only one M. pneumonie-positive ptient ws less thn 5 yers old (ged 4 yers). Figure 1 Percentge of clinicl community surveillnce smples from ptients ged under 15 yers positive for Mycoplsm pneumonie determined by qpcr, Englnd nd Wles, October 2005 Jnury 2012 (totl of 33 positive in 1,354 smples) Percentge of positive smples / / / / / /12 All yers Totl < Age (yers) qpcr: quntittive rel-time polymerse chin rection. The number of positive smples nd totl number of smples per yer were 7 of 98 in 2005/06, 2 of 120 in 2006/07, 1 of 134 in 2007/08, 3 of 249 in 2008/09, 2009 not tested, 7 of 609 in 2010/11, 13 of 144 in 2011/12, giving totl of 33 positive in 1,354 smples for ll yers nlysed. Error brs indicte the 95% CI for the percentges. Excludes October (week 42) 2009 to Jnury (week 3) 2010 when smpling ws not performed due to the influenz A(H1N1)pdm09 pndemic. 2

3 Of the 144 ptients nlysed, 62 were mle nd 79 femle (sex ws not specified for three ptients). Of the 13 M. pneumonie-positive ptients, 5 were mle nd 8 femle. M. pneumonie type nd mcrolide resistnce Smples tht were positive by qpcr were exmined for M. pneumonie type nd mcrolide resistnce. Multiocus vrible number tndem repet nlysis (MLVA) typing by frgment nlysis, which hs previously been used to type M. pneumonie strins [7,11], ws used to nlyse nucleic cid extrcts of clinicl smples in our study; culture isoltion of M. pneumonie ws not undertken. MLVA typing ws lso performed on nine dditionl M. pneumonie-positive respirtory smples tht were submitted to the lbortory during October 2011 to Jnury Genetic diversity ws clculted using Hunter nd Gstons vrition of Simpson s diversity index [12]. The presence of muttions previously ssocited with mcrolide resistnce ws exmined by mplifiction nd sequencing of 720-bse pir (bp) frgment of the 23S rrna gene using the primers MpnMR2063F (5 -ATCTCTTGACTGTCTCGGC-3 ) nd MpnMR2617R (5 -TACAACTGGAGCATAAGAGGTG-3 ) [13]. MLVA nlysis of eight of the 13 qpcr-positive community surveillnce smples nd the nine M. pneumonie-positive respirtory smples tht were submitted to the lbortory during October 2011 to Jnury 2012 showed totl of nine distinct strin types: seven of known MLVA type (type E (n=1), type M (n=4), type P (n=2), type S (n=1), type T (n=1), type U (n=2)nd type Z (n=3)) nd two puttive novel types (profile 4,4,5,7,3 (n=2) nd 5,3,5,7,3 (n=1)) (Figure 2). A full MLVA profile could not be obtined for the other five qpcr-positive community surveillnce smples, probbly becuse of the low levels of M. pneumonie nucleic cid in these smples. The strin type most frequently found in the 17 smples ws MLVA-M (n=4), which ws lso the most prevlent strin type in Englnd nd Wles in 2010 nd hs been found in Frnce (in 1997, 1999, 2000 nd 2006), Germny (in 1995 nd 2000) nd Jpn (in 2000 to 2003) [5,11]. Comprison of the Hunter Gston diversity index (DI) indicted tht both popultions in October to Jnury 2010/11 nd 2011/12 were similrly diverse (2010 DI: 0.93; 95% CI: , 2011 DI: 0.91, 95% CI: ). A full-length sequence of the 720 bp frgment of the 23S rrna gene contining ll four loci ssocited with mcrolide resistnce (2063, 2064, 2067 nd 2618) ws obtined from 12 of the 13 qpcr-positive community surveillnce smples. No muttions in these loci ssocited with mcrolide resistnce were identified in Tble 3 Percentge of clinicl community surveillnce smples positive for Mycoplsm pneumonie determined by qpcr per week for children ged under 15 yers, Englnd nd Wles, October (week 42) Jnury (week 3) (totl of 33 positive in 1,354 smples) Percentge of smples positive for M. pneumonie Week Number 2005/ / / / / / ( ) 0.0 ( ) 0.0 ( ) 0.0 ( ) 0.0 ( ) 0.0 ( ) ( ) 0.0 ( ) 0.0 ( ) 20.0 ( ) 0.0 ( ) 0.0 ( ) ( ) 0.0 ( ) 0.0 ( ) 0.0 ( ) 13.3 ( ) 0.0 ( ) ( ) 0.0 ( ) 0.0 ( ) 12.5 ( ) 0.0 ( ) 0.0 ( ) ( ) 12.5 ( ) 0.0 ( ) 0.0 ( ) 0.0 ( ) 25.0 ( ) ( ) 0.0 ( ) 0.0 ( ) 0.0 ( ) 5.9 ( ) 7.7 ( ) ( ) 0.0 ( ) 0.0 ( ) 0.0 ( ) 0.0 ( ) 0.0 ( ) ( ) 0.0 ( ) 0.0 ( ) 0.0 ( ) 2.1 ( ) 0.0 ( ) ( ) 0.0 ( ) 4.8 ( ) 2.4 ( ) 0.0 ( ) 16.7 ( ) ( ) 0.0 ( ) 0.0 ( ) 0.0 ( ) 0.0 ( ) 10.5 ( ) ( ) 0.0 ( ) 0.0 ( ) 0.0 ( ) 0.0 ( ) 15.4 ( ) ( ) 0.0 ( ) 0.0 ( ) 0.0 ( ) 0.0 ( ) 18.2 ( ) ( ) 7.7 ( ) 0.0 ( ) 0.0 ( ) ( ) 0.0 ( ) ( ) 0.0 ( ) 0.0 ( ) 0.0 ( ) 0.0 ( ) 0.0 ( ) All weeks 4.5 ( ) 1.7 ( ) 0.7 ( ) 1.2 ( ) 1.2 ( ) 9.0 ( ) positive smples/totl number of smples 7/98 2/120 1/134 3/249 7/609 13/144 qpcr: quntittive rel-time polymerse chin rection. Shded cells represent weeks when M. pneumonie ws detected. Excludes October (week 42) 2009 to Jnury (week 3) 2010 when smpling ws not performed due to the influenz A(H1N1)pdm09 pndemic. 3

4 these smples. For the remining qpcr-positive community surveillnce smple, sequence informtion could not be obtined, presumbly due to low levels of M. pneumonie nucleic cid. Discussion The level of M. pneumonie infection in the qpcrbsed community surveillnce of children ged under 16 yers from October 2011 to Jnury 2012 ws 9.0%, rising to 14.3% in the 5 14 yer-olds. This is considerbly higher thn tht in the sme months from previous yers from 2005 to 2011 (1.7%) [5]. Detectble M. pneumonie infection ws found by qpcr in children ged from 4 to 14 yers nd ws bsent from those ged under 4 yers in the 2011/12 study period. As qpcr ws not performed on specimens from dults, the level of dults with detectble M. pneumonie DNA could not be scertined. However, M. pneumonie-positive lbortory reports collted from regionl lbortories were received on dult ptients during this period nd the ge profile ws consistent with tht of ll reports received from 1975 to The lst period showing lrge pek of detectble M. pneumonie infection by qpcr ws winter 2005/06, in which the infection ws detected in 6% of 5 14 yer-olds ttending generl prctitioners with respirtory signs. In the study period reported here (winter 2011/12), n even greter number of children of this ge group were infected (14.3%), indicting t lest one in seven children with respirtory signs ttending generl prctitioners were infected with M. pneumonie. In similr period in 2010/11 (week to week ), 11 differing MLVA types were detected in 15 clinicl smples with MLVA-M being the most prevlent in Englnd nd Wles [7]. Within the study period reported here (week to week ), seven MLVA types were identified, four of which were MLVA-M. The smple number is too low to specify the exct diversity of the popultion or to investigte the ssocition of prticulr types with clinicl severity. Nonetheless, it is interesting tht clonl strins were not detected. Two puttive new profiles were obtined but confirmtion of these pprently novel MLVA types will require isoltion of the strins. The typing method used here ws originlly described by DéGrnge et l., in which stbility of five isoltes ws determined over 10 pssges, indicting tht the M. pneumonie MLVA type is reltively stble [11]. Clonl spred of M. pneumonie does occur, however. In fct, Pereyre et l., recently described the detection of M. pneumonie MLVA-type 3,4,5,7,2 in seven children ttending primry school in Frnce [14]. In our study, ptients were from vriety of loctions in Englnd nd Wles nd, similr to our findings lst yer [5], the dt do not support the hypothesis tht Figure 2 Minimum spnning trees for Mycoplsm pneumonie MLVA types detected in Englnd nd Wles, October Jnury 2011/12 (n=17) nd 2010/11 (n=16) 5,3,5,7,3* 4,4,5,7,3* P T 5,3,6,6 2 P 4,4,5,7 2 T 27 3,4,6, ,4,5, E 2,4,5,7 2 C 2,3,6,6 2 C E U S M S 5,3,5,6,2 U 5,4,5,7,2 M 4,3,5,6,2 P P 4,4,5,7 2 E J J 3,4,5,7 2 U B M B 2,3,5,6 2 M 4,3,5,6,2 E 2,4,5,7 2 Z U 5,4,5,7,2 Z 7,4,5,7,2 2011/ /11 MLVA: Multiocus vrible number tndem repet nlysis. Trees were derived from the five MLVA lleles [11]. Ech circle represents unique MLVA type. The size of ech circle illustrtes the proportion of isoltes with tht MLVA type (the smllest circle in ech tree represents one isolte). Solid lines seprte single locus vrints nd dotted lines seprte double locus vrints. The sterisks mrk the two puttive novel MLVA types. 4

5 single strin type of M. pneumonie ws responsible for this observed increse in infection in Englnd nd Wles. MLVA typing discrimintes well between M. pneumonie-positive specimens. In fct, there is high diversity of types in the popultion nd it does not pper tht few clonl types dominte in circultion. It would be of vlue to hve consistent typing methodology for M. pneumonie strins in use interntionlly, with dtbse of types similr to those for other bcteril species. It would lso be interesting to type strins from other countries during the sme time period to determine how strins differ geogrphiclly during periods of incresed infection. Mcrolide resistnce is becoming n incresing problem in other countries [15]; despite the low smple number, no resistnce ws detected in ny of the qpcrpositive smples from Englnd nd Wles nlysed during the study period. 10. Pitcher D, Chlker VJ, Shepprd C, George RC, Hrrison TG. Rel-time detection of Mycoplsm pneumonie in respirtory smples with n internl processing control. J Med Microbiol. 2006;55(Pt 2): Dégrnge S, Cznve C, Chrron A, Renudin H, Bébér C, Bébér CM. Development of multiple-locus vrible-number tndem-repet nlysis for moleculr typing of Mycoplsm pneumonie. J Clin Microbiol. 2009;47(4): Hunter PR, Gston MA. Numericl index of the discrimintory bility of typing systems: n ppliction of Simpson s index of diversity. J Clin Microbiol. 1988;26(11): Li X, Atkinson TP, Hgood J, Mkris C, Duffy LB, Wites KB. Emerging mcrolide resistnce in Mycoplsm pneumonie in children: detection nd chrcteriztion of resistnt isoltes. Peditr Infect Dis J. 2009:28(8): Pereyere S, Renudin H, Chrron A, Bébér C. Clonl spred of Mycoplsm pneumonie in primry school, Bordeux, Frnce. Emerg Infect Dis. 2012:18 (2): Bébér C, Pereyre S, Peuchnt O. Mycoplsm pneumonie: susceptibility nd resistnce to ntibiotics. Future Microbiol. 2011; 6(4): Acknowledgments The uthors would like to thnk the Birminghm Reserch Unit of the Royl College of Generl Prctitioners, HPA Influenz Group nd Joy Field, HPA Helth Protection Services. References 1. Lenglet A, Herrdor Z, Mgiorkos AP, Leitmeyer K, Coulombier D, Europen Working Group on Mycoplsm pneumonie surveillnce. Surveillnce sttus nd recent dt for Mycoplsm pneumonie infections in the Europen Union nd Europen Economic Are, Jnury Euro Surveill. 2012;17(5):pii= Avilble from: eurosurveillnce.org/viewarticle.spx?articleid= Polkowsk A, Hrjunpää A, Toikknen S, Lpplinen M, Vuento R, Vuorinen T, et l.. Incresed incidence of Mycoplsm pneumonie infection in Finlnd Euro Surveill. 2012;17(5):pii= Avilble from: eurosurveillnce.org/viewarticle.spx?articleid= Blystd H, Ånestd G, Vestrheim DF, Mdsen S, RønningK. Incresed incidence of Mycoplsm pneumonie infection in Norwy Euro Surveill. 2012;17(5):pii= Avilble from: spx?articleid= Uldum SA, Bngsborg JM, Ghrn-Hnsen B, Ljung R, Mølvdgrd M, Føns Petersen R, et l. Epidemic of Mycoplsm pneumonie infection in Denmrk, 2010 nd Euro Surveill. 2012;17(5):pii= Avilble from: Chlker VJ, Stocki T, Mentsti M, Fleming D, Sdler C, Ellis J, et l. Mycoplsm pneumonie infection in primry cre investigted by rel-time PCR in Englnd nd Wles. Eur J Clin Microbiol Infect Dis. 2011;30(7): Nguipdop Djomo, P. Contribution to understnding the dynmics of Mycoplsm pneumonie infections in Englnd nd Wles [disserttion]. London: London School of Hygiene nd Tropicl Medicine, University of London; Chlker VJ, Stocki T, Mentsti M, Fleming D, Hrrison T. Incresed incidence of Mycoplsm pneumonie infection in Englnd nd Wles in 2010: multiocus vrible number tndem repet nlysis typing nd mcrolide susceptibility. Euro Surveill. 2011;16(19):pii= Avilble from: eurosurveillnce.org/viewarticle.spx?articleid= Helth Protection Agency (HPA). HPA influenz surveillnce grphs. London: HPA; 2 Feb [Accessed 8 Februry 2012]. Avilble from: HPAweb_C/ #pge3 9. Helth Protection Agency (HPA). Lbortory reports to HPA Colindle of infections due to Mycoplsm pneumonie, Englnd nd Wles by dte of report (4 weekly). London: HPA. [Accessed 9 My 2011]. Avilble from: