Importance of tissue sampling, laboratory methods, and patient characteristics for detection of Pneumocystis in autopsied lungs of non-immunosuppressed individuals

REVIEW Impo ance o issue sampling, labo a o y me hods, and pa ien cha ac e is ics o de ec ion o Pneumocys is in au opsied lungs o non-immunosupp essed indi iduals S. L. Va gas 1 &C. Ponce 1 &R. Bus aman e 1 &E. Calde ón 2 &G. Ne ez 3 &Y. De A mas 4 & O. Ma os 5 &R. F. Mille 6,7 &M. J. Gallo 8 Recei ed: 26 Feb ua y 2017 /Accep ed: 3 May 2017 /Published online: 5 June 2017 #The Au ho (s) 2017. This a icle is an open access publica ion Abs ac To unde s and he epidemiological signi icance o Pneumocys is de ec ion in a lung issue sample o non- immunosupp essed indi iduals, we examined sampling p o- cedu es, labo a o y me hodology, and pa ien cha ac e is ics o au opsy se ies epo ed in he li e a u e. Numbe o issue specimens, DNA-ex ac ion p ocedu es, age and unde lying diagnosis highly in luence yield and a e c i ical o unde s and yield di e ences o Pneumocys is among epo s o pulmo- na y coloniza ion in immunocompe en indi iduals. In oduc ion I is well-known ha he ungus Pneumocys is ji o ecii dis- plays opism o he lungs and causes se e e pneumonia in pa ien s immunocomp omised by HIV in ec ion o o he causes o immunosupp ession [1]. Howe e , in he b oad con- ex , Pneumocys is pneumonia (PCP) is an excep ional e en wi hin he gene al popula ion; while i is inc easingly e iden P. ji o ecii coloniza ion is o widesp ead occu ence and poin s o he need o unde s and signi icance o his o ganism beyond he pneumonia [2–4]. The mos common signi icance o his mild in ec ion, e med Bcoloniza ion^, is he human- o- human ansmission o he ungus, meaning immunocompe- en indi iduals a e likely pa icipan s in he ci cula ion o his pa hogen in he communi y [2,5,6]. T ansmission s udies can be done using non-in asi e espi a o y samples. Howe e , beyond ansmission s udies, Pneumocys is is no longe con- side ed a commensal, and cla i ica ion o any pa hogenic ole o P. j i o e ci i in he causa ion o lung disease in immunocom- pe en hos s would g ea ly bene i om a clea e unde s and- ing o he epidemiology o , and issue dis ibu ion o Pneumocys is in human lung specimens. A pa hogenic ole is sugges ed by he associa ion o P. ji o ecii and inc eased se e i y o ch onic obs uc i e pulmona y disease (COPD), by he de ec ion o inc eased mucus associa ed wi h P. ji o ecii in he lungs o in an s dying unexpec edly in he communi y, and by he ecen ly desc ibed link be ween P. ji o ecii coloniza- ion and as hma [6–8]. De ec ion o Pneumocys is in human lungs is di icul as he o ganism will no g ow in mic obial *S. L. Va gas s a [email protected] 1 P og ama de Mic obiología y Micología, Ins i u o de Ciencias Biomédicas, Facul ad de Medicina, Uni e sidad de Chile, Independencia 1027, 8380453 San iago, Chile 2 Cen o de In es igación Biomédica en Red de Epidemiología y Salud Pública (CIBERESP) and Ins i u o de Biomedicina de Se illa, Hospi al Uni e si a io Vi gen del Rocío/CSIC/Uni e sidad de Se illa, Se ille, Spain 3 Labo a o y o Pa asi ology and Mycology, B es Uni e si y Hospi al, & Uni e si y o B es , GEIHP, EA 3142 B es , F ance 4 Hospi al Mic obiology Depa men , Ins i u e o T opical Medicine BPed o Kou í^Pa hology Depa men , Ins i u e o T opical Medicine BPed o Kou í^Hospi al, Ha ana, Cuba 5 Unidade de Pa asi ología Médica, G upo de P o ozoá ios Opo unis as/VIH e Ou os P o ozoa ios, Global Heal h and T opical Medicine, Ins i u o de Higiene e Medicina T opical, Uni e sidade NOVA de Lisboa, 1349-008 Lisbon, Po ugal 6 Resea ch Depa men o In ec ion and Popula ion Heal h, Ins i u e o Global Heal h, Uni e si y College London, Mo ime Ma ke S ee , London WC1E 6BT, UK 7 Clinical Resea ch Depa men , London School o Hygiene and T opical Medicine, London, UK 8 Se icio Médico Legal, A . La Paz 1012, 8380454 San iago, Chile Eu J Clin Mic obiol In ec Dis (2017) 36:1711–1716 DOI 10.1007/s10096-017-3006-8 cul u e, adop s a cha ac e is ically ocal dis ibu ion in he lungs, and equi es a speci ic sea ch using DNA ampli ica ion echniques and/o speci ic s ains ha need ained expe ienced mic oscopis s o iden i y he cys ic and he mo e abundan ophic biological o ms. The high p e alence o P. ji o ecii wi hin he gene al popula ion, and new associa ions wi h hu- man disease ha a e being desc ibed, unde sco e he impo - ance o op imizing issue sampling and s anda dizing diag- nos ic me hodology, which a e needed o ecognize he epide- miology and o unde s and any ole o his ungal o ganism in lung disease. A ecen publica ion desc ibing he p e alence o P. ji o ecii coloniza ion in lungs o he gene al popula ion in Tu key [3], using DNA ampli ica ion echniques, con i ms p e ious epo s de ec ing Pneumocys is in he au opsied lungs o immunocompe en indi iduals om he gene al pop- ula ion dying in he communi y in Chile [2,5] and in he Uni ed S a es [4], and mo i a ed us o compa e he sampling and diagnos ic p ocesses unde lying he di e en yields in Pneumocys is de ec ion be ween hese s udies (Table 1). Unde s anding de ec ion me hodology is pi o al o compa e epidemiology be ween coun ies, and will lead o imp o ed diagnos ic me hodologies, o eliable collabo a i e s udies, and ul ima ely o each a be e unde s anding o disease. Ma e ials and me hods Lung sampling echnique The Chilean se ies examined be ween 2 and 15 (median 7) lung issue samples, each weighing 0.5 g, ep esen ing up o 3% o he weigh o he igh uppe lobe. Pneumocys is-neg- a i e samples we e con i med as nega i e a e analysis o 7% o lung weigh . Magne ic agi a ion wi h indi idually s e ilized magne s was used o homogenize samples [2,6]. Samples we e cen i uged a 2900 g o 10 min. Özkoç e al. [3]ana- lyzed a single 1 g sample om he igh uppe lobe o each pa ien . These we e mechanically homogenized, and cen i- uged a a speed o 690 g o 10 min. Bea d e al. [4] ob ained ou samples ( wo om each uppe lobe) and cen i uged a 14000 g o 5–7 min. The in luence o he lowe cen i uga- ion o ce used by Özkoç e al. on hei lowe yield o de ec - ing Pneumocys is DNA is di icul o in e p e . Howe e , o ces o e 1000 g a e gene ally used. When jus he i s o all lung samples was analyzed, esul s in Chilean samples show ha Pneumocys is DNAwas de ec - ed in 16 (29%) o 55 adul s wi h iolen dea hs, and in 6 (31.5%) o 19 adul s dying om medical condi ions (Fig. 1). This yield o de ec ion o Pneumocys is om jus he i s analyzed lung sample is s ill highe when compa ed o he 12% and 27% epo ed by Özkoç e al. in samples om Tu key o iolen dea hs o o dea hs om medical condi ions, espec i ely (Table 1). Da a om Bea d e al. is no a ailable ega ding he yield om a single o mo e han one lung sample in he US s udy. Me hod used o ex ac DNA Va gas e al. used he QIAmp DNA ex ac ion ki (QIAGEN), Özkoç e al. used he Mache y-Nagel ex ac ion ki , and Bea d e al. used he Wiza d Genomic DNA pu i ica ion ki (P omega). I is no known how hese DNA ex ac ion p o- cesses pe o m ela i e o he QIAmp ki . Hugge e al. showed ha he amoun o P. ji o ecii DNA ex ac ed om BAL luid samples was dependan on which ex ac ion ki was used; he yield using QIAmp was highe han ha using DNAeasy [9]. QIAmp eco e ed mo e DNA han he P omega ki o speci ically de ec ing Mycobac e ium lep ae on issue samples [10]. In none o he h ee s udies ha a e compa ed we e ex ac ed DNA yield o DNA quali y pa am- e e s documen ed using A260/280 (yield pu i y) o A260/230 (quali y/con amina ion) a ios. Ampli ica ion eac ions in he Chilean se ies we e un using human β-globin as an in e nal con ol o moni o o DNA-inhibi ion and li e issue as a P. ji o ecii-nega i e con ol o moni o o c oss con amina- ion. Howe e , no assessmen s we e made o moni o o DNA yield o quali y pa ame e s in any o he manusc ip s e iewed in his a icle. P omega, Quiagen, and Mache ey- Nagel DNA ex ac ion ki s yield amoun s o DNA ha may a y depending on elusion olumes acco ding o hei manu- ac u e manuals. In e es ingly, compa isons o he DNA amoun eco e ed om he same b onchoal eola la age sam- ples om immunocomp omised pa ien s ex ac ed by hese ki s as done by Hugue e al. using DNA-easy and QIAamp a o ed he QIAamp ki o e he DNA-easy ki (12.5- old yield e sus 2- old yield) [9]. The po en ial impac o hese di e ences in diagnos ic sensi i i y is unknown and may e- lec he bu den o Pneumocys is in he sample. Whe he de- ec ion o a e y low and ocal bu den o Pneumocys is o gan- isms, as in lungs om immunocompe en adul s, is imp o ed using a ki ha pe o ms a o ably in ex ac ing a highe amoun DNA, as indica ed in he manu ac u e manuals, e- mains o be de e mined. We ha e compa ed P omega and Quiagen ki s in he same nasopha yngeal aspi a e samples om immunocompe en in an s ha ha e a highe Pneumocys is bu den han adul s, and, al hough DNA was no measu ed, diagnos ic esul s we e consis en o nes ed- PCR in hese in an s (Ponce, CA; unpublished). DNA ampli ica ion p o ocol S udies om Chile, Tu key, and he Uni ed S a es ampli ied DNA using he mul i-copy gene m LSU RNA. Conside a ion o quan i a i e PCR p o ocols should be gi en in u u e s ud- ies as hey will p o ide a be e es ima e o he amoun o 1712 Eu J Clin Mic obiol In ec Dis (2017) 36:1711–1716 P. ji o ecii DNA in a gi en sample. Howe e , he epidemio- logical and diagnos ic ele ance o P. ji o ecii quan i a ion will need o be documen ed because a gi en pulmona y bu - den o o ganisms may ha e a di e en meaning in di e en pa ien con ex s. Fo example, non-HIV pa ien s wi h PCP ha e pa adoxically a lowe bu den o P. ji o ecii o ganisms and mo e se e e pneumonia han pa ien s wi h HIV- ela ed PCP, he e o e indica ing ha Pneumocys is disease is hos dependen and disease se e i y does no co ela e wi h Pneumocys is bu den. P. ji o ecii coloniza ion o unde e - mined bu den has been associa ed wi h old age, o inc eased se e i y o bac e ial pneumonia in non-immunosup essed pa- ien s [11]. A majo di icul y in in e p e ing quan i a i e PCR is he simila bu den le els ha can o e lap in pa ien s ha a e colonized and in pa ien s wi h PCP [12]. Age o in an s s udied Among in an s, Va gas e al. [5,6,13,14] and La sen e al. [15] ha e p e iously shown ha de ec ion a es o P. ji o ecii a y by age; wi h a peak a 2–5 mon hs o age and declining he ea e . In he s udy by Özkoç e al., he ages o in an s <12 mon hs o age a e no gi en, bu wo o 11 in an s died 1–2 days a e bi h, hus in e ing ha hey would be unlikely o ha e had ime o become hea ily colonized wi h P. ji o ecii. Mode o diagnosis o dea h Al hough a dec eased CD4+ T-cell lymphocy e coun is he s onges p edic o o Pneumocys is suscep ibili y, espi a o y disease appea s o in luence he de ec ion a e o Pneumocys is among he immunocompe en popula ion in hese s udies. Respi a o y disease was diagnosed only in he se ies om Tu key, whe e indi iduals dying om medical condi ions had signi ican ly inc eased de ec ion o P. ji o ecii DNA han hose dying om o he causes. Immunosupp essi e illnesses we e no diagnosed [2,3]. Discussion Amoun o lung issue and issue dis ibu ion The smalle amoun o lung issue examined may pa ly ex- plain he lowe yield epo ed by Özkoç e al. when compa ed o esul s om Chile o om he Uni ed S a es. Howe e , p e ious publica ions also iden i y dis ibu ion o P. ji o ecii in lungs is he e ogeneous [16,17], and a s udy o immuno- compe en pa ien s wi h COPD shows ha Pneumocys is may be mo e abundan in he lowe lobes o he lung [7]. The ocal dis ibu ion o Pneumocys is de e mines he need o sample mo e han one si e o de ec coloniza ion. Impo an ly, in he Chilean se ies in adul s, no new posi i es we e de ec ed a e se en samples we e analyzed (Fig. 1). S udies in in an s om Chile analyzed up o six samples in in an s o aling he same 3% o lung issue weigh as in adul s. These s udies used a 0.2-g issue sample pe lung lobe sam- pling he h ee igh lung lobes. Those in an s ha ing a leas one posi i e sample we e conside ed posi i e, and nega i e in an s we e sampled a second ime o con i ma ion ob aining 0.4 g om he igh uppe lobe. A o al o 20% o Pneumocys is-nega i e in an s on i s sample changed o pos- i i e a e analysis o he second sample, p o iding a inal Pneumocys is de ec ion a e in in an s o 82% [6]. O in e es , in e -lobe consis ency be ween he h ee lobes was 59% when only i s sampling analysis was conside ed, sugges ing all lobes need o be s udied [6]. A mo e ecen se ies analyzing one, wo, o h ee indi idual lung issue samples o 0.4 g each adding up o 3% o he uppe igh lung lobe weigh gi e consis en nes ed-PCR de ec ion esul s o each sample, and nega i e esul s do no change a e analysis o addi ional issue (Ponce e al., unpublished). In e -loba consis ency is being e alua ed. The e o e, he Pneumocys is de ec ion yields in au opsy samples om Tu key, and om bo h Chile and he Uni ed S a es need o be in e p e ed in he pe spec i e o he numbe Table 1 De ec ion o P. ji o ecii DNA om au opsy lung issue o immune compe en adul s and in an s Re e ence Geog aphical loca ion Tu key Chile Uni ed S a es Re . [3]Re .[2]Re .[4] Pa ien g oup No PCR posi i es o P.ji o ecii DNA/No sampled (%) Adul s wi h cause o dea h due o iolence, suicide, o acciden 13/111 (12) 34/55 (62) NA Adul s wi h cause o dea h due o an unde lying medical condi ion 20/73 a (27) 15/19 b (79) NA In an s, age < 12 mon hs 5/11 (43) 105/128 (82) 58/58 (100) PCR polyme ase chain eac ion, NA no applicable a Diagnoses: ca dio ascula condi ions including myoca dial in a c ion (n= 48), pulmona y in ec ion, edema (n= 14), suba achnoid hemo hage (n=7), pe ica dial amponade (n= 2), epilep ic seizu es (n= 1), and in es inal obs uc ion (n=1) b Diagnoses: myoca dial in a c ion (n= 13), ca diac amponade (n= 1), s oke (n= 1), suba achnoid hemo hage (n= 1), diges i e hemo hage (n=1), pe i oni is (n= 1), and in es inal obs uc ion (n=1) Eu J Clin Mic obiol In ec Dis (2017) 36:1711–1716 1713 o samples analyzed. They con i m ha he p e alence o P. ji o ecii in au opsy lung ma e ial om immunocompe en adul s is high, ocal in dis ibu ion, and wi h a e y low bu den o Pneumocys is o ganisms as e idenced by 1714 Eu J Clin Mic obiol In ec Dis (2017) 36:1711–1716 immuno luo escence mic oscopy examina ion in he Chilean se ies [2], and by he nega i e esul s o a p e ious s udy using a less sensi i e, single- ound PCR echnique in pos -mo em lungs om non-immunosupp essed indi iduals 15 o 70 yea s o age om he Uni ed Kingdom [18]. The lowe bu den o pulmona y coloniza ion by Pneumocys is in non-immunosupp essed adul indi iduals makes i unlikely ha non-in asi e sampling will eplace he s udy o lung specimens o de ec ion o pulmona y coloniza- ion in he u u e. S udies using immunosupp essed animals sugges a co ela ion o Pneumocys is-DNA esul s be ween non-in asi e samples like nasopha yngeal aspi a es and lung issue samples [19–21]. Howe e , in human adul s, a single diagnos ic nasopha yngeal aspi a e sample may de ec only a ac ion o Pneumocys is uppe ai way ca ie s, and sampling he uppe ai ways mo e han once may inc ease he diagnos ic yield o Pneumocys is [22,23]. This means ha a single up- pe ai way specimen in adul s is no necessa ily ep esen a i e o lung in ec ion. By con as , i has been documen ed ha Pneumocys is-DNA in non-in asi e samples om non- immunocomp omised humans may ep esen nasal ca iage acqui ed om an ex e nal sou ce o con agion, indica ing ei- he ansien o long- e m coloniza ion in he uppe ai ways [23]. S udies ha compa e he yield o nasopha yngeal aspi- a e samples, b onchoal eola la age samples, and lung sam- ples in he same indi idual pa ien s will be di icul o pe o m and a e lacking. Nasopha yngeal aspi a e samples in heal hy immunocompe en adul s ha e an app oxima e yield o 20% [24]. A s udy o b onchoal eola la age samples in immuno- compe en indi iduals unde going b onchoscopy as pa o a diagnos ic wo k-up o possible espi a o y disease in he Uni ed Kingdom, documen ed ha 18% we e colonized wi h P. ji o ecii [25]. The yield o lung issue samples is highe as documen ed in immunocompe en adul s in Chile and Tu key, and de ec ion a e a ied depending on sampling p ocedu es. They documen ed an a e age o 65% P. ji o ecii-posi i e cases in a Chilean se ies a e analysis o a median o se en specimens o 0.5 g each pe lung o amoun o 7% o o al lung weigh , and an a e age o 18.5% o P. ji o ecii-posi i e cases in he se ies om Tu key a e analysis o 1 g o lung issue om a single specimen. This sugges s ha , simila as in non- in asi e sampling [22], he s udy o mul iple au opsy lung samples inc eases he yield and is he e o e necessa y o ec- ognize he p e alence o he mild and ocal pulmona y in ec- ion by Pneumocys is in he non-immunosupp essed adul . Fu u e au opsy s udies in non-immunosupp essed adul s should con inue o speci y he mode o diagnosis o dea h, and include he numbe o pulmona y samples analyzed, hei o al weigh , si e o o igin, p ecau ions o a oid con amina- ion, DNA ex ac ion me hod, and gene a ge ampli ied, as essen ial in o ma ion o compa ison and ep oducibili y o esul s. Fu u e s udies in au opsied in an lungs should dis- close in an ages in addi ion o he abo e. Compliance wi h e hical s anda ds All p ocedu es pe o med in s ud- ies in ol ing human pa icipan s we e in acco dance wi h he e hical s anda ds o he ins i u ional and/o na ional esea ch commi ee and wi h he 1964 Helsinki decla a ion and i s la e amendmen s o compa able e hical s anda ds. P e ious wo k conduc ed in Chile ha is epo ed he e in g ea e de ail (Fig. 1) was app o ed by he espec i e E hics Commi ee (see [2,14]). Fo his ype o s udy o mal consen is no equi ed. Funding This e iew was suppo ed by he Fondo Nacional de Desa ollo Cien í ico y Tecnológico (FONDECYT-Chile) g an numbe 1140412 (S. Va gas), and by CONICYT in he con ex o ERANe LAC G an ELAC2014/HID-0254 (S. Va gas, E. Calde ón, and o he s). Con lic o in e es s The au ho s decla e ha hey ha e no con lic s o in e es . Open Access This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0 In e na ional License (h p:// c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided you gi e app o- p ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons license, and indica e i changes we e made. Re e ences 1. Mille RF, Huang L, Walze PD (2013) Pneumocys is pneumonia associa ed wi h human immunode iciency i us. Clin Ches Med 34(2):229–241. doi:10.1016/j.ccm.2013.02.001 2. Ponce CA, Gallo M, Bus aman e R, Va gas SL (2010) Pneumocys is coloniza ion is highly p e alen in he au opsied lungs o he gene al popula ion. Clin In ec Dis 50(3):347–353. doi:10.1086/649868 3. Özkoç S, Köke M, Önde M, DelibaşSB (2016) P e alence o Pneumocys is ji o ecii coloniza ion in au opsy cases in Tu key. J Med Mic obiol 65(10):1152–1157. doi:10.1099/jmm.0.000337 4. Bea d CB, Fox MR, Law ence GG, Gua ne J, Hanzlick RL, Huang L e al (2005) Gene ic di e ences in Pneumocys is isola es eco - e ed om immunocompe en in an s and om adul s wi h AIDS: epidemiological implica ions. J In ec Dis 92(10):1815–1818. doi: 10.1086/497381 5. Va gas SL, Ponce CA, Hughes WT, Wake ield AE, Wei z JC, Donoso S e al (1999) Associa ion o p ima y Pneumocys is ca inii in ec ion and sudden in an dea h synd ome. Clin In ec Dis 29(6): 1489–1493. doi:10.1086/313521 6. Va gasSL,PonceCA,GalloM,Pe ezF,As o gaJF,Bus aman eRe al (2013) Nea -uni e sal p e alence o Pneumocys is and associa ed in- c ease in mucus in he lungs o in an s wi h sudden unexpec ed dea h. Clin In ec Dis 56(2):171–179. doi:10.1093/cid/cis870 Fig. 1 De ec ion o P. ji o ecii DNA in au opsy lungs om adul s, using n-PCR om mul iple lung samples ob ained om he igh uppe lobe. Ho izon al axis shows he numbe o 0.4 g lung samples analyzed. Ve ical axis numbe s ep esen indi idual adul s. aFi y- i e adul s dy- ing in he communi y om iolence, acciden o suicide. bNine een adul s dying in he communi y om an unde lying medical cause (ex- panded om Re . [2]). Posi i e image posi i e o Pneumocys is-DNA, Nega i e image Nega i e o Pneumocys is-DNA, NP New posi i e in- di iduals ha we e de ec ed a e he analysis o he 1s , 2nd, 3 d, ...e c. lung sample. This shows ha in adul s om he gene al popula ion dying in he communi y he e we e no new posi i es o changes in de ec ion esul s a e he analysis o se en samples R Eu J Clin Mic obiol In ec Dis (2017) 36:1711–1716 1715 7. Si am S, Sciu ba FC, Luch LA, Zhang Y, Duncan SR, No is KA e al (2011) Dis ibu ion o Pneumocys is ji o ecii in lungs om colonized COPD pa ien s. Diagn Mic obiol In ec Dis 71(1):24–28. doi:10.1016/j.diagmic obio.2011.05.008 8. Eddens T, Camp ield BT, Se ody K, Manni ML, Ho ne W, Elsegeiny W e al (2016) A no el CD4+ T cell-dependen mu ine model o Pneumocys is-d i en as hma-like pa hology. Am J Respi C i Ca e Med 194(7):807–820. doi:10.1164/ ccm.201511-2205OC 9. Hugge JF, Taylo MS, Kocjan G, E ans HE, Mo is-Jones S, Gan V e al (2008) De elopmen and e alua ion o a eal- ime PCR assay o de ec ion o Pneumocys is ji o ecii DNA in b onchoal e- ola la age luid o HIV-in ec ed pa ien s. Tho ax 63(2):154–159. doi:10.1136/ hx.2007.081687 10. Ruiz-Fuen es JL, Diaz A, En enza AE, F ion Y, Sua ez O, To es P e al (2015) Compa ison o ou DNA ex ac ion me hods o he de ec ion o Mycobac e ium lep ae om Ziehl-Neelsen-s ained mi- c oscopic slides. In J Mycobac e iol 4(4):284–289. doi:10.1016/j. ijmyco.2015.06.005 11. Helweg-La sen J, Jensen JS, Dohn B, Ben ield TL, Lundg en B (2002) De ec ion o Pneumocys is DNA in samples om pa ien s suspec ed o bac e ial pneumonia—a case-con ol s udy. BMC In ec Dis 2:28. doi:10.1186/1471-2334-2-28 12. Damiani C, Le Gal S, Da Cos a C, Vi maux M, Ne ez G, To e A (2013) Combined quan i ica ion o pulmona y Pneumocys is ji o ecii DNA and se um (1→3)-β-d-glucan o di e en ial diag- nosis o Pneumocys is pneumonia and Pneumocys is coloniza ion. J Clin Mic obiol 51(10):3380–3388. doi:10.1128/JCM.01554-13 13. Va gas SL, Ponce CA, Gal ez P, Iba a C, Haas EA, Chadwick AE e al (2007) Pneumocys is is no a di ec cause o sudden in an dea h synd ome. Pedia In ec Dis J 26(1):81–83. doi:10.1097/01. in .0000247071.40739. d 14. Va gas SL, Ponce CA, Luchsinge V, Sil a C, Gallo M, Lopez R e al (2005) De ec ion o Pneumocys is ca inii . sp. hominis and i uses in p esumably immunocompe en in an s who died in he hospi al o in he communi y. J In ec Dis 191(1):122–126. doi:10. 1086/426451 15. La sen HH, on Lins ow ML, Lundg en B, Hogh B, Wes h H, Lundg en JD (2007) P ima y Pneumocys is in ec ion in in an s hospi alized wi h acu e espi a o y ac in ec ion. Eme g In ec Dis 13(1):66–72. doi:10.3201/eid1301.060315 16. Amb ose HE, Ponce CA, Wake ield AE, Mille RF, Va gas SL (2001) Dis ibu ion o Pneumocys is ca inii . sp. hominis ypes in he lung o a child dying o Pneumocys is pneumonia. Clin In ec Dis 33(9):e100–e102. doi:10.1086/322690 17. Helweg-La sen J, Lundg en B, Lundg en JD (2001) He e ogenei y and compa men aliza ion o Pneumocys is ca inii . sp. hominis geno ypes in au opsy lungs. J Clin Mic obiol 39(10):3789–3792. doi:10.1128/JCM.39.10.3789-3792.2001 18. Pe e s SE, Wake ield AE, Sinclai K, Milla d PR, Hopkin JM (1992) A sea ch o Pneumocys is ca inii in pos -mo em lungs by DNA ampli ica ion. J Pa hol 166(2):195–198. doi:10.1002/ pa h.1711660217 19. Chouk i F, Alioua el M, Meno i J, To e A, Gan ois N, Ga in YJ e al (2011) Dynamics o Pneumocys is ca inii ai shedding du ing expe imen al pneumocys osis. J In ec Dis 203(9):1333–1336. doi: 10.1093/in dis/ji 018 20. Linke MJ, Rebholz S, Collins M, Tanaka R, Cushion MT (2003) Nonin asi e me hod o moni o ing Pneumocys is ca inii pneumo- nia. Eme g In ec Dis 9(12):1613–1616. doi:10.3201/eid0912. 030270 21. Meno i J, Emmanuel A, Bouchekouk C, Chabe M, Chouk i F, Po ie M e al (2013) E idence o ai bo ne exc e ion o Pneumocys is ca inii du ing in ec ion in immunocompe en a s. Lung in ol emen and an ibody esponse. PLoS One 8(4): e62155. doi:10.1371/jou nal.pone.0062155 22. Va gas SL, Piza o P, Lopez-Viey a M, Nei a-A iles P, Bus aman e R, Ponce CA (2010) Pneumocys is coloniza ion in olde adul s and diagnos ic yield o single e sus pai ed nonin asi e espi a o y sampling. Clin In ec Dis 50(3):e19–e21. doi:10.1086/649869 23. Va gas SL, Ponce CA, Giglio i F, Ulloa AV, P ie o S, Munoz MP e al (2000) T ansmission o Pneumocys is ca inii DNA om a pa ien wi h P. ca inii pneumonia o immunocompe en con ac heal h ca e wo ke s. J Clin Mic obiol 38(4):1536–1538 24. Med ano FJ, Mon es-Cano M, Conde M, de la Ho a C, Respaldiza N, Gasch A e al (2005) Pneumocys is ji o eciii in gene al popula- ion. Eme g In ec Dis 11:245–250. doi:10.3201/eid1102.040487 25. Maskell NA, Waine DJ, Lindley A, Peppe ell JC, Wake ield AE, Mille RF e al (2003) Asymp oma ic ca iage o Pneumocys is ji o eci in subjec s unde going b onchoscopy: a p ospec i e s udy. Tho ax 58(7):594–597 1716 Eu J Clin Mic obiol In ec Dis (2017) 36:1711–1716