Pneumocystis pneumonia in South African children diagnosed by molecular methods
BMC Research Notes
Pneumocystis pneumonia in South African children diagnosed by molecular methods
Brenda M Morrow 0 2
Catherine M Samuel 1
Marco Zampoli 0 2
Andrew Whitelaw 1
Heather J Zar 0 2
0 Department of Paediatics and Child Health, Red Cross War Memorial Children's Hospital (RCWMCH), University of Cape Town, 5th Floor Institute of Child Health Building , Klipfontein Road, Rondebosch 7700, Cape Town , South Africa
1 Division of Medical Microbiology, National Health Laboratory Services, University of Cape Town , Cape Town , South Africa
2 Department of Paediatics and Child Health, Red Cross War Memorial Children's Hospital (RCWMCH), University of Cape Town, 5th Floor Institute of Child Health Building , Klipfontein Road, Rondebosch 7700, Cape Town , South Africa
Background: Pneumocystis pneumonia (PCP) is an important cause of hospitalization and mortality in HIV-infected children. However, the incidence of PCP has been underestimated due to poor sensitivity of diagnostic tests. The use of polymerase chain reaction (PCR) for pneumocystis has enabled more reliable diagnosis. This study describes the incidence, clinical features and outcome of PCP in South African children diagnosed using PCR. Methods: A prospective study of children hospitalised in South Africa with suspected PCP was done from November 2006 to August 2008. Clinical, laboratory and radiological information were collected. Lower respiratory tract specimens were obtained for PCP immunofluorescence (IF), real- time PCR for pneumocystis, bacterial and mycobacterial culture. Nasopharyngeal aspirates were taken for immunofluorescence (IF), real-time PCR for pneumocystis and PCR for respiratory viruses. A blood specimen for bacterial culture and for cytomegalovirus PCR was taken. Children were followed for the duration of their hospitalisation and the outcome was recorded. Results: 202 children [median (interquartile range, IQR) age 3.2 (2.1- 4.6) months] were enrolled; 124 (61.4%) were HIV infected. PCP was identified in 109 (54%) children using PCR, compared to 43 (21%) using IF and Grocott staining (p < 0.0001). Most PCP cases (88, 81%) occurred in HIV-infected children. All 21 cases (19%) occurring in HIV- negative children had another risk factor for PCP. On logistic regression, predictive factors for PCP were HIV infection, lack of fever, high respiratory rate and low oxygen saturation whilst cotrimoxazole prophylaxis was protective (OR 0.24; 95% CI 0.1 to 0.5; p < 0.002). The case fatality of children with PCP was higher than those without PCP (32.1% versus 17.2%; relative risk 1.87; 95% confidence interval (CI) 1.11 - 3.15). Amongst HIV-infected children, a CD4 less than 15% was the only independent predictor of mortality. Conclusions: The diagnostic yield for PCP is more than 2.5 times higher on PCR than other detection methods. PCP is a very common cause of severe hypoxic pneumonia and is associated with high mortality in HIV-infected African infants.
Pneumocystis pneumonia; HIV; Children; Prophylaxis; PCR; Diagnosis; Incidence
Pneumocystis pneumonia (PCP) is a major cause of
morbidity and mortality in HIV infected infants [
Approximately 29 – 67% of respiratory related deaths among
African HIV infected children have been associated with
], and in-hospital case-fatality rates range from
20 – 63% [
]. South African studies have reported
that the prevalence of PCP ranges from 10% to 49% among
antiretroviral naïve HIV infected children hospitalized with
]. PCP has also been shown to occur in
HIV uninfected infants, mostly with an underlying
predisposing factor such as HIV exposure or malnutrition
The use of highly active antiretroviral therapy (HAART)
has dramatically reduced the incidence of PCP in
developed countries [
]. However PCP remains a common
cause of hospitalization and mortality in HIV-infected
South African children [
]. In a recent study, 21% of
children admitted to a tertiary paediatric hospital with acute
severe hypoxic pneumonia were found to have PCP despite
a well- functioning paediatric HIV program . In previous
studies, immunofluorescence (IF) or silver staining of
respiratory secretions have been used for the diagnosis of
PCP. Such methods have been reported to be insensitive
for diagnosis, potentially leading to under recognition of
].We recently reported that real-time
polymerase chain reaction (PCR) is more sensitive than IF for the
diagnosis of PCP when used on lower [induced sputum
(IS) or non-bronchoscopic broncho-alveolar lavage (BAL)]
or upper respiratory tract [nasopharyngeal aspirate (NPA)]
specimens. The yield for PCP from upper and lower
respiratory tract specimens was similar by PCR [
The aim of this study was to describe the incidence,
clinical features and outcome of PCP in children when
diagnosed with PCR.
This was a prospective study of consecutive children
hospitalized for acute hypoxic pneumonia at Red Cross
War Memorial Children’s Hospital in Cape Town, South
Africa from November 2006 to August 2008. Inclusion
criteria were an acute onset of respiratory illness
requiring hospitalization, defined as age specific tachypnoea,
hypoxia and diffuse lung disease not associated with
]. Children were excluded if they had
received treatment for PCP in the preceding two weeks, if
they had been on PCP therapy for the acute illness for
more than 48 hours or if informed consent was not
obtainable. The study was approved by the Research and
Ethics Committee of the Faculty of Health Sciences at
the University of Cape Town. Written informed consent
for participation in the study was obtained from each
child’s parent or legal guardian.
Clinical, radiological and laboratory data were recorded
including symptoms, signs and oxygen saturation. The
HIV status of a child (if unknown) was confirmed using
whole-blood HIV deoxyribonucleic acid PCR (Amplicor
HIV-1 DNA test version 1.5, Roche Diagnostics, GmbH,
Mannheim, Germany) in those younger than 18 months,
or an HIV enzyme-linked immunosorbent assay (Architect
HIV Ag/Ab Combo ELISA, Abbott Laboratories, Abbott
Park, IL) in older children. CD4 counts and HIV viral
loads were done in all children newly diagnosed with HIV
and in those in whom HAART was commenced. A full
blood count and serum lactate dehydrogenase (LDH) was
done on admission.
An upper respiratory tract (NPA) and a lower respiratory
tract (LRT) specimen (IS or BAL in intubated patients)
were obtained in a standardized manner, as described
]. Respiratory specimens were submitted for
detection of P. jirovecii by direct IF using a monoclonal
antibody (IF: Detect IF PC, Axis-Shield, UK) and Grocott
staining. Real time PCR were done on stored
histopathological specimens, as described by Samuel et al. .
Investigation for additional pathogens included
“inhouse” respiratory viral shell vial culture and rapid viral
antigen detection (murine FITC-conjugated anti- RSV or
adenovirus monoclonal, Chemicon, Temecula, CA, USA)
on respiratory specimens, bacterial blood culture and
bacterial and mycobacterial culture on IS or BAL specimens.
Blood specimens were also sent for qualitative whole blood
nested cytomegalovirus (CMV) PCR (Super-Therm, JMR
Holdings, Kent, UK). CMV pneumonia was defined as
positive CMV PCR on a blood specimen as well as
detection of CMV on a LRT sample.
Children received standard therapy including oxygen,
broad spectrum antibiotics and intravenous
cotrimoxazole (trimethoprim-sulfamethoxazole) and oral
corticosteroids (prednisone 1–2 mg/kg with tapered doses for
up to 21 days) as per national guidelines [
children were started on HAART according to national
Continuous data were tested for normality using the
Shapiro- Wilks test. Descriptive statistics, Mann- Whitney
U tests for continuous data, and chi- square tests for
categorical data (Yates- corrected chi-square tests where
values in the cells were <10) were performed using
STATISTICA data analysis software system (version 8,
StatSoft, Inc. 2004). Forward stepwise logistic regression
analyses, to determine predictive variables for the
dichotomous outcomes of PCP and mortality, were
performed using STATA (version 10.0, Statcorp, Texas,
USA). Model variables were selected if they were
associated with the outcome of interest on univariate analysis
and according to biological plausibility. Variables found
to be significant when analysed jointly were included in
the final model. Weight- for- age and height- for- age Z
(or standard deviation) scores were calculated using the
Microsoft Office Excel (Microsoft Corporation 2003)
addin ImsGrowth Program (version 2.12, Medical Research
Council UK, 2002 – 2005) (15). The WHO categorises
weight- for age and height- for age scores ≤2 as
representing moderate under-nutrition and scores ≤3 representing
]. A 95% significance level was chosen.
Two hundred and two children [92 (45.5%) male] were
enrolled with a median (interquartile range, IQR) age
of 3.2 (2.1 – 4.6) months. HIV results were available for
200 children; 124 (61.4%) were HIV infected; 34 (16.8%)
were HIV exposed but uninfected and 42 (20.8%) were
HIV unexposed. Seventy of the HIV exposed or infected
children (44.3%) had been in the Prevention of Mother
to Child Transmission (PMTCT) program, but only 29
(18.4%) were receiving cotrimoxazole prophylaxis. Five
(4%) HIV infected children were on HAART; 72 (58%)
were started on HAART during hospitalization, at a
median (IQR) of 10 (7 – 14.5) days after admission. One
hundred and nine (54%) patients received gancyclovir
for presumed or proven CMV infection.
Most children were under- nourished with median (IQR)
weight- for age and height- for age Z scores of −2.5 (−4.3
to −1.5) and −2.3 (−3.9 to −0.9) respectively. The most
common presenting features were cough (85.1%),
vomiting (31.2%), diarrhoea (21.8%), or poor feeding (24.3%);
these did not differ by HIV status.
Pneumocystis jirovecii was detected in 43 (21.3%) children
by IF and/or Grocott staining and in 109 (54.0%) children
using PCR (p < 0.0001) on 107 induced sputum and 97
BAL specimens. No child was positive by IF and negative
by PCR. Eighty- seven children (79.8%) with PCP were
HIV infected (Table 1). Risk factors amongst the 21 HIV
uninfected children with PCP included malnutrition in 12
(57.1%) children, HIV exposure in 5 (23.8%); a history of
prematurity in 5 (23.8%); congenital cardiac disease in 2
(9.5%); and primary immune deficiency and post-
transplant immunosuppression in one child each. Clinical
features distinguishing children with PCP from those without
PCP were absence of fever, higher respiratory rate and
lower oxygen saturation on admission (Table 1).
Presenting symptoms n (%)
HIV infected n (%)
HIV exposed, uninfected
Weight for age Z score
Height for age Z score
Duration of symptoms (days )
Subcostal recessions n (%)
SpO2 (%) in room air (n = 146)
CD4% in HIV infected children
Lactate dehydrogenase (u/l)
Respiratory rate on admission (breaths per minute)
Continuous data are median (interquartile range).
n = 109
Co-infection with CMV was common in children with
PCP. The prevalence of CMV pneumonia or viraemia
was higher in children with PCP compared to those
without PCP (Table 2). In contrast other respiratory
viruses were more commonly identified in children
without PCP (Table 2). There was no difference in the rate
of bacteraemia or culture confirmed tuberculosis
between the two groups.
Predictive factors for PCP
LDH and SpO2 were not included in the multiple
regression analyses due to missing data.In the final model
cotrimoxazole prophylaxis was found to be protective for PCP;
whilst HIV infection, CMV viraemia, lack of fever and
tachypnoea were associated with PCP (Table 3).
There was no difference between children with PCP
compared to those without PCP in terms of PICU admission;
ventilation requirements and duration; or length of
hospital and PICU stay. The in-hospital mortality was 35
(32.1%) in children with PCP compared to 16 (17.2%) in
those without PCP (relative risk 1.87; 95% CI 1.11 – 3.15;
p = 0.02). Multiple regression controlling for age, HIV
infection, PCP and cotrimoxazole prophylaxis showed that
only HIV infection was predictive of mortality (OR 3.7,
95% CI 1.5 – 9.0; p = 0.004). In a separate model of 115
HIV infected children with complete data, only CD4
count <15% was significantly associated with mortality
(OR 3.6, 95% CI 1.4 – 9.0; p = 0.006) (Table 4). None of
the five HIV exposed uninfected infants with PCP died.
PCP was diagnosed in more than half of children
hospitalised with hypoxic pneumonia using molecular
techniques, more than double that diagnosed by IF or
Grocott staining on the same respiratory samples [
The incidence of PCP reported in prior studies based on
IF staining of respiratory secretions may therefore be a
large underestimate [
]. The study indicates
that PCP in HIV-infected infants is even more of a
concern than was previously recognised, and emphasises the
need to strengthen paediatric HIV programs including
early use of cotrimoxazole prophylaxis and HAART
according to current global guidelines [
CMV-cytomegalovirus; CI- confidence interval. Lactate dehydrogenase and
oxygen saturation were not included due to missing data.
Cotrimoxazole prophylaxis and HAART had been
instituted in only a minority of eligible children despite the
free availability of HAART and PMTCT programs in
this area of South Africa. Reassuringly, cotrimoxazole
prophylaxis was found to be highly effective for preventing
PCP, supporting the widespread use of this cost-effective
]. Although this study was not
designed to investigate implementation of national policies
for paediatric HIV or adherence to HAART or
cotrimoxazole, the results indicate that the HIV program is still
not functioning appropriately, with potentially devastating
consequences. Further research to investigate the
underlying reasons for failure of the PMTCT and HAART
programs in this setting is needed.
The in- hospital case fatality rate for children with
PCP of 32% is within the range reported in other African
], and is also similar to that reported eight
years previously from the same study site .
A minority of HIV uninfected children also developed
PCP. Consistent with prior reports, a number of risk
factors were identified [
] including malnutrition
and HIV exposure [
]. HIV exposed infants
may be at increased risk of PCP due to impaired
immunity, exposure to P. jirovecii from an HIV infected
mother or adult, or poor protection from maternal
]. All HIV exposed but uninfected
infants with proven PCP survived, consistent with
previous reports of better outcome in HIV uninfected
compared to HIV infected infants [
]. With strengthening
of PMTCT programs, HIV exposed but uninfected
infants are an increasingly important group who may be at
risk for PCP. The use of cotrimoxazole prophylaxis in
this group deserves further consideration given the
increasing number of case reports of PCP in HIV-exposed
uninfected infants and this growing population [
Clinical and laboratory measures associated with PCP
were similar to previous reports [
elevated LDH may be a non-specific marker of lung injury
, these results suggest that there should be a high
index of suspicion of PCP in those children with raised
LDH admitted to hospital with severe pneumonia,
particularly when there are other known risk factors for
As expected, co-infection with P. jirovecii and other
pathogens, particularly viruses, was common [
Similar to previous studies, CMV coinfection occurred most
frequently, in a third of children with PCP [
This may also reflect severe immunosuppression as CMV
pneumonia has been associated with moderate or severe
immunosuppression in HIV-infected infants [
Limitations of this study include lack of a control group.
Several adult studies have reported carriage of P. jirovecii
as detected by a positive PCR on respiratory specimens
], however, positive PCR results in our study are
unlikely to represent colonisation given the presentation
of severe pneumonia, the young age of infants and the
protective effect of cotrimoxazole prophylaxis [
were unable to use response to treatment as a means of
confirming PCR diagnosis as all children were treated for
presumptive PCP based on clinical signs. The lack of a
gold standard for PCP diagnosis makes testing of any new
diagnostic modality challenging, especially as the organism
cannot be easily cultured. This was a single- centre study
of a selected population admitted to a tertiary hospital;
therefore results may not be generalisable to children from
different geographical areas and with different clinical
disease spectra. Further studies of PCR based diagnosis in
children with non-HIV immunosuppression are needed.
PCP remains a common cause of severe hypoxic
pneumonia and is associated with high mortality in HIV-infected
African infants. New molecular diagnost ic methods
indicate that the burden of PCP in this populat ion has been
under -est imated in the past.
The authors declare that they have no competing interests.
BM recruited patients, acquired and analysed data, and drafted the
manuscript. HZ was responsible for study conception and design, clinical
supervision and obtaining funding. AW was the laboratory supervisor,
contributed to the study design and obtained funding. MZ recruited patients
and acquired data. CS performed the molecular investigations. All authors
contributed to the final manuscript and have read and approved of it.
This research was supported by an NHLS Research Trust grant; the National
Research Foundation, South Africa; ASTRA-Zeneca Respiratory Award from
the South African Thoracic Society and the Medical Research Council of
Southern Africa. We thank Louise LeSai for her assistance with enrolling
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