Improved detection of Pneumocystis jirovecii in upper and lower respiratory tract specimens from children with suspected pneumocystis pneumonia using real-time PCR: a prospective study
Catherine M Samuel
0
Andrew Whitelaw
0
Craig Corcoran
2
Brenda Morrow
1
Nei-Yuan Hsiao
2
Marco Zampoli
1
Heather J Zar
1
0
Division of Medical Microbiology, University of Cape Town and National Health Laboratory Service
,
Cape Town
,
South Africa
1
Department of Paediatrics and Child Health, University of Cape Town, Red Cross War Memorial Children's Hospital
,
Cape Town
,
South Africa
2
Division of Clinical Virology, University of Cape Town and National Health Laboratory Service
,
Cape Town
,
South Africa
Background: Pneumocystis pneumonia (PCP) is a major cause of hospitalization and mortality in HIV-infected African children. Microbiologic diagnosis relies predominantly on silver or immunofluorescent staining of a lower respiratory tract (LRT) specimens which are difficult to obtain in children. Diagnosis on upper respiratory tract (URT) specimens using PCR has been reported useful in adults, but data in children are limited. The main objectives of the study was (1) to compare the diagnostic yield of PCR with immunofluorescence (IF) and (2) to investigate the usefulness of upper compared to lower respiratory tract samples for diagnosing PCP in children. Methods: Children hospitalised at an academic hospital with suspected PCP were prospectively enrolled. An upper respiratory sample (nasopharyngeal aspirate, NPA) and a lower respiratory sample (induced sputum, IS or bronchoalveolar lavage, BAL) were submitted for real-time PCR and direct IF for the detection of Pneumocystis jirovecii. A control group of children with viral lower respiratory tract infections were investigated with PCR for PCP. Results: 202 children (median age 3.3 [inter-quartile range, IQR 2.2 - 4.6] months) were enrolled. The overall detection rate by PCR was higher than by IF [180/349 (52%) vs. 26/349 (7%) respectively; p < 0.0001]. PCR detected more infections compared to IF in lower respiratory tract samples [93/166 (56%) vs. 22/166 (13%); p < 0.0001] and in NPAs [87/183 (48%) vs. 4/183 (2%); p < 0.0001]. Detection rates by PCR on upper (87/183; 48%) compared with lower respiratory tract samples (93/166; 56%) were similar (OR, 0.71; 95% CI, 0.46 - 1.11). Only 2/30 (6.6%) controls were PCR positive. Conclusion: Real-time PCR is more sensitive than IF for the detection of P. jirovecii in children with PCP. NPA samples may be used for diagnostic purposes when PCR is utilised. Wider implementation of PCR on NPA samples is warranted for diagnosing PCP in children.
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Background
Pneumocystis pneumonia (PCP), caused by Pneumocystis
jirovecii, is an important opportunistic infection in
HIVinfected children [1,2]. The incidence of PCP in
developed countries has declined since the introduction of
highly active anti-retroviral therapy and use of
chemoprophylaxis. However, PCP remains a major cause of
hospitalization and mortality in HIV-infected children in
low or middle income countries, [1,3-5] with reported
incidence rates of 10 - 49%, [1,3,6] and in-hospital
casefatality rates of 20 - 63% [1,3,4,6]. Apart from HIV
infection, there are other factors that predispose
children to developing PCP including malnutrition, other
immune deficiencies or HIV exposure. Untreated, the
case fatality rate in children with PCP approximates
100% [1,3,4,6]. However, diagnosis can be difficult as
clinical and radiological findings are non-specific.
Therefore, a rapid, accurate laboratory diagnosis is
important for timely use of appropriate medication.
Detection of P. jirovecii is hampered by the lack of a
sustainable in-vitro culture method [7]. Standard
laboratory diagnostic methods are microscopic examination of
a lower respiratory tract sample with Gomori Grocotts
methenamine silver nitrate stain or the more sensitive
immunofluorescence assay (IF) [8] on bronchoalveolar
lavage (BAL) or induced sputum (IS) specimens. Using
microscopy, the yield from IS has been reported to be
similar compared to that from BAL [9]. However,
sputum induction in children is not widely performed,
requires staff trained to do the procedure and may
result in clinical deterioration or nosocomial
transmission of respiratory pathogens. Diagnosis using a
noninvasive sample such as a nasopharyngeal aspirate
(NPA) is, therefore, desirable.
The clinical sensitivity from a NPA has been reported
to be low and variable when microscopy is utilised
[4,5,10]. The development of polymerase chain reaction
(PCR) techniques has provided a more reliable
diagnostic method. In adult studies, PCR is as specific as and
more sensitive than microscopy for diagnosis when
performed on respiratory specimens, including oral washes
[7,11-21]. In a study of oropharyngeal washes from
HIV-infected adult patients, P. jirovecii
DNA-amplification had a sensitivity of 44% using a nested PCR
protocol compared to trans-bronchial biopsy, [16] increasing
to 90% when touch-down real-time PCR was utilised
[7,11]. Real-time PCR also allows for quantification of
the organism load and with application of cutoff values,
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