Humoral Immune Responses to Pneumocystis jirovecii Antigens in HIV-Infected and Uninfected Young Children with Pneumocystis Pneumonia
Walzer PD (2013) Humoral Immune Responses to Pneumocystis jirovecii Antigens in HIV-Infected and
Uninfected Young Children with Pneumocystis Pneumonia. PLoS ONE 8(12): e82783. doi:10.1371/journal.pone.0082783
Humoral Immune Responses to Pneumocystis jirovecii Antigens in HIV-Infected and Uninfected Young Children with Pneumocystis Pneumonia
Kpandja Djawe 0
Kieran R. Daly 0
Linda Levin 0
Heather J. Zar 0
Peter D. Walzer 0
Marta Feldmesser, Albert Einstein College of Medicine, United States of America
0 1 Department of Environmental Health, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America, 2 Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America, 3 Research Service, Veterans Affairs Medical Center , Cincinnati , Ohio, United States of America, 4 Department of Pediatrics and Child Health, University of Cape Town , Cape Town , South Africa
Background: Humoral immune responses in human immunodeficiency virus (HIV)-infected and uninfected children with Pneumocystis pneumonia (PcP) are poorly understood. Methods: Consecutive children hospitalized with acute pneumonia, tachypnea, and hypoxia in South Africa were investigated for PcP, which was diagnosed by real-time polymerase chain reaction on lower respiratory tract specimens. Serum antibody responses to recombinant fragments of the carboxyl terminus of Pneumocystis jirovecii major surface glycoprotein (MsgC) were analyzed. Results: 149 children were enrolled of whom 96 (64%) were HIV-infected. PcP occurred in 69 (72%) of HIV-infected and 14 (26%) of HIV-uninfected children. HIV-infected children with PcP had significantly decreased IgG antibodies to MsgC compared to HIV-infected patients without PcP, but had similar IgM antibodies. In contrast, HIV-uninfected children with PcP showed no change in IgG antibodies to MsgC, but had significantly increased IgM antibodies compared to HIV-uninfected children without PCP. Age was an independent predictor of high IgG antibodies, whereas PcP was a predictor of low IgG antibodies and high IgM antibodies. IgG and IgM antibody levels to the most closely related MsgC fragments were predictors of survival from PcP. Conclusions: Young HIV-infected children with PcP have significantly impaired humoral immune responses to MsgC, whereas HIV-uninfected children with PcP can develop active humoral immune responses. The children also exhibit a complex relationship between specific host factors and antibody levels to MsgC fragments that may be related to survival from PcP.
Funding: This work was supported by the National Institutes of Health Grant R01 HL090335 and the Department of Veterans Affairs, the National
Research Foundation, South Africa; an ASTRA-Zeneca Respiratory Award from the South African Thoracic Society and the Medical Research Council of
South Africa. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: ASTRA-Zeneca gave a research grant to the South African Thoracic Society (SATS) and the grant was administered by SATS.
ASTRA-Zeneca had nothing how SATS administered the grant or spent the money. ASTRA-Zeneca had nothing to do with any other relevant declarations
relating to employment, consultancy, patents, products in development, or marketed products etc). We also confirm that this grant does not alter our
adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in our guide for authors.
These authors contributed equally to this work.
Pneumocystis jirovecii is an opportunistic pulmonary
pathogen of worldwide distribution. Primary P. jirovecii infection
is acquired during the first few months of life, and is either
asymptomatic or a self-limited infection [1,2].
Seroepidemiological studies have shown that by 2-3 years of
age, most healthy children have been infected with the
organism [2-6]. P. jirovecii remains a major cause of
lifethreatening pneumonia (termed PcP) in children who are
immunocompromised by human immunodeficiency virus (HIV)
infection, cancer, or other disorders. This is especially true in
children in low or middle income countries where the disease
occurs in 10% to 49% of HIV-infected children hospitalized for
pneumonia with an in-hospital mortality rate of 20% to 63%
The diagnosis of PcP has traditionally been made by the
demonstration of the organism by histologic or
immunofluorescent staining in specimens that have been
carefully obtained from the respiratory tract. It is likely that this
method underestimates the true incidence of PcP, particularly
in areas with limited laboratory facilities [1-14]. Recent studies
in adults with and without PcP have shown that the polymerase
chain reaction (PCR), particularly real time (RT)-PCR, is more
sensitive than microscopy in detecting P. jirovecii and may also
distinguish colonization from active disease [15-21]. We have
obtained similar results with the use of RT-PCR in the
diagnosis of PcP and tuberculosis in young children [22-24].
HIV-infected children with PcP have markedly decreased
CD4+ cell counts and broad defects in cellular and humoral
function, as illustrated by their low serum antibody levels to
common infectious agents and to immunizations [25-28].
HIVuninfected children, who are exposed to HIV but remain
HIVnegative, have been reported to be at greater risk for
developing PcP than HIV-uninfected, unexposed children
[29,30]; however, the reasons for the difference are unclear
Little is known about the role of specific immune responses
to P.jirovecii in HIV-infected children with or without PcP. Over
the past decade, the development of recombinant antigens has
begun to change this picture. The major surface glycoprotein
(Msg) of P.jirovecii plays a central role in the interaction of the
organism with the host; contains protective B and T cell
epitopes; is encoded by a multi-gene family, and is capable of
antigenic variation [35-39]. We have developed 3 recombinant
fragments (MsgA, MsgB, MsgC1) that span the length of a
single Msg isoform , and analyzed their reactivity in both
adult and pediatric populations [6,40-48]. MsgC1, which
contains the carboxyl terminus and is the most conserved part
of Msg, showed the most promise; thus, 3 variants (MsgC3,
MsgC8, and MsgC9) were developed to better characterize the
reactivity of the antibodies [40-48].
The aims of this study were: 1) to characterize the IgG and
IgM antibody responses to MsgC fragments in HIV-infected
and HIV-uninfected children hospitalized with PcP (PcP+) and
other causes of pneumonia (PcP-); 2) to identify specific host
factors that are independent predictors of these antibody
levels; 3) to determine if any of the antibody responses are
independent predictors of mortality from PcP.
Materials and Methods
A prospective study was conducted of consecutive children
admitted to the Red Cross War Memorial Childrens Hospital,
Cape Town, South Africa, with hypoxic pneumonia from Nov
2006 to Aug 2008 . Clinical criteria for suspected PCP were
an acute onset (<2 weeks) of a respiratory illness; the presence
of age-specific tachypnea and hypoxia (arterial oxygen
saturation <92% in room-air); bilateral lung disease (not
associated with wheezing); and the presence of a risk factor for
PCP (e.g. HIV-infection, malnutrition, immunosuppressive
therapy). These criteria were established to ensure that
subjects were seriously ill with pneumonia and had significant
risk factors for PCP. Initial specimens were obtained within the
first 48 hours of admission. Exclusion criteria included
treatment of PcP in the preceding 2 weeks or treatment for
PCP for the current admission for more than 48 hours.
Blood specimens were collected at enrollment for HIV testing
(if status was unknown), CD4 cell measurement, and a serum
specimen was frozen for further analysis. A child was defined
as HIV-infected if he/she had a positive HIV PCR (Roche) and
was younger than 18 months or a positive HIV ELISA for
antibodies (Abbott) in older children. HIV exposure was defined
as HIV-seropositive by ELISA and negative by HIV PCR.
Upper respiratory tract (URT) specimens were obtained from
nasopharyngeal aspirates (NPAs) and lower respiratory tract
(LRT) specimens by induced sputum (IS) or bronchoalveolar
lavage (BAL) in a standard manner within 48 hours of
admission. The specimens were examined for the presence of
P. jirovecii by RT-PCR and microscopic techniques (direct
immunofluorescence (IF) using a monoclonal antibody, silver
staining) and other organisms as described (23). The data
showed that RT-PCR was far more sensitive than microscopic
techniques in detecting P. jirovecii with good specificity. Other
organisms that were found included viruses (e.g.
cytomegalovirus (CMV), respiratory viruses) and bacteria (e.g.
Staphylococcus aureus, Mycobacterium tuberculosis).
Treatment of PcP included trimethoprim-sulfamethoxazole
(TMP-SMX) and corticosteroids; other antimicrobial drugs, or
antiretroviral drugs were at the discretion of the treating
physician. The overall in-hospital mortality rate was 25%, and
the case-fatality rate was also significantly higher in PcP
patients (40%) than in non-PcP patients (21%). Written
informed consent was obtained from a parent or legal guardian.
Ethical approval of the study was obtained from the Research
and Ethics Committee of the Faculty of Health Sciences,
University of Cape Town and the University of Cincinnati
Institutional Review Board
The DNA fragments containing genes encoding recombinant
MsgC1, C3, C8, and C9 fragments were prepared via PCR
using DNA isolated from P. jirovecii- infected lung or cloned
msg genes as templates as described [40-42]. Each of the 4
MsgC fragments included approximately 425 amino acids. The
fragments exhibited 83 to 99% homology at the nucleotide level
and 77 to 99% homology at the putative amino acid level .
MsgC3 and MsgC9 were the most closely related fragments
with just 3 amino acid substitutions.
The IgG ELISA was performed as previously described
[40-42]. Serum specimens to be analyzed and the standard
reference serum were tested against each MsgC fragment. The
standard specimens were obtained by testing banks of sera
from blood donors and HIV-infected patients. The standard
serum for each antigen consisted of a pool of 4 to 6 serum
specimens with high antibody reactivity to that antigen. The
standard serum was defined as having a value of 100 U in 100
l of a 1:100 dilution. We used the same standard pools
throughout the study, and as a further measure to ensure
consistency between assays, we titrated subsequent standard
pools against those initial standards. From the standard pool,
we generated a standard curve for each Msg construct on each
day the assay was used. We used this curve to calculate the
units of reactivity to the Msg construct. We diluted test serum
samples at 1:100 to 1:200 to fit the linear portion of the curves.
Taking into account the dilution, we then calculated units of
IgM antibodies were analyzed in a similar manner except
that an anti-IgM (-specific) antibody was used . Variations
in the assay results were analyzed using a control serum and
were measured for MsgC1 on a per-plate, daily, and 4 day
basis; the coefficients of variance (CV) were 3.6 to 7%, 4.8 to
7.4%, and 13.3%, respectively .
Data Analysis and Statistics
In the first part of the analysis, we characterized the children
as follows: HIV-infected with PcP (PcP+), HIV-infected with
other causes of pneumonia (PcP-), HIV-uninfected with PcP
(HIV-uninfected/PcP+), and HIV-uninfected without PcP
(HIVuninfected/PcP-). We used medians (interquartile range) or
counts (percent) to describe continuous and discrete
characteristics, respectively. In the second part of the analysis,
quantile regression analysis was employed to compare
antibody levels among groups and to determine independent
risk factors associated with antibody levels. In our previous
publications, means of log-transformed antibody responses to
Msg fragments were modeled using Tobit regression, as the
transformed uncensored responses approximated a truncated
lognormal distribution [6,41-45]. In the present study, however,
although some subjects had antibody levels below the limit of
detection and the values were censored to 1, we chose to use
Quantile regression. This strategy was primarily due to the
large number of extremely high antibody responses which
precluded modeling the mean response, even after
logtransformation. Quantile regression is a non-parametric method
which models the relation between a set of independent
variables and conditional quantiles (percentiles) of the
dependent variable . Quantile regression has also been
used to analyze immunological data with high frequency of
undetectable results or non-detects . Eilers et al. showed
that quantile regression permitted groups to be compared and
meaningful linear trends could be computed even though more
than 50% of the data was composed of non-detects. In the
present study, 29% of antibody responses to MsgC1, 32% to
MsgC3, 16% to MsgC8, and 19% to MsgC9 were censored.
Since non-censored antibody levels in the present study
were in the upper quartiles, we compared the groups in the
75th and 90th quantiles, and then determined the factors
associated with these conditional percentiles. In the final stage
of the analysis, we determined predictors of PCP-related
mortality using Cox proportional hazard models. The analysis
included HIV+ children with PCP and HIV- children with PCP.
SAS for Windows, version 9.2 (SAS Institute, Cary, NC) was
used to carry out all statistical analyses, and a 5% significance
level was assumed, unless stated otherwise.
Demographic and Clinical Characteristics
Of the 202 children originally enrolled (17), 149 (73%) were
included in the present study: 96 (64%) were HIV-infected of
whom 69 (72%) had PcP+ (Table 1). These children differed
significantly from the HIV-infected/PcP- children in their
younger age; lower proportion receiving PcP prophylaxis;
greater level of immunosuppression as measured by CD4+ cell
count; and greater lung damage as evidenced by higher LDH
levels. There was also a trend towards higher mortality.
Of the 53 HIV-uninfected patients, 21 (40%) were
HIVexposed. Of the exposed patients, only 3 (14%) had PcP,
whereas 11 (34%) of the 32 unexposed patients had PcP.
There were no significant differences in demographic or clinical
characteristics between HIV-uninfected/PcP+ and
HIVuninfected/PcP- children (Table 1).
HIVAntigens Percentile PcP+ (N=69) PcP- (N=27) PcP+ (N=14) PcP- (N=39)
MsgC1 75th 1.00 [0.0] 76.78 [16.0] 3.35 [17.2] 14.35 [12.0]
MsgC1 90th 3.88*[6.4] 121.59* [17.0] 23.97 [46.3] 76.01[25.2]
MsgC3 75th 1.00*[2.3] 174.27*[62.2] 24.27 [48.9] 4.37 [2.8]
MsgC3 90th 18.53* [18.0] 250.90* [55.6] 141.26 [72.7] 32.14 [27.2]
MsgC8 75th 1.00 [0.0] 21.77 [49.1] 3.85 [20.3] 1.00 [0.4]
MsgC8 90th 1.00* [8.0] 287.47* [130.8] 36.25 [53.0] 4.77 [16.0]
MsgC9 75th 1.00 [0.3] 3.10 [31.9] 16.10 [55.2] 1.00 [2.5]
MsgC9 90th 8.86 [10.9] 181.03 [134.9] 153.89 [79.7] 13.27 [12.9]
* The groups are significantly different. SE = Standard error. HIV+ = HIV-infected.
HIV- = HIV-uninfected
HIVAntigens Percentile PcP+ (N=69) PcP- (N=27) PcP+ (N=14) PcP- (N=39)
MsgC1 75th 49.54 [10.7] 50.97 [42.3] 109.64 [41.0] 20.66 [9.0]
MsgC1 90th 100.72 [14.0] 202.93 [91.3] 126.94 [19.0] 88.60 [41.1]
MsgC3 75th 10.76 [3.6] 9.55 [2.7] 13.21 [10.1] 1.48 [1.8]
MsgC3 90th 27.65 [5.6] 18.62 [6.9] 36.15 [9.7] 7.66 [2.6]
MsgC8 75th 10.84 [3.8] 9.70 [4.1] 18.27 [9.7] 1.21 [0.9]
MsgC8 90th 27.65 [4.2] 15.80 [12.3] 33.02 [9.9] 5.37 [6.3]
MsgC9 75th 6.41 [2.2] 5.60 [1.6] 18.72* [11.3] 4.06* [1.8]
MsgC9 90th 17.73 [3.3] 10.02 [2.4] 25.20 [31.7] 8.14 [3.5]
* The groups are significantly different. SE = Standard error. HIV+ = HIV-infected.
HIV- = HIV-uninfected SE:
Serum IgG and IgM Antibody Levels
HIV-infected/PcP+ patients had significantly lower IgG
antibody levels than HIV-infected/PcP- patients to MsgC1 in
the 90th percentile; to MsgC3 in the 75th and 90th percentiles;
and to MsgC8 in the 90th percentile (Table 2). In contrast, no
significant differences were seen in IgG antibody levels to the
MsgC fragments between HIV-uninfected/PcP+ and
HIVuninfected/PcP- children or between HIV-infected/PcP+ and
HIV-uninfected/PcP+ children (Table 2). There were also no
significant differences in IgG antibody levels between all
HIVinfected and all HIV-uninfected children (data not shown).
No significant differences were found in IgM antibody levels
to any of the MsgC constructs among HIV-infected/PcP+ or
HIV-infected/PcP- subjects (Table 3). By contrast,
HIVuninfected/PcP+ patients had significantly higher IgM antibody
levels than HIV-uninfected/PcP- patients to MsgC1, C8, and C9
in the 75th percentile (Table 3). We were unable to compare
antibody levels between HIV-uninfected, exposed, PcP+ and
PcP- children due to the small number (3) of PcP+ children.
Predictors of IgG and IgM Antibody Responses
Regression analyses were performed to find correlations
between host factors and antibody responses. Of the IgG
antibody responses, age was a predictor of high antibody
levels to MsgC1 in the 75th percentile and PcP was a predictor
of low antibody levels to MsgC1 in the 75th and the 90th
percentiles (Table 4). Age was also a predictor of high antibody
levels to MsgC3, MsgC8, and MsgC9 in the 75th percentiles. Of
the IgM antibody responses, PcP was a predictor of high
antibody levels to MsgC3 in the 75th and 90th percentiles, and to
MsgC9 in the 90th percentile (Table 5).
IgG and IgM Antibody Responses as Predictors of
Antibody levels to each MsgC construct, weight, and age
were analyzed as predictors of PcP-related mortality using HIV
+ with PcP and HIV- with PcP and the results were expressed
as the Adjusted Mortality Risk Ratios (RR). Of the IgG
antibodies, only the antibody level to MsgC3 approached
significance: RR 0.58, 95%CI [0.34-1.00] p= 0.05. Of the IgM
antibodies, only the antibody level to MsgC9 was significant:
RR 0.64, 95% CI [0.41-0.99] p< 0.05. Thus, the IgG antibody
level to MsgC3 and IgM antibody level to MsgC9 were
associated with a 36.7% and 39% decrease in PcP-related
mortality, respectively. In the sensitivity analysis using only HIV
+ children with PCP, the effect of CD4+ cell count on mortality
was not statistically significant.
This study has shown that hospitalized HIV-infected children
with PcP (PcP+) had significantly lower serum IgG antibodies
to recombinant MsgC fragments than HIV-infected children with
pneumonia due to other causes (PcP-). By contrast,
HIVuninfected PcP+ children had significantly higher IgM
antibodies to these fragments than HIV-uninfected PcP
patients. Age was a predictor of high IgG antibodies, whereas
PcP was a predictor of low IgG antibodies but high IgM
antibodies. IgG antibody levels to MsgC3 and IgM antibody
levels to MsgC9 also were associated with a reduced mortality
Studies of the humoral and cellular immune responses to a
specific respiratory pathogen in any patient with pneumonia
depend on accurate etiological diagnosis. Recent reports
showing that RT-PCR is more sensitive than microscopic
analysis in diagnosing PcP in adults [15-21] and children 
are important because they increase the number of subjects
available for analysis and can also distinguish P. jiroveci
colonization from disease. HIV-infected children not only have
broad immune defects ranging from low CD4 counts to B-cell
dysregulation with hyperimmunoglobulinemia, but also have
poor antibody responses to specific infectious agents or
immunizations [25-28,51,52]. Impaired placental transfer of
antibodies from HIV-infected mothers is an important
contributor to these poor specific antibody responses, as has
been shown with viral, bacterial, and parasitic antigens [53,54].
Since maternal antibodies are the main source of protective
humoral immune responses during the first 6 months of life, it is
not surprising that many of these infections occur during this
Our HIV-infected/PcP+ children exhibited many of the
characteristics described above, including low CD4+ cell
counts, development of PcP at age 3-4 months, severe
disease, elevated LDH levels, low fraction of patients receiving
chemoprophylaxis, and a high mortality rate. These children
also had very low serum IgG antibody levels to the MsgC
fragments, and were unable to develop an active IgM antibody
responses that could distinguish them from
In contrast to these young HIV-infected children, adult
HIVinfected patients with active PcP or a previous episode of PcP
whom we have studied display higher antibody levels to the
MsgC fragments than HIV-infected patients who never had PcP
or healthy adults . HIV-infected adults hospitalized with
active PcP had significantly higher levels of IgM and IgG
antibody levels than HIV-infected adults hospitalized with other
causes of pneumonia at the time of diagnosis, and the
differences in antibody levels were maintained until 3-4 weeks
later . The positive predictive values (PPV) for IgG and IgM
antibody levels rose from 71.5% and 79.3% at admission to
100% and 89.8%, respectively, at 3-4 weeks. It is likely that
most HIV-infected adults have fully developed immune systems
and repeated exposures to P. jirovecii throughout their lives
before they develop PcP, whereas young HIV-infected children
have immature immune systems and lesser cumulative
exposure to P. jirovecii.
The MsgC fragments used in the present and previous
reports exhibited a high degree homology, and thus have
shared as well as unique antigenic determinants. If one MsgC
fragment elicits a good antibody response, other fragments
also usually elicit good responses; on the other hand, the
MsgC fragments react independently and also have unique
epitopes (Refs 41-43). Our previous studies in HIV-infected
adults have identified specific host and environmental factors
that are independent predictors of antibody levels to one or
more of the MsgC fragments [42-44]. Thus, current PcP,
previous episode of PcP, age, failure to take PcP
chemoprophylaxis, and geographic location have been
associated with increased IgG and /or IgM antibody levels,
whereas smoking and high LDH levels have been associated
with decreased IgG and/or IgM antibody levels [6,43-45].
The present study showed age was independently
associated with increased IgG antibody levels, but not to IgM
antibody levels. It is likely that similar to antibody responses to
other commonly encountered organisms, IgG antibody levels
increase on repeated exposure to P. jirovecii antigens, as
children grow and mature. On the other hand, PcP was
associated with low IgG antibody levels to MsgC1, which
probably reflects the decreased IgG antibody levels that are
maternally derived in the HIV-infected/PcP+ patients. The
association of PcP with high IgM antibody levels to MsgC3 and
MsgC9 probably reflects the antibody response to PcP in the
HIV-uninfected/PcP+ children and the fact that MsgC3 and
MsgC9 are closely related.
HIV-uninfected exposed children in the present study had
similar, though less severe immune defects (including antibody
responses to specific antigens) than HIV-infected children
[31-34]. Low antibody levels in HIV-infected mothers and
decreased placental transmission of antibodies in HIV-infected
exposed infants have played an important role. Yet, when
tested at age 16 months following vaccination, antibody
responses in HIV- uninfected exposed children were as high as
or higher than those in the unexposed children .
Our HIV-uninfected/PcP+ children had serum IgG antibody
levels to the MsgC fragments that were similar to the antibody
levels in HIV-uninfected/PcP- patients. However, the
HIVuninfected/PcP+ children exhibited significantly greater IgM
antibody responses to the MsgC fragments than the
HIVuninfected/PcP- children. This finding suggests that
HIVuninfected children can develop an active antibody response to
PcP. We were surprised to find that only 3 (14%)
HIVuninfected, exposed children developed PcP, compared with
11 (34%) of unexposed children. The reasons for this are
unclear, but risk factors other than HIV infection may have
played a role in the development of PcP. Case reports have
suggested that HIVuninfected exposed children are also at
increased risk for the development of PcP or other lower
respiratory infections [29,30,34].
Some experimental studies have shown that Msg contains
protective B and T cell antigenic determinants [36-38], but
other reports using different models did not confirm these
observations . A recent prospective study we conducted of
550 adult HIV-infected and HIV-uninfected patients hospitalized
with cough >2 weeks in Kampala, Uganda showed that lower
serum IgM antibody responses to MsgC3 and MsgC8 were
both associated with increased in-hospital mortality (48). In the
current study, we found that increased IgG antibody levels to
MsgC3 and IgM antibody levels to MsgC9 were associated with
a reduction in PcP-related mortality. Taken together, the results
of these two studies have led us to hypothesize that these
antibodies are protective or contribute to the protection from
This study has several limitations. We did not determine
whether the defect in PcP antibodies was limited to this
organism or whether it was part of a broader global defect in
antibody production associated with HIV infection. Further
studies of the functional properties of these antibodies and of
the serologic responses to other infectious agents are needed.
Another limitation of this study is that it is a cross-sectional
study. Longitudinal studies in HIV-infected children are needed
to better understand the immune responses to this organism
over time, and the factors that affect these responses. The
present report only analyzed systemic antibody responses.
Further analysis of local (respiratory tract) antibody responses
and interactions between native Msg variants present in the
cell wall of P. jirovecii would be of interest. Although we found
RT-PCR to be sensitive and specific for PcP, further studies in
other pediatric populations are needed. The small number of
HIV-uninfected, exposed children with PcP precluded
evaluation of the effects of HIV exposure on development of
PcP. Additional investigation of larger numbers of these
individuals would be very helpful.
In summary, this study provides new information about the
humoral immune responses of young HIV-infected and
HIVuninfected children to P. jirovecii antigens, and suggests future
areas for investigation.
Conceived and designed the experiments: KD KRD LL HJ PW.
Performed the experiments: KD KRD HJ. Analyzed the data:
KD KRD LL HJ PW. Contributed reagents/materials/analysis
tools: KRD LL HJ PW. Wrote the manuscript: KD LL HJ PW.
1. Larsen HH , von Linstow ML , Lundgren B , Hgh B , Westh H et al. ( 2007 ) Primary pneumocystis infection in infants hospitalized with acute respiratory tract infection . Emerg Infect Dis 13 : 66 - 72 . doi:10.3201/ eid1301.060315. PubMed: 17370517 .
2. Vargas SL , Hughes WT , Santolaya ME , Ulloa AV , Ponce CA et al. ( 2001 ) Search for primary infection by Pneumocystis carinii in a cohort of normal, healthy infants . Clin Infect Dis 32 : 855 - 861 . doi: 10.1086/319340. PubMed: 11247708 .
3. Meuwissen JH , Tauber I , Leeuwenberg AD , Beckers PJ , Sieben AD ( 1977 ) Parasitologic and serologic observations of infection with Pneumocystis in humans . J Infect Dis 136 ( 1 ): 43 - 49 . PubMed: 328785 .
4. Pifer LL , Hughes WW , Stagno S , Woods D. ( 1978 ) (200l) Pneumocystis carinii infection: evidence for high prevalence in normal and immunosuppressed children . Pediatrics 61 ( 1 ): 35 - 41 . PubMed: 400818 .
5. Peglow SL , Smulian AG , Linke MJ , Crisler J , Phair JWM et al. ( 1990 ) Serologic responses to Pneumocystis carinii antigens in health and disease . J Infect Dis 161 ( 2 ): 296 - 306 . doi:10.1093/infdis/161.2.296. PubMed: 2299209 .
6. Djawe K , Daly K , Vargas S , Santolaya ME , Ponce CA et al. ( 2010 ) Seroepidemiological study of Pneumocystis jirovecii infection in healthy infants in Chile using recombinant fragments of the P. jirovecii major surface glycoprotein . Int J Infect Dis 14 ( 12 ): e1060 - 6 .
7. Zar HJ , Dechaboon A , Hanslo D , Apolles P , Magnus KG et al. ( 2000 ) Pneumocystis carinii pneumonia in South African children infected with human immunodeficiency virus . Pediatr Infect Dis J 19 : 603 - 607 . doi: 10.1097/ 00006454 - 200007000 -00004. PubMed: 10917216 .
8. Graham SM , Mtitimila EL , Kamanga HS , Walsh AL , Hart CA et al. ( 2000 ) Clinical presentation and outcome of Pneumocystis carinii pneumonia in Malawian children . Lancet 355 ( 9201 ): 369 - 373 . doi: 10.1016/ S0140-6736(98)11074-7 . PubMed: 10665557 .
9. Ruffini DD , Madhi SA ( 2002 ) The high burden of Pneumocystis carinii penumonia in African HIV-1-infected children hospitalized for severe pneumonia . AIDS 16 ( 1 ): 105 - 112 . doi: 10.1097/ 00002030 - 200201040 -00013. PubMed: 11741168 .
10. Madhi SA , Cutland C , Ismail K , O'Reilly C , Mancha A et al. ( 2002 ) Ineffectiveness of trimethoprim-sulfamethoxazole prophylaxis and the importance of bacterial and viral cocoinfections in African children with Pneumocystis carinii pneumonia . Clin Infect Dis 35 ( 9 ): 1120 - 1126 . doi: 10.1086/343049. PubMed: 12384847 .
11. Wonodi CB , Deloria-Knoll M , Feikin DR , DeLuca AN , Driscoll AJ et al. ( 2012 ) Pneumonia Methods Working Group and PERCH Site Investigators . Evaluation of risk factors for severe pneumonia in children: the Pneumonia Etiology Research for Child Health Study . Clin Infect Dis 54 Suppl 2 : S124 - S131 . doi:10.1093/cid/cir1067. PubMed: 22403226 .
12. Scott JA , Wonodi C , Mosi JC , Deloria-Knoll M , DeLuca AN et al. ( 2012 ) Pneumonia Methods Working Group . The definition of pneumonia, the assessment of severity, and clinical standardization in the Pneumonia Etiology Research for Child Health study . Clin Infect Dis 54 Suppl 2 : S109 - S116 . doi:10.1093/cid/cir1065. PubMed: 22403224 .
13. Murdoch DR , O'Brien KL , Driscoll AJ , Karron RA , Bhat N ( 2012 ) Pneumonia Methods Working Group; PERCH Core Team. Laboratory methods for determining pneumonia etiology in children . Clin Infect Dis 54 Suppl 2 : S146 - S152 . doi:10.1093/cid/cir1073. PubMed: 22403229 .
14. Bhat N , O'Brien KL , Karron RA , Driscoll AJ , Murdoch DR ( 2012 ) Pneumonia Methods Working Group. Use and evaluation of molecular diagnostics for pneumonia etiology studies . Clin Infect Dis 54 Suppl 2 : S153 - S158 . doi:10.1093/cid/cir756. PubMed: 22403230 .
15. Huggett JF , Taylor MS , Kocjan G , Evans HE , Morris-Jones S et al. ( 2008 ) Development and evaluation of a real-time PCR assay for detection of Pneumocystis jirovecii DNA in bronchoalveolar lavage fluid of HIV-infected patients . Thorax 63 ( 2 ): 154 - 159 . PubMed: 17693588 .
16. Alanio A , Desoubeaux G , Sarfati C , Hamane S , Bergeron A et al. ( 2011 ) Real-time PCR assay-based strategy for differentiation between active Pneumocystis jirovecii pneumonia and colonization in immunocompromised patients . Clin Microbiol Infect 17 ( 10 ): 1531 - 1537 . doi:10.1111/j.1469- 0691 . 2010 .03400.x. PubMed: 20946413.
17. McTaggart LR , Wengenack NL , Richardson SE ( 2012 ) Validation of the MycAssay Pneumocystis kit for detection of Pneumocystis jirovecii in bronchoalveolar lavage specimens by comparison to a laboratory standard of direct immunofluorescence microscopy, real-time PCR, or conventional PCR . J Clin Microbiol 50 ( 6 ): 1856 - 1859 . doi:10.1128/JCM. 05880- 11 . PubMed: 22422855 .
18. Tia T , Putaporntip C , Kosuwin R , Kongpolprom N , Kawkitinarong K et al. ( 2012 ) A highly sensitive novel PCR assay for detection of Pneumocystis jirovecii DNA in bronchoalveloar lavage specimens from immunocompromised patients . Clin Microbiol Infect 18 ( 6 ): 598 - 603 . doi: 10.1111/j.1469- 0691 . 2011 .03656.x. PubMed: 21951463.
19. Seah C , Richardson SE , Tsui G , Yu B , Thornback J et al. ( 2012 ) Comparison of the FXG: RESP (Asp+) real-time PCR assay with direct immunofluorescence and calcofluor white staining for the detection of Pneumocystis jirovecii in respiratory specimens . Med Mycol 50 ( 3 ): 324 - 327 . doi:10.3109/13693786.2011.598878. PubMed: 21859386 .
20. Botterel F , Cabaret O , Foulet F , Cordonnier C , Costa JM et al. ( 2012 ) Clinical significance of quantifying Pneumocystis jirovecii DNA by using real-time PCR in bronchoalveolar lavage fluid from immunocompromised patients . J Clin Microbiol 50 ( 2 ): 227 - 231 . doi: 10.1128/JCM.06036- 11 . PubMed: 22162560 .
21. Fillaux J , Berry A ( 2013 ) Real-time PCR assay for the diagnosis of Pneumocystis jirovecii pneumonia . Methods Mol Biol 943 : 159 - 170 . doi: 10.1007/978- 1 - 60327 - 353 -4_ 11 . PubMed: 23104289 .
22. Morrow BM , Hsaio NY , Zampoli M , Whitelaw A , Zar HJ ( 2010 ) Pneumocystis pneumonia in South Aftican children with and without human immunodeficiency virus infection in the era of highly active antiretroviral . Pediatr Infect Dis J 29 : 535 - 539 . PubMed: 20072079 .
23. Samuel CM , Whitelaw A , Corcoran C , Morrow B , Hsiao NY et al. ( 2011 ) 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 . BMC Infect Dis 11 : 329. doi:10.1186/ 1471 - 2334 - 11 -329. PubMed: 22123076 .
24. Zar HJ , Workman L , Isaacs W , Munro J , Black F et al. ( 2012 ) Rapid molecular diagnosis of pulmonary tuberculosis in children using nasopharyngeal specimens . Clin Infect Dis 55 ( 8 ): 1088 - 1095 . doi: 10.1093/cid/cis598. PubMed: 22752518 .
25. Eley BS , Hughes J , Potgieter S , Keraan M , Burgess J et al. ( 1999 ) Immunological manifestations of HIV-infected children . Ann Trop Paediatr 19 : 3 - 7 . PubMed: 10605514 .
26. Lyamuya EF , Matee MI , Kasubi M , Scheutz F ( 1999 ) Immunoglobulin profile in HIV-1 infected children in Dar es Salaam . East Afr Med J 76 : 370 - 375 . PubMed: 10520363 .
27. Bernstein LI , Ochs HD , Wedgwood RJ , Rubinstein A ( 1985 ) Defective humoral immunity in pediatric acquired immune deficiency syndrome . J Pediatr 107 : 352 - 357 . doi:10.1016/ S0022-3476(85)80505-9 . PubMed: 4032129 .
28. Nair N , Moss WJ , Scott S , Mugala N , Ndhlovu ZM et al. ( 2009 ) HIV-1 infection in Zambian children impairs the development and avidity maturation of measles virus-specific immunoglobulin G after vaccination and infection . J Infect Dis 200 : 1031 - 1038 . doi: 10.1086/605648. PubMed: 19702505 .
29. Chintu C , Mudenda V , Lucas S , Nunn A , Lishimpi K et al. ( 2002 ) Lung diseases at necropsy in African children dying from respiratory illnesses: a descriptive necropsy study . Lancet 360 : 985 - 990 . PubMed: 12383668 .
30. McNally LM , Jeena PM , Lalloo U , Nyamande K , Gajee K et al. ( 2005 ) Probable mother to infant transmission of Pneumocystis jiroveci from an HIV-infected woman to her HIV-uninfected infant . AIDS 19 : 1548 - 1549 . doi:10.1097/01.aids. 0000183941 .67730.3a. PubMed: 16135912.
31. Blanche S , Rouzioux C , Moscato ML , Veber F , Mayaux MJ et al. ( 1989 ) A prospective study of infants born to women seropositive for human immunodeficiency virus type 1 . N Engl J Med 320 : 1643 - 1648 . doi: 10.1056/NEJM198906223202502. PubMed: 2657430 .
32. De Martino M , Tovo PA , Galli L , Gabiano C , Cozzani S et al. ( 1991 ) Prognostic significance of immunologic changes in 675 infants perinatally exposed to human immunodeficiency virus . J Pediatri 119 : 702 - 709 . doi:10.1016/S0022-3476(05)80283- 5 .
33. Bunders M , Pembrey L , Kuijpers T , Newell ML ( 2010 ) Evidence of impact of maternal HIV infection on immunoglobulin levels in HIVexposed uninfected children . AIDS Res Hum Retroviruses 26 : 967 - 975 . doi:10.1089/aid.2009.0241. PubMed: 20718630 .
34. Jones CE , Naidoo S , De Beer C , Esser M , Kampmann B et al. ( 2011 ) Maternal HIV infection and antibody responses against vaccinepreventable diseases in uninfected infants . JAMA 305 : 576 - 584 . doi: 10.1001/jama.2011.100. PubMed: 21304083 .
35. Walzer PD ( 1999 ) Immunological features of Pneumocystis carinii infection in humans . Clin Diagn Lab Immunol 6 : 14 - 55 . PubMed: 9874657 .
36. Kovacs JA , Powell F , Edman JC , Lundgren B , Martinez A et al. ( 1993 ) Multiple genes encode the major surface glycoprotein of Pneumocystis carinii . J Biol Chem 268 ( 8 ): 6034 - 6040 . PubMed: 8449961 .
37. Gigliotti F , Hughes WT ( 1988 ) Passive immunoprophylaxis with specific monoclonal antibody confers partial protection against Pneumocystis carinii pneumonia in animal models . J Clin Invest 81 : 1666 - 1668 . doi: 10.1172/JCI113503. PubMed: 2454947 .
38. Theus SA , Andrews RP , Steele P , Walzer PD ( 1995 ) Adoptive transfer of lymphocytes sensitized to the major surface glycoprotein of Pneumocystis carinii confers protection in the rat . J Clin Invest 95 : 2587 - 2593 . doi:10.1172/JCI117960. PubMed: 7769101 .
39. Theus SA , Smulian AG , Steele P , Linke MJ , Walzer PD ( 1998 ) Immunization with the major surface glycoprotein of Pneumocystis carinii elicits a protective response . Vaccine 16 : 1149 - 1157 . doi: 10.1016/ S0264-410X(98)80113-8 . PubMed: 9682373 .
40. Stringer JR ( 2007 ) Antigenic variation in Pneumocystis . J Eukaryot Microbiol 54 : 8 - 13 . doi:10.1111/j.1550- 7408 . 2006 .00225.x. PubMed: 17300510.
41. Daly KR , Koch J , Levin L , Walzer PD ( 2004 ) Enzyme-linked immunosorbent assay and serologic responses to Pneumocystis jiroveci . Emerg Infect Dis 10 : 848 - 854 . doi:10.3201/eid1005.030497. PubMed: 15200818 .
42. Daly KR , Koch JV , Shire NJ , Levin L , Walzer PD ( 2006 ) Human immunodeficiency virus-infected patients with prior Pneumocystis pneumonia exhibit increased serologic reactivity to several major surface glycoprotein clones . Clin Vaccine Immunol 13 : 1071 - 1078 . doi: 10.1128/CVI.00140- 06 . PubMed: 17028210 .
43. Daly K , Koch J , Respaldiza N , De la Horra C , Montes-Cano MA et al. ( 2009 ) Geographical variation in serological responses to Pneumocystis jirovecii major surface glycoprotein antigens . Clin Microbiol Infect 15 : 937 - 942 . doi:10.1111/j.1469- 0691 . 2009 .02716.x. PubMed: 19416292.
44. Walzer PD , Djawe K , Levin L , Daly KR , Koch J et al. ( 2009 ) Long-term serologic responses to the Pneumocystis jirovecii major surface glycoprotein in HIV-positive individuals with and without P. jirovecii infection . J Infect Dis 199 : 1335 - 1344 . doi:10.1086/597803. PubMed: 19301979 .
45. Gingo MR , Lucht L , Daly KR , Djawe K , Norris KA et al. ( 2011 ) Serologic responses to pneumocystis proteins in HIV patients with and without Pneumocystis jirovecii penumonia . J Acquir Immune Defic Syndr 57 : 190 - 196 . doi:10.1097/QAI.0b013e3182167516. PubMed: 21372726.
46. Djawe K , Huang L , Daly KR , Levin L , Koch J et al. ( 2010 ) Serum antibody levels to the Pneumocystis jirovecii major surface glycoprotein in the diagnosis of P. jirovecii pneumonia in HIV-infected patients . PLOS ONE 5: e14259. doi:10.1371/journal.pone.0014259. PubMed: 21151564.
47. Tipirneni R , Daly KR , Jarlsberg LG , Koch JV , Swartzman A et al. ( 2009 ) Healthcare worker occupation and immune response to Pneumocystis jirovecii . Emerg Infect Dis 15 : 1590 - 1597 . doi:10.3201/ eid1510.090207. PubMed: 19861050 .
48. Crothers K , Justice A , Daly K , Rimland D , Goetz M et al. ( 2011 ) Decreased serum antibody response to recombinant pneumocystis antigens in HIV-infected and uninfected current smokers . Clin Vaccine Immunol 18 : 380 - 386 . doi:10.1128/CVI.00421- 10 . PubMed: 21191078 .
49. Seymour J , McNamee P , Scott A , Tinelli M ( 2010 ) Shedding new light onto the ceiling and floor? A quantile regression approach to compare EQ-5D and SF-6D responses . Health Econ 10 : 683 - 696 .
50. Paul HC , Esther R , Huub FJ , Roy GV ( 2012 ) Quantile regression for the statistical analysis of immunological data with many non-detects . BMC Immunol 7;13:37
51. Blount RJ , Jarlsberg LG , Daly KR , Djawe K , Fong S et al. ( 2012 ) Serologic responses to recombinant Pneumocystis jirovecii major surface glycoprotein in HIV-positive and HIV-negative individuals in Uganda with respiratory symptoms . PLOS ONE 7(12): e51545 . doi: 10.1371/journal.pone.0051545. PubMed: 23284710 .
52. Italian Register for HIV Infection in Children (2004) Combined antiretroviral therapy reduces hyperimmunoglobulinemia in HIV-1 infected children . AIDS 18 (1423-8)
53. De Moraes-Pinto MI , Almeida AC , Kenj G , Filgueiras TE , Tobias W et al. ( 1996 ) Placental transfer and maternally acquired neonatal IgG immunity in human immunodeficiency virus infection . J Infect Dis 173 : 1077 - 1084 . doi:10.1093/infdis/173.5.1077. PubMed: 8627057 .
54. De Moraes-Pinto MI , Verhoeff F , Chimsuku L , Milligan PJ , Wesumperuma L et al. ( 1998 ) Placental antibody transfer: influence of maternal HIV infection and placental malaria . Arch Dis Child Fetal Neonatal Ed 79 : F202 - F205 . doi:10.1136/fn.79.3.F202. PubMed: 10194992.
55. Gigliotti F , Wiley JA , Harmsen AG ( 1998 ) Immunization with Pneumocystis carinii gpA is immunogenic but not protective in a mouse model of P. carinii pneumonia . Infect Immun 66 : 3179 - 3182 . PubMed: 9632583 .