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.
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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-
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esul s a e he analysis o se en samples
R
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