The Turkish Journal of Pediatrics
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Prematurity and protracted mechanical ventilation as risk factors for Pneumocystis jiroveci infection in HIV-negative neonates in an intensive care unit
Agnieszka Kordek1, Lidia Kołodziejczyk2, Małgorzata Adamska3, Bogumiła Skotarczak3, Beata Łoniewska1, Beata Pawlus1, Wanda Kuźna-Grygiel2, Jacek Rudnicki1, Ryszard Czajka1
1Clinic of Obstetrics and Perinatology, Pomeranian Medical University, Poland
2Department of Biology and Medical Parasitology, Pomeranian Medical University, Poland
3Department of Genetics, Szczecin University, Szczecin, Poland
|Kordek A, Kołodziejczyk L, Adamska M, Skotarczak B, Łoniewska
B, Pawlus B, Kuźna-Grygiel W, Rudnicki J, Czajka R. Prematurity and protracted
mechanical ventilation as risk factors for Pneumocystis jiroveci infection in HIVnegative
neonates in an intensive care unit. Turk J Pediatr 2007; 49: 158-164.|
This work was undertaken to elucidate some aspects of the epidemiology of
Pneumocystis pneumonia (PP). We studied 42 mechanically ventilated, human
immunodeficiency virus (HIV)-negative, severely ill neonates treated at an
intensive care unit. The study group included 40 premature neonates and
two mature neonates with lethal congenital defects. Progressive respiratory
dysfunction in PP necessitated mechanical ventilation. Infection was usually
noticeable on the 22nd day of life or after 12 days of ventilation. The usual
manifestations included apnea, pallor, copious frothy sputum, seizures, and
feeding difficulties. The diagnosis was established by detecting Pneumocystis
jiroveci cysts in bronchial lavage fluid specimens (88.1% sensitivity). PP
was managed with cotrimoxazole and pentamidine combination therapy
administered over 14 days. No clinical improvement was noted in four
neonates and three of them died during therapy. Prematurity and protracted
mechanical ventilation are two risk factors for P. jiroveci infection in severely
ill neonates in an intensive care unit.
Pneumocystis jiroveci, newborn infant, pneumonia, mechanical ventilation, prematurity
|Pneumocystis jiroveci, previously known as
Pneumocystis carinii forma specialis hominis, is
an atypical fungus responsible for Pneumocystis
pneumonia (PP) in patients with immune deficits
that accompany acquired immunodeficiency
syndrome (AIDS), immunosuppression,
transplantation, or steroid therapy[1,5].Also
at increased risk of P. jiroveci infection are
severely ill and immature neonates whose
cellular (CD4+ cells) and humoral (interferon)
responses are compromised[6,7].|
Molecular studies have revealed that the
genus Pneumocystis is a heterogeneous group
of opportunistic fungi with exceptional host
specificity8. This feature of Pneumocystis
is linked with differences as to phenotype
and genotype[9,10]. Infections in humans are caused by P. jiroveci[1,2]. Healthy humans can
serve as a reservoir for the pathogen[11,12].
Vargas et al. identified P. carinii DNA in nasopharyngeal secretions of healthy infants
and Chabé et al. demonstrated the ability
of Pneumocystis to replicate in the lungs of
immunocompetent hosts. Thus, transmission
of the pathogen among medical personnel and
patients is likely and transmission from the
mother to the neonate remains possible[11,12,15]. Extrapulmonary sites of pneumocystosis in
the liver, lymph nodes, muscles, and rarely
in kidneys, thymus and pancreas have been
reported in terminally ill AIDS patients[16,18].
The literature on PP in children without
coexisting human immunodeficiency virus
(HIV) infection is scarce[19,21]. Therefore, we decided to undertake a clinical observational
study of P. jiroveci infections in HIV-negative,
severely ill, mechanically ventilated neonates.
|Material and Methods |
|We analyzed 234 severely ill, mechanically
ventilated neonates at the Clinic of Obstetrics
and Perinatology, Pomeranian Medical University,
between 1999 and 2003. Neonates were assigned
to two groups: A (n=42) with PP, and B
(n=192) without PP. Assignment was based
on microscopic examination of bronchial lavage
fluid and additionally on clinical symptoms,
chest X-ray, and laboratory findings.|
Intrauterine bacterial infection was recognized
during the first 72 hours of life based on maternal
history [chorioamnionitis defined as intrapartum
fever >38ºC, tachycardia (maternal ≤100 beats/
min; fetal ≤160 beats/min), uterine tenderness, purulent or foul smell of amniotic fluid or
increased total leukocyte count (>15x109/L)],
clinical symptoms in the neonate (skin color:
pallor, jaundice, cyanosis; respiratory dysfunction:
apnea, tachypnea >60/min, grunting, nasal flaring, intercostal or sternal retractions,
need for high ventilator settings or oxygen;
cardiovascular symptoms: brady/tachycardia, poor
peripheral perfusion, hypotension; neurologic
symptoms: hypotonia, irritability, lethargy,
seizures; gastrointestinal dysfunction: abdominal
distension, green or bloody residuals, vomiting;
temperature instability), and/or positive blood
culture. Nosocomial infections were diagnosed
when appearing after three days of life. Pneumonia
was diagnosed in neonates presenting with
characteristic findings in chest X-ray (diffuse,
asymmetric or localized, alveolar or interstitial
disease) and with respiratory distress.
Diagnostic criteria for respiratory distress
syndrome (RDS) included oxygen supplementation
exceeding 40%, demand for alveolar
surfactant, typical chest radiograph (uniform
reticulogranular pattern and air bronchograms),
and no evidence of systemic infection in
Diagnosis of bronchopulmonary dysplasia
(BPD) was made when the infant still required
oxygen after 28 days of age or at 36 weeks of
postconceptional age, and continued to show
signs of respiratory problems at that time.
Chest X-ray film may facilitate the diagnosis
by demonstrating lungs that resemble ground
glass (RDS) or appear spongy as in BPD.
Infection (bacterial pneumonia and sepsis)
was further confirmed with cultures and
biochemical tests of the blood. We determined
the concentration of C-reactive protein (CRP
values <10 mg/L were considered normal),
leukocyte (>25 or <5x109/L), and platelet
counts (<100x1012/L), and the immature to total neutrophil ratio (I:T <0.2).
In all cases, bronchial lavage fluid specimens
were stained with Giemsa reagent and searched
for P. jiroveci cysts at the start and after 7-10
days of treatment. Microscopy was done by the
same, highly-experienced person. Specimens
were collected during routine nursing care of
the ventilated airways.
We also searched for P. carinii DNA in the
lavage fluid but the result had no impact
on the diagnosis. Polymerase chain reaction
(PCR) was done with 130 randomly selected
samples which were stored at -24ºC prior to
the diagnosis or microscopic examination.
DNA was isolated with the Masterpure DNA
purification kit – Epicentre. Single PCR was
performed with the PC41/PC22 primer pair.
Product size was 420 bp consistent with
the targeting by these primers of the small
ribosome subunit 18S rRNA gene sequence.
The following conditions were applied: initial
denaturation at 94ºC for 5 minutes; 40 cycles consisting of denaturation at 94ºC for 1 minute,
annealing at 52°C for 1 minute, extension
at 72ºC for 1 minute, and final extension
at 72ºC for 5 minutes. We also performed
nested PCR with pAZ102H/pAZ102E and
pAZ102X/pAZ102Y primers targeting the
mitochondrial large subunit (mtLSU) rRNA
gene sequence. Product size with these primer
pairs was 346 bp and 263 bp, respectively. We used 40 cycles of denaturation at 94ºC
for 1.5 minutes, annealing at 50ºC for 1.5
minutes and extension at 72ºC for 2 minutes;
other PCR conditions were the same as for
PC41/PC22 primers. Positive control was done
with DNA isolated from the lung of inoculated
rat and supplied by the Polish Institute of
Hygiene in Warsaw. Amplification products
were electrophoresed in 2% agarose gel and
stained with ethidium bromide.
Statistics were done with χ2-test and Student's t-test and the level of significance was taken
|Pneumocystis pneumonia was diagnosed in 42
(17.9%) neonates (group A). The remaining
neonates served as controls (group B). The
mothers were Caucasian whites, with an
average or good socio-economic status, normal
body weight, no immune impairment or
systemic disease, HIV-negative, and not on
Two children with PP were born at term
(40 and 41 weeks of gestation, 2250 and
3000 g), but both had lethal congenital defects
(trisomy 18; complex defect of the central
nervous system with tracheo-esophageal fistula)
and required mechanical ventilation from birth
till death (68 and 30 days, respectively). The
remaining children in this group were born
We found that both groups of neonates
(Table I) differed as to maturity and weight
at birth. For these reasons, the study and
control groups differed significantly as to
duration of mechanical ventilation and hospital
stay. Immaturity and low birth weight are
factors predisposing to serious complications
postnatally. Neonates with PP were less
mature, had a lower birth weight, and more
often demanded steroids to accelerate lung
maturation. In this group, there were more
cases of RDS necessitating treatment with exogenous surfactant, and likewise, criteria
for BPD were more often fulfilled. Bacterial
and fungal infections in the form of sepsis
or pneumonia were more often diagnosed in
| ||Table I: Demographic and Clinical Data of Mechanically Ventilated Neonates|
The initial suspicion of PP was based on clinical
findings (dyspnea, pallor, copious frothy or
mucous sputum, episodes of apnea, feeding
difficulties, absence of weight gain, seizures,
pulmonary bleeding) and radiologic evidence
of interstitial pneumonia. PP was confirmed in
37 out of 42 neonates (sensitivity = 88.1%,
specificity = 100%) by the presence of cysts
in bronchial lavage fluid. In the remaining
five cases with negative staining, the clinical
course and radiologic evidence were clearly in
favor of the diagnosis. Worth noting is the fact
that bronchial fluid was obtained after all five
neonates were started on cotrimoxazole. Thus,
the final diagnosis of PP was confirmed by
positive microscopic examination of bronchial
lavage fluid and positive response to specific
None of the 130 samples of bronchial fluid
from mechanically ventilated neonates tested
positive for P. jiroveci DNA.
In group A, mechanical ventilatory support was
started in the first day of life and continued
over three days in 33 neonates (78.6%). Five
premature neonates (11.9%) with RDS I/IIº received a single dose of surfactant postnatally
and were ventilated during less than 24 h.
In the remaining four cases, symptoms of
dyspnea appeared during the first 10 days of
life. In group B, 99 neonates (52%) required
ventilation during less than three days. PP led
to further deterioration of respiratory function
in all neonates. All neonates with PP required
continuous positive airway pressure (CPAP) or
intermittent mandatory ventilation (IMV).
Infection with P. jiroveci was diagnosed between
the 6th and 58th day of life (mean 22±12) or
between day 6 and day 27 of ventilation (mean
12±8). PP was the first pulmonary infection in
five neonates. In three cases, PP coincided with
bacterial infection [Klebsiella oxytoca (n:1),
Serratia marcescens (n:2)]. Initial symptoms
of P. jiroveci infection included apnea and
reduced saturation recorded with the oximeter.
Symptoms appeared either spontaneously or
during nursing procedures, were unaccompanied
by slowing of heart action, and were absent
in four neonates only. Cyanosis or pallor was usually seen in spite of normal hematology,
saturation, or oxygen partial pressure (paO2)
in arterial blood. Frothy or mucous exudate
appeared in the airways in large or very large
quantities. Pulmonary bleeding of varying
intensity was noted in eight neonates. Due
to aggravating symptoms of dyspnea, 12 out
of 13 previously extubated neonates required
re-intubation and assisted ventilation and one
required nasal CPAP. The remaining neonates developed PP during mechanical ventilation for
RDS, BPD or infection (bacterial or fungal).
When infection coincided with mechanical
ventilation, an upregulation of the respirator was
necessary. Seizures appeared or intensified in 16
neonates. Feeding difficulties, regurgitation, slow
gastric emptying, and flatulence were noted in
29 cases (Table II).
| ||Table II: Clinical Findings in the Course of
Biochemical determinants of infection
(leukocyte, neutrophil, and platelet counts,
I:T ratio) were normal. CRP was modestly
elevated (between 18.2 and 29.8 mg/L)
and thrombocytopenia (85 and 53x109/L)
was determined in two children. In both
cases, however, pneumocystosis coincided
with bacterial infection. Radiologic evidence
of interstitial pneumonia was obtained in
37 neonates. Diffuse infiltrates were seen in
Bacteriologically confirmed intrauterine
infection was diagnosed in 27 neonates of both
groups [positive blood cultures: Streptococcus
agalactiae (n=7), Enterococcus faecalis (n=5),
Escherichia coli (n=9), Staphylococcus aureus
(n=2), Staphylococcus epidermidis (n=4)].
Nosocomial infections were caused by Serratia
marcescens (n=5), Klebsiella pneumoniae (n=7),
Klebsiella oxytoca (n=8), and Staphylococcus
haemolyticus, methicillin-resistant Staphylococcus
aureus (MRSA) (n=12). Cultures were negative
in the remaining cases and the diagnosis
of bacterial infection was based on clinical
symptoms, characteristic laboratory findings,
and response to antibiotics (cephalosporins,
penicillin, aminoglycosides, imipenem,
vancomycin). Fungal sepsis caused by Candida
albicans (n=6) or Candida glabrata (n=6) was
diagnosed only in neonates with extremely low
birth weight chronically on antibiotics and was
managed with fluconazole.
Combined treatment with cotrimoxazole and
pentamidine was continued for 14 days in
most cases. Cotrimoxazole 40 mg/kg/24h
was administered in two doses. A single
4 mg/kg dose of pentamidine was infused during
30 minutes. Microscopic examination was
repeated after 7-10 days in five neonates,
disclosing the presence of P. jiroveci. Treatment
was continued and a negative result of
microscopy was demonstrated after 28 days.
Clinical improvement was noted in 38 neonates
after 8.7±2.2 days of therapy (range: 5 to
12 days). In spite of treatment and negative
results of microscopy, four neonates failed
to improve clinically, showing symptoms of
chronic lung disease or other pathology. Three
of these neonates died - one (2.4%) due to
massive pulmonary hemorrhage evidently
associated with pneumocystosis, and the other
two (mature) due to congenital defects.
|The risk of P. jiroveci infection is of particular
concern in immature or sick neonates. Infection
in a Neonatal Intensive Care Unit may be
sporadic among children with primary or
secondary immune deficiency but may also
take the form of an epidemic[25,26]. Sources of
infection remain unclear. Stringer reported
on long-term colonization by Pneumocystis
of immunocompetent hosts. Although
Pneumocystis DNA has been detected in air
samples, person-to-person transmission remains
the most probable route of infection. According
to Stringer and Dei-Cas[27,7], symptomatic
infection in adults may be the outcome of
colonization during infancy or early childhood
by genetically variant Pneumocystis strains.
Vargas et al. suggested that Pneumocystis
lacks the ability to colonize the airways. Thus,
PP would be the result of a recent infection.
Infection is airborne but the need for isolation
of infected patients remains disputable.|
Dutz et al. found pneumocystosis in infants
aged 10 to 24 weeks. In the present study,
symptoms of P. jiroveci infection appeared
on the average after 20 days from birth.
This rapid course of pneumocystosis may be
attributed to immaturity of our neonates and
of their immune system, surfactant deficiency,
or coexisting infection. Iatrogenic causes, like
protracted mechanical ventilation, antibiotics,
and steroids, may also be considered. The wellknown
predilection of P. jiroveci for cells of
the alveoli explains why respiratory symptoms
dominate. Growth of Pneumocystis in the
infected host is inhibited when the lung contains
normal amounts of surfactant and conversely, alterations in the composition and quantity
of surfactant contribute to PP. Congenital
deficiency of surfactant was noted in 90.5% of
neonates in our PP group and in only 43.2%
of the control group. Apparently, immaturityrelated
surfactant deficiency implicated in RDS,
as well as protracted mechanical ventilation and
oxygen supplementation (leading to BPD), are
important risk factors for PP.
Intermittent apnea was the first symptom of
PP. Copious amounts of frothy sputum are
the result of hypertrophy and proliferation
of type II alveolar cells and accumulation of
macrophages and eosinophils in the alveolar
lumen. Vargas et al. observed sudden infant
death associated with P. carinii infection.
Extrapulmonary symptoms include diarrhea and
weight loss. We observed feeding difficulties
and gastrointestinal disorders.
Pneumocystis pneumonia is usually managed
with cotrimoxazole, pentamidine, or both.
Dautzenberg et al. and Jules- Elysee
et al. reported on the effectiveness of
pentamidine inhalation. Berrington et al.found cotrimoxazole to be effective in all
50 infants with primary immune deficiency.
PP is a serious complication in severely ill
patients and is marked by a high mortality
rate. Identification of high-risk groups and
effective prevention remain part of the clinical
approach to PP. Cotrimoxazole was found
ineffective in preventing PP among HIVpositive
patients and children of HIV-positive
mothers[4,35].In our experience, administration
of cotrimoxazole to manage an unknown
infection will produce false-negative results in
bronchial fluid histopathology done to confirm
pneumocystosis. However, we always used
combination therapy to manage PP.
Our single and nested PCR proved of little
value for the diagnosis of P. carinii infection.
Sing et al. reviewed 27 papers on the diagnosis
of PP with traditional microscopy and PCR
applied to broncho-alveolar lavage fluid, but
failed to find any distinct advantage of PCR.
They demonstrated that single or nested PCR
has a marginal advantage over microscopy,
even in HIV-positive patients. Of greater
importance for the diagnosis of PP are the
quantity, quality, and moment of specimen
collection. Perhaps our disappointing results
with PCR were due to the small volume of
fluid that can be obtained from an immature
neonate. Nevez et al. suggested that the
lungs are colonized by too few P. jiroveci
organisms for a reliable application of PCR. It
should also be emphasized that Pneumocystis
strains demonstrate marked host specificity.
Until recently, the human pathogen P. jiroveci
was believed to be a strain of P. carinii[2,8].Durand-Joly et al. convincingly demonstrated
that transmission of pneumocystosis from
animal to man is impossible. Nevertheless, a
review of the literature revealed that the same
PCR protocol is used to detect P. carinii and
P. jiroveci[37,40]. One may presuppose that there
is no acceptable PCR protocol for the detection
of P. jiroveci and that the investigator is left
with sequencing as the only way to confirm or exclude amplicon specificity. Further advances
in this field await the development of a highly
sensitive and specific PCR method that will
provide for a rapid and reliable detection of
P. jiroveci DNA in bronchial lavage fluid from
premature neonates. When routinely available,
this test shall certainly facilitate the diagnosis
of P. jiroveci infection.
In conclusion, we believe that prematurity
and protracted mechanical ventilation are risk
factors for P. jiroveci infection in severely ill
neonates in an intensive care unit.
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