Med. Mycol. J.

Med.
Vol. Mycol.
57E, E 111J.−
Vol. 57（No.
E 116, 20164）
, 2016 E 111
ISSN 2185 − 6486
5
Review

Recent Advances in the Diagnosis of Pneumocystis Pneumonia

ઃ,઄,અ આ ઃ,઄,અ ઃ,઄,અ
Yinggai Song , Yi Ren , Xiaowen Wang and Ruoyu Li

Department of Dermatology, Peking University First Hospital

Research Center for Medical Mycology, Peking University
Beijing Key Laboratory of Molecular Diagnosis on Dermatoses
'
Beijing Tropical Medical Research Institute, Beijing Friendship Hospital, Capital Medical University

ABSTRACT
Pneumocystis jirovecii is a prototypical opportunistic pathogen, causing an asymptomatic or mild
infection in normal hosts and fulminating pneumonia（Pneumocystis pneumonia, PCP）in immunocom-
promised hosts. PCP is a leading cause of morbidity and mortality in immunocompromised patients such
as AIDS patients. Microscopic detection of cysts and trophic forms of P. jirovecii in respiratory
secretions is simple and useful but may underestimate the P. jirovecii infection. Conventional
polymerase chain reaction（PCR）and nested PCR increase the sensitivity and specificity to identify
PCP and provide an approach to discriminate PCP from pulmonary P. jirovecii colonization, but the
targeted genes and cut-off value from quantitative real-time PCR remain to be determined. Serum（1-3） -
β-D-glucan level and the specific serum antibody titer are ancillary indicators for PCP diagnosis. The
successful cultivation of P. jirovecii in vitro is an important progress for PCP research. The diagnosis of
PCP relies on the combination of these laboratory examinations as well as the clinical presentations.
Key words： （1-3）-β -D-glucan, CuFi-8 cells, ELISA, major surface glycoprotein, Pneumocystis jirovecii,
Pneumocystis pneumonia, polymerase chain reaction

this fungus during childhood, but healthy adults

Introduction are asymptomatic carriers of the fungus . PCP
2）

may develop from the reactivation of latent

Pneumocystis spp. are extracellular, host-
specific, and yeast-like parasitic fungi virtually
restricted to lung tissues of many species of
rodents, horses, and primates. Pneumocystis was
discovered in 1909, detected in rats in 1910, and
classified as Pneumocystis carinii in 1912. In the
first decade of the 20th century, the human
pathogen of Pneumocystis was classified as a
separate species from the rodent form（P.
carinii）, and was renamed as Pneumocystis
1）
jirovecii . Pneumocystis pneumonia（PCP）is an
opportunistic infection caused by P. jirovecii in
immunocompromised humans, especially in indi-
viduals infected with HIV or undergoing che-
motherapy. Epidemiologically, ~95% of the world-
wide population are believed to be infected with
infection, but de novo exposure to PCP patients
or P. jirovecii carriers through person-to-person
3−6）
transmission may also occur . PCP remains one
of the important complications in AIDS patients
and HIV-uninfected immunocompromised patients
including patients with malignancies and organ
transplantation. Better diagnostic tools are
needed. In recent years, the promising diagnostic
methods such as molecular biology allowed
detection of P. jirovecii in sputum and serologic
test for the differentiation between PCP and
pulmonary P. jirovecii colonization have made the
diagnosis of PCP less invasive and more
3, 7, 8）
accurate . This article will review the recent
advances in the laboratory diagnosis of PCP.

Address for correspondence : Ruoyu Li

Department of Dermatology, Peking University First Hospital
E-mail : mycolab@126.com
 E 112
Microscopic detection of P. jirovecii in respira-
tory tract specimens
Microscopic examination of Pneumocystis in
respiratory tract specimens after staining with
dyes or antibodies is an important approach for
PCP diagnosis. Currently, the most commonly
employed staining techniques include the methe-
namine silver stain which stains the cyst wall, the
modified Giemsa stain which stains the parasite at
all life cycle stages, the nonspecific fluorescent
stain with calcofluor white, and the immunof-
luorescent stain using specific monoclonal anti-
bodies.
The sensitivity of staining methods for the
induced sputum samples ranges 55-78%. Positive
results in bronchoalveolar lavage fluid（BALF）
and lung biopsy are considered as the “gold
9）
standard” for PCP diagnosis . The sensitivities of
immunofluorescent antibodies against surface
antigens of P. carinii or P. jirovecii are higher than
10）
those using dye staining methods .
The accuracy of microscopic approaches is
closely dependent on the skill of observers. The
diagnosis can also be hampered in patients using
highly active antiretroviral therapy and PCP
chemoprophylaxis which may lead to low burden
of P. jirovecii.
In vitro culture of P. jirovecii
Culturing P. jirovecii has been attempted for
several decades but without successful results.
The lack of a reliable in vitro culture system
hampered the comprehension of P. jirovecii
pathogenicity and epidemiology. In 2014, Schildgen
et al. reported that P. jirovecii could be cultured in
a permanent three-dimensional air-liquid interface
11）
culture system formed by CuFi-8 cells .
CuFi-8 cell is a differentiated pseudostratified
airway epithelial cell line. When the pseudostrati-
fied epithelia grow and a mucous layer on the cell
surface forms, the cells become polarized. The
cultured CuFi-8 cells on the air side of the air-
liquid interface are inoculated with 10-150μl BALF.
Five days later, the cells and basal medium are
harvested and tested for P. jirovecii. P. jirovecii
can be detected in the basal culture medium by
quantitative real-time PCR（qPCR） , which indi-
cates that P. jirovecii is able to grow in this culture
system, and must have actively passed through
CuFi-8 cells, collagen, and a pore size of 4μm in
Medical Mycology Journal Volume 57, Number 4, 2016
filter membrane into the culture medium. The
culture of P. jirovecii will enable us to study its life
cycle, drug sensitivity and environmental factors
on its growth.
The role of serum（1-3）
-β-D-Glucan（BG）in
the diagnosis of PCP
Serum BG may be one of the useful serologic
markers for establishing PCP diagnosis. Serum
measurement of BG is based on the level of this
polysaccharide that is present within the cell wall
12）
of Pneumocystis and other fungi . BG is one of
the best characterized host response factors of
Pneumocystis. Numerous studies have demon-
strated that purified Pneumocystis BG stimulates
expression of inflammatory cytokines and chemo-
13, 14）
kines in vitro . In addition, BG in the cell wall of
Pneumocystis stimulates pulmonary inflammation.
The clinical usefulness of BG in serum and BALF
as a potential marker of PCP have been
15, 16）
evaluated . Treatment of PCP with BG synth-
ase inhibitors such as anidulafungin in animal
models provides an alternative therapeutic
17）
strategy for PCP .
Several studies using BG assay obtained a
sensitivity of 90-100% and a specificity of 88-96%
for PCP at various cutoff values depending on the
18, 19）
BG kit and the population . There is a higher
cutoff value for the diagnosis of PCP than for the
diagnosis of invasive aspergillosis or
19, 20）
candidiasis , but the optimal cutoff values have
not been determined for these diseases. Damiani
et al. found that infants with PCP had higher
serum BG of 184~2,710 pg/ml, and that the serum
BG levels（applying a threshold of 100 pg/ml）
combined with qPCR for mtLSUrRNA gene in
3
BALF（applying lower cutoff value of 1.6 × 10 and
4
upper cut off value of 2 × 10 copies/l）could
differentiate PCP from P. jirovecii coloniza-
21, 22）
tion . One study indicated that BG level
correlated with organism burden, but most others
did not find a relationship of serum BG with
organism burden, PCP severity, and response to
23, 24）
therapy . Serum BG is also increased in other
fungal infections such as Aspergillus, it is there-
fore used as an ancillary test in patients with a
high suspicion of PCP. In patients who have a
clinical presentation and radiological findings
compatible with PCP, an elevated serum BG
together with an appropriate copy number of
P. jirovecii DNA in respiratory samples is the
15）
stronger indication for the diagnosis of PCP .
 Med. Mycol. J. Vol. 57（No. 4）
, 2016
Serum anti-major surface glycoprotein（Msg）
antibody level
Msg protein is a highly glycosylated protein of
90-120 kDa encoded by multiple genes with an
estimation of 50-100 copies/P. jirovecii. Switch of
Msg gene expression gives rise to multiple Msg
25）
isoforms and antigenic variations . Msg contains
shared and species specific antigenic determi-
nants, elicits humoral and cellular protective
immune responses, and plays a central role in the
interaction between Pneumocystis and host .
Recombinant Msg proteins offer valuable anti-
gens to develop novel immunological assays . In
a series of studies utilizing recombinant Msg
fragments that cover several parts of the
polypeptide, MsgA（amino acid residues 15-119）
MsgB（amino acid residue 729-2282）, and MsgC
（amino acid residues 2015-3332） , Walzer et al.
developed western blot and ELISA that suggested
27, 28）
epidemiological significance . Recombinant
Msg fragments at the C-terminus were recognized
more frequently by serum specimens than those
29, 30）
at the N-terminus . HIV-infected patients with
PCP elicited greater responses against MsgC
than did HIV-infected patients without PCP or with
pneumonia due to other causes, and a prior
episode of PCP also presented a higher titer of
27, 31）
antibodies against MsgC fragments
bodies against MsgA, MsgB and MsgC were
detected in about 84% healthy blood donors,
limiting its usefulness in the diagnosis of PCP.
Serum antibodies against MsgC in PCP patients
increased to a peak level 3-4 weeks after recov-
26, 27）
ery from PCP . Quantitative ELISA found that
about 10% of healthy persons showed an increase
of ≥ 4-fold titers against Msg during one year of
observation, suggesting that they had experi-
29）
enced an infection of P. jirovecii .
Conventional and nested PCR for the detection
of P. jirovecii DNA
Amplification of P. jirovecii specific DNA se-
quences by PCR may increase the detection
sensitivity. The targeted P. jirovecii DNA seg-
ments for amplification include internally tran-
scribed spacer（ITS） , and the genes of 18S rRNA,
5S rRNA, mitochondrial large subunit rRNA
（mtLSUrRNA） , dihydrofolate reductase, Msg,
cdc2, and heat shock protein 70（HSP70）. The
targeted sequence for PCR is preferentially the
mtLSUrRNA gene using pAZ102-E and pAZ102-H
E 113
primers. Fan et al. conducted a systematic review
and found that the sensitivity and specificity
values of PCR in BALF for diagnosis of PCP were
98. 3% and 91. 0%, respectively, and the positive
and negative likelihood ratios were 10. 894 and
32）
0.018, respectively .
Nested PCR increases the sensitivity without
33）
loss of specificity . The nested PCR for
mtLSUrRNA gene was found to be more sensitive
and useful than conventional PCR targeting Msg
34）
gene in detecting PCP . The primer pairs for the
1）
nested PCR targeting mtLSUrRNA gene included
the outer pair of primers pAZ102-E and pAZ102-H
26）
and the inner pair of primers pAZ102-X and
pAZ102-Y. Nested PCR could detect P. jirovecii
DNA in BALF two to three orders of magnitude
35）
, more dilute than conventional PCR .
Sequencing of amplified DNA fragments is
useful to identify P. jirovecii strain and genotype,
providing a unique way for tracking the fungus in
human populations. Amplification of selected
polymorphic sequences such as ITS in P. jirovecii
genome has become a method to genotype P.
36）
jirovecii strain that may relate to its virulence .
ITS can be used as a genetic marker to distinguish
new infection and reactivation of latent
37）
infection . However, changes of ITS have been
detected in a single episode of disease. Mutations
. Anti- in P. jirovecii dihydropteroate synthase gene
detected by PCR are associated with sulfa drug
resistance and has been used for monitoring the
treatment effectiveness and also for typing P.
38）
jirovecii isolates in AIDS patients . Microsatellite
genotyping on noninvasive samples（oropharyn-
geal washes）may aid in studying the molecular
39）
epidemiology of this pathogen .
qPCR for the detection of P. jirovecii DNA
qPCR is a new and cost-effective method for
the detection and quantification of P. jirovecii in
samples, monitoring therapeutic effectiveness,
and distinguishing colonization from pneumonia in
40, 41）
P. jirovecii infections . For BALF from HIV-
infected patients with or without PCP, qPCR
targeting HSP70 gene in P. jirovecii to quantify P.
jirovecii DNA showed a clinical sensitivity of 98%
and specificity of 96%; by contrast, the clinical
sensitivity was 97% and the specificity was 68%
when conventional PCR targeting mtLSUrRNA
42）
gene was used . Matsumura et al. found that the
sensitivity and specificity for discriminating defi-
 E 114
nite PCP from colonization was 100.0% and 80.0%,
respectively, at a cutoff value of 1,300
15）
copies/ml . Juliano et al. reported that the
specificity was increased to 100% with the com-
bination of the two qPCR assays targeting
dihydropteroate synthase and Msg in oropharyn-
39）
geal washes . However, Flori et al. found over-
lapped cutoff values between samples obtained
43）
from potential carriers and proven PCP . In most
cases, qPCR could greatly increase specificity
with a good sensitivity to perform a diagnosis of
PCP, however, qPCR cannot diagnose all cases of
44, 45）
PCP for the presence of grey zone . Further
studies should focus on standardization of qPCR
and determination of the optimal cutoff value to
discriminate PCP from P. jirovecii colonization for
wide use of this method in clinical practice.
In summary, the accurate diagnosis of PCP is
still a challenge. It is crucial to develop a less
invasive and more accurate diagnosis strategy
for PCP. The combination of clinical diagnosis,
etiological diagnosis, molecular diagnosis and
serological tests is necessary. The successful
cultivation of P. jirovecii in vitro will influence the
fields of diagnostic microbiology and clinical
treatment of PCP.
Conflict of Interest
All authors declare no conflict of interest.
References
1）Stringer JR, Beard CB, Miller RF, Wakefield AE: A
new name （Pneumocystis jiroveci） for
Pneumocystis from humans. Emerg Infect Dis 8:
891-896, 2002.
2）Morris A, Norris KA: Colonization by Pneumocy-
stis jirovecii and its role in disease. Clin Microbiol
Rev 25: 297-317, 2012.
3）Ponce CA, Gallo M, Bustamante R, Vargas SL:
Pneumocystis colonization is highly prevalent in
the autopsied lungs of the general population. Clin
Infect Dis 50: 347-353, 2010.
4）de Boer MG, de Fijter JW, Kroon FP: Outbreaks
and clustering of Pneumocystis pneumonia in
kidney transplant recipients: a systematic review.
Med Mycol 49: 673-680, 2011.
5）Lanaspa M, OʼCallaghan-Gordo C, Machevo S,
Madrid L, Nhampossa T, Acacio S, de la Horra C,
Friaza V, Campano E, Alonso PL, Calderón EJ,
Roca A, Bassat Q: High prevalence of Pneumocy-
stis jirovecii pneumonia among Mozambican chil-
dren < 5 years of age admitted to hospital with
clinical severe pneumonia. Clin Microbiol Infect 21:
Medical Mycology Journal Volume 57, Number 4, 2016
1018.e9-e15, 2015.
6）Valade S, Azoulay E, Damiani C, Derouin F, Totet A,
Menotti J: Pneumocystis jirovecii airborne trans-
mission between critically ill patients and health
care workers. Intensive Care Med 41: 1716-1718,
2015.
7）Catherinot E, Lanternier F, Bougnoux ME, Lecuit M,
Couderc LJ, Lortholary O: Pneumocystis jirovecii
pneumonia. Infect Dis Clin North Am 24: 107-138,
2010.
8）Carmona EM, Limper AH: Update on the diagnosis
and treatment of Pneumocystis pneumonia. Ther
Adv Respir Dis 5: 41-59, 2011.
9）Silva RM, Bazzo ML, Borges AA: Induced sputum
versus bronchoalveolar lavage in the diagnosis of
pneumocystis jiroveci pneumonia in human im-
munodeficiency virus-positive patients. Braz J
Infect Dis 11: 549-553, 2007.
10）Kaur R, Wadhwa A, Bhalla P, Dhakad MS:
Pneumocystis pneumonia in HIV patients: a di-
agnostic challenge till date. Med Mycol 53: 587-592,
2015.
11）Schildgen V, Mai S, Khalfaoui S, Lüsebrink J,
Pieper M, Tillmann RL, Brockmann M, Schildgen O:
Pneumocystis jirovecii can be productively cul-
tured in differentiated CuFi-8 airway cells. MBio 5:
e01186-01114, 2014.
12）Morris AM, Masur H: A serologic test to diagnose
pneumocystis pneumonia: are we there yet? Clin
Infect Dis 53: 203-204, 2011.
13）Carmona EM, Lamont JD, Xue A, Wylam M, Limper
AH: Pneumocystis cell wall beta-glucan stimulates
calcium-dependent signaling of IL-8 secretion by
human airway epithelial cells. Respir Res 11: 95,
2010.
14）Carmona EM, Kottom TJ, Hebrink DM, Moua T,
Singh RD, Pagano RE, Limper AH: Glycosphingoli-
pids mediate pneumocystis cell wall β -glucan
activation of the IL-23/IL-17 axis in human dendritic
cells. Am J Respir Cell Mol Biol 47: 50-59, 2012.
15）Matsumura Y, Ito Y, Iinuma Y, Yasuma K, Yamamo-
to M, Matsushima A, Nagao M, Takakura S,
Ichiyama S: Quantitative real-time PCR and the（1
→ 3） -β -D-glucan assay for differentiation be-
tween Pneumocystis jirovecii pneumonia and
colonization. Clin Microbiol Infect 18: 591-597, 2012.
16）Costa JM, Botterel F, Cabaret O, Foulet F,
Cordonnier C, Bretagne S: Association between
circulating DNA, serum（1 → 3）-β -D-glucan, and
pulmonary fungal burden in Pneumocystis
pneumonia. Clin Infect Dis 55: e5-8, 2012.
17）Cushion MT, Linke MJ, Ashbaugh A, Sesterhenn T,
Collins MS, Lynch K, Brubaker R, Walzer PD:
Echinocandin treatment of pneumocystis pneumo-
nia in rodent models depletes cysts leaving
trophic burdens that cannot transmit the infection.
PLoS One 5: e8524, 2010.
18）Watanabe T, Yasuoka A, Tanuma J, Yazaki H,
Honda H, Tsukada K, Honda M, Gatanaga H,
Teruya K, Kikuchi Y, Oka S: Serum（1 → 3）beta-D-
 Med. Mycol. J. Vol. 57（No. 4）
, 2016
glucan as a noninvasive adjunct marker for the
diagnosis of Pneumocystis pneumonia in patients
with AIDS. Clin Infect Dis 49: 1128-1131, 2009.
19）Desmet S, Van Wijngaerden E, Maertens J,
Verhaegen J, Verbeken E, De Munter P, Meersse-
man W, Van Meensel B, Van Eldere J, Lagrou K:
Serum（1-3）-beta-D-glucan as a tool for diagnosis
of Pneumocystis jirovecii pneumonia in patients
with human immunodeficiency virus infection or
hematological malignancy. J Clin Microbiol 47:
3871-3874, 2009.
20）Cuétara MS, Alhambra A, Chaves F, Moragues MD,
Pontón J, del Palacio A: Use of a serum（1 → 3）-
beta-D-glucan assay for diagnosis and follow-up
of Pneumocystis jiroveci pneumonia. Clin Infect
Dis 47: 1364-1366, 2008.
21）Damiani C, Le Gal S, Lejeune D, Brahimi N, Virmaux
M, Nevez G, Totet A: Serum（1 → 3）-beta-D-glucan
levels in primary infection and pulmonary col-
onization with Pneumocystis jirovecii. J Clin
Microbiol 49: 2000-2002, 2011.
22）Damiani C, Le Gal S, Da Costa C, Virmaux M, Nevez
G, Totet A: Combined quantification of pulmonary
Pneumocystis jirovecii DNA and serum（1 → 3） -β-
D-glucan for differential diagnosis of pneumocy-
stis pneumonia and Pneumocystis colonization. J
Clin Microbiol 51: 3380-3388, 2013.
23）de Boer MG, Gelinck LB, van Zelst BD, van de
Sande WW, Willems LN, van Dissel JT, de Jonge R,
Kroon FP: β-D-glucan and S-adenosylmethionine
serum levels for the diagnosis of Pneumocystis
pneumonia in HIV-negative patients: a prospec-
tive study. J Infect 62: 93-100, 2011.
24）Held J, Koch MS, Reischl U, Danner T, Serr A:
Serum（1 → 3） -β -D-glucan measurement as an
early indicator of Pneumocystis jirovecii pneumo-
nia and evaluation of its prognostic value. Clin
Microbiol Infect 17: 595-602, 2011.
25）Stringer JR, Keely SP: Genetics of surface antigen
expression in Pneumocystis carinii. Infect Immun
69: 627-639, 2001.
26）Daly KR, Fichtenbaum CJ, Tanaka R, Linke MJ, Oʼ
Bert R, Thullen TD, Hui MS, Smulian AG, Walzer
PD: Serologic responses to epitopes of the major
surface glycoprotein of Pneumocystis jiroveci
differ in human immunodeficiency virus-infected
and uninfected persons. J Infect Dis 186: 644-651,
2002.
27）Daly KR, Huang L, Morris A, Koch J, Crothers K,
Levin L, Eiser S, Satwah S, Zucchi P, Walzer PD:
Antibody response to Pneumocystis jirovecii
major surface glycoprotein. Emerg Infect Dis 12:
1231-1237, 2006.
28）Walzer PD, Djawe K, Levin L, Daly KR, Koch J,
Kingsley L, Witt M, Golub ET, Bream JH, Taiwo B,
Morris A: Long-term serologic responses to the
Pneumocystis jirovecii major surface glycopro-
tein in HIV-positive individuals with and without P.
jirovecii infection. J Infect Dis 199: 1335-1344, 2009.
29）Bishop LR, Kovacs JA: Quantitation of anti-
E 115
Pneumocystis jiroveci antibodies in healthy per-
sons and immunocompromised patients. J Infect
Dis 187: 1844-1848, 2003.
30）Blount RJ, Jarlsberg LG, Daly KR, Worodria W,
Davis JL, Cattamanchi A, Djawe K, Andama A,
Koch J, Walzer PD, Huang L; International HIV-
Associated Opportunistic Pneumonias（IHOP）
Study: Serologic responses to recombinant
Pneumocystis jirovecii major surface glycopro-
tein among Ugandan patients with respiratory
symptoms. PLoS One 7: e51545, 2012.
31）Djawe K, Huang L, Daly KR, Levin L, Koch J,
Schwartzman A, Fong S, Roth B, Subramanian A,
Grieco K, Jarlsberg L, Walzer PD: Serum antibody
levels to the Pneumocystis jirovecii major surface
glycoprotein in the diagnosis of P. jirovecii
pneumonia in HIV + patients. PLoS One 5: e14259,
2010.
32）Fan LC, Lu HW, Cheng KB, Li HP, Xu JF: Evaluation
of PCR in bronchoalveolar lavage fluid for diagno-
sis of Pneumocystis jirovecii pneumonia: a bivari-
ate meta-analysis and systematic review. PLoS
One 8: e73099, 2013.
33）Özkoç S, Bayram Delibaş S: Investigation of
Pneumocystis jirovecii pneumonia and coloniza-
tion in iatrogenically immunosuppressed and
immunocompetent patients. Mikrobiyol Bul 49:
221-230, 2015.
34）Gupta R, Mirdha BR, Guleria R, Kumar L, Saman-
taray JC, Agarwal SK, Kabra SK, Luthra K:
Diagnostic significance of nested polymerase
chain reaction for sensitive detection of
Pneumocystis jirovecii in respiratory clinical
specimens. Diagn Microbiol Infect Dis 64: 381-388,
2009.
35）Tia T, Putaporntip C, Kosuwin R, Kongpolprom N,
Kawkitinarong K, Jongwutiwes S: A highly sensi-
tive novel PCR assay for detection of Pneumocy-
stis jirovecii DNA in bronchoalveloar lavage
specimens from immunocompromised patients.
Clin Microbiol Infect 18: 598-603, 2012.
36）Totet A, Pautard JC, Raccurt C, Roux P, Nevez G:
Genotypes at the internal transcribed spacers of
the nuclear rRNA operon of Pneumocystis
jiroveci in nonimmunosuppressed infants without
severe pneumonia. J Clin Microbiol 41: 1173-1180,
2003.
37）Helweg-Larsen J, Lee CH, Jin S, Hsueh JY,
Benfield TL, Hansen J, Lundgren JD, Lundgren B:
Clinical correlation of variations in the internal
transcribed spacer regions of rRNA genes in
Pneumocystis carinii f. sp. hominis. AIDS 15:
451-459, 2001.
38）Durand-Joly I, Chabé M, Soula F, Delhaes L, Camus
D, Dei-Cas E: Molecular diagnosis of Pneumocy-
stis pneumonia. FEMS Immunol Med Microbiol 45:
405-410, 2005.
39）Juliano JJ, Barnett E, Parobek CM, Taylor SM,
Meshnick SR, Stone S, Chang E, Fong S, Huang L:
Use of Oropharyngeal Washes to Diagnose and
 E 116
Genotype Pneumocystis jirovecii. Open Forum
Infect Dis 2: ofv080, 2015.
40）Botterel F, Cabaret O, Foulet F, Cordonnier C,
Costa JM, Bretagne S: Clinical significance of
quantifying Pneumocystis jirovecii DNA by using
real-time PCR in bronchoalveolar lavage fluid
from immunocompromised patients. J Clin Micro-
biol 50: 227-231, 2012.
41）Fauchier T, Hasseine L, Gari-Toussaint M,
Casanova V, Marty PM, Pomares C: Detection of
Pneumocystis jirovecii by quantitative PCR to
differentiate colonization and Pneumonia in
immunocompromised HIV-positive and HIV-
negative patients. J Clin Microbiol 54: 1487-1495,
2016.
42）Huggett JF, Taylor MS, Kocjan G, Evans HE,
Morris-Jones S, Gant V, Novak T, Costello AM,
Zumla A, Miller RF: Development and evaluation of
a real-time PCR assay for detection of Pneumocy-
stis jirovecii DNA in bronchoalveolar lavage fluid
Medical Mycology Journal Volume 57, Number 4, 2016
of HIV-infected patients. Thorax 63: 154-159, 2008.
43）Maillet M, Maubon D, Brion JP, François P, Molina
L, Stahl JP, Epaulard O, Bosseray A, Pavese P:
Pneumocystis jirovecii（Pj）quantitative PCR to
differentiate Pj pneumonia from Pj colonization in
immunocompromised patients. Eur J Clin Microbiol
Infect Dis 33: 331-336, 2014.
44）Flori P, Bellete B, Durand F, Raberin H, Cazorla C,
Hafid J, Lucht F, Sung RT: Comparison between
real-time PCR, conventional PCR and different
staining techniques for diagnosing Pneumocystis
jiroveci pneumonia from bronchoalveolar lavage
specimens. J Med Microbiol 53: 603-607, 2004.
45）Mühlethaler K, Bögli-Stuber K, Wasmer S, von
Garnier C, Dumont P, Rauch A, Mühlemann K,
Garzoni C: Quantitative PCR to diagnose
Pneumocystis pneumonia in immunocompromised
non-HIV patients. Eur Respir J 39: 971-978, 2012.