Comparison between real-time PCR, conventional PCR and different staining techniques for diagnosing Pneumocystis jiroveci pneumonia from bronchoalveolar lavage specimens
Between January 2002 and July 2003, 173 bronchoalveolar lavage (BAL) specimens from 150 patients (19 HIV-infected and 131 non-HIV-infected patients) were evaluated for identification of Pneumocystis jiroveci (formerly known as Pneumocystis carinii f. sp. hominis) using staining techniques, conventional PCR (mtLSUrRNA gene) and real-time PCR (MSG gene). Test results were compared to Pneumocystis pneumonia (PCP) confirmed by typical clinical findings and response to treatment. Sensitivity and specificity of the techniques were 60 and 100 % for staining (where either one or both techniques were positive), 100 and 87.0 % for conventional PCR and 100 and 84.9 % for real-time PCR, respectively. The use of a concentration of 103 copies of DNA per capillary of BAL as a cut-off (determined by real-time PCR) increased specificity from 84.9 to 98.6 % without reducing the sensitivity of the technique. This technique is rapid (<3 h) and therefore of major interest in differentiating between asymptomatic carriage and PCP. A BAL specimen with <103 copies per capillary of Pneumocystis-specific DNA is more likely to indicate a chronic carrier state, but in such cases follow-up is required to ensure that the patient is not in the early stage of an active PCP.
CaliendoA. M.,
HewittP. L.,
AllegaJ. M.,
KeenA.,
RuoffK. L.,
FerraroM. J.1998; Performance of a PCR assay for detection of Pneumocystis carinii from respiratory specimens. J Clin Microbiol 36:979–982
CartwrightC. P.,
NelsonN. A.,
GillV. J.1994; Development and evaluation of a rapid and simple procedure for detection of Pneumocystis carinii by PCR. J Clin Microbiol 32:1634–1638
CostaJ. M.,
ErnaultP.,
GautierE.,
BretagneS.2001; Prenatal diagnosis of congenital toxoplasmosis by duplex real-time PCR using fluorescence resonance energy transfer hybridization probes. Prenat Diagn 21:85–88[CrossRef]
CreganP.,
YamamotoA.,
LumA.,
VanDerHeideT.,
MacDonaldM.,
PulliamL.1990; Comparison of four methods for rapid detection of Pneumocystis carinii in respiratory specimens. J Clin Microbiol 28:2432–2436
Helweg-LarsenJ.,
JensenJ. S.,
DohnB.,
BenfieldT. L.,
LundgrenB.2002; Detection of Pneumocystis DNA in samples from patients suspected of bacterial pneumonia – a case−control study. BMC Infect Dis 2:28http://www.biomedcentral.com/1471-2334/2/28
LarsenH. H.,
MasurH.,
KovacsJ. A. & 7 other authors; 2002; Development and evaluation of a quantitative, touch-down, real-time PCR assay for diagnosing Pneumocystis carinii pneumonia. J Clin Microbiol 40:490–494[CrossRef]
LeighT. R.,
GazzardB. G.,
RowbottomA.,
CollinsJ. V.1993; Quantitative and qualitative comparison of DNA amplification by PCR with immunofluorescence staining for diagnosis of Pneumocystis carinii pneumonia. J Clin Pathol 46:140–144[CrossRef]
LipschikG. Y.,
GillV. J.,
LundgrenJ. D.,
AndrawisV. A.,
NelsonN. A.,
NielsenJ. O.,
OgnibeneF. P.,
KovacsJ. A.1992; Improved diagnosis of Pneumocystis carinii infection by polymerase chain reaction on induced sputum and blood. Lancet 340:203–206[CrossRef]
NevezG.,
RaccurtC.,
JounieauxV.,
Dei-CasE.,
MazarsE.1999; Pneumocystosis versus pulmonary Pneumocystis carinii colonization in HIV-negative and HIV-positive patients. AIDS 13:535–536[CrossRef]
NininE.,
HamidouM.,
GermaudP.,
MorinO.,
MilpiedN.,
RaffiF.1998; Pneumocystose pulmonaire chez les patients non VIH – étude rétrospective de 31 cas. Presse Méd 27:244–249 (in French
SingA.,
RoggenkampA.,
AutenriethI. B.,
HeesemannJ.1999; Pneumocystis carinii carriage in immunocompetent patients with primary pulmonary disorders as detected by single or nested PCR. J Clin Microbiol 37:3409–3410
SingA.,
TrebesiusK.,
RoggenkampA.,
RussmannH.,
TybusK.,
PfaffF.,
BognerJ. R.,
EmmingerC.,
HeesemannJ.2000; Evaluation of diagnostic value and epidemiological implications of PCR for Pneumocystis carinii in different immunosuppressed and immunocompetent patient groups. J Clin Microbiol 38:1461–1467
TakahashiT.,
GotoM.,
EndoT.,
NakamuraT.,
YusaN.,
SatoN.,
IwamotoA.2002; Pneumocystis carinii carriage in immunocompromised patients with and without human immunodeficiency virus infection. J Med Microbiol 51:611–614
WakefieldA. E.,
PixleyF. J.,
BanerjiS.,
SinclairK.,
MilllerR. F.,
MoxonE. R.,
HopkinJ. M.1990; Detection of Pneumocystis carinii with DNA amplification. Lancet 336:451–453[CrossRef]
WakefieldA. E.,
GuiverL.,
MillerR. F.,
HopkinJ. M.1991; DNA amplification on induced sputum samples for diagnosis of Pneumocystis carinii pneumonia. Lancet 337:1378–1379[CrossRef]
WeigM.,
KlinkerH.,
BognerB. H.,
MeierA.,
GrossU.1997; Usefulness of PCR for diagnosis of Pneumocystis carinii pneumonia in different patient groups. J Clin Microbiol 35:1445–1449
ZamanM. K.,
WootenO. J.,
SuprahmanyaB.,
AnkobiahW.,
FinchP. J.,
KamholzS. L.1988; Rapid noninvasive diagnosis of Pneumocystis carinii from induced liquefied sputum. Ann Intern Med 109:7–10[CrossRef]
Comparison between real-time PCR, conventional PCR and different staining techniques for diagnosing Pneumocystis jiroveci pneumonia from bronchoalveolar lavage specimens