Therapeutic Potential of Caspofungin Combined with Trimethoprim-Sulfamethoxazole for Pneumocystis Pneumonia: A Pilot Study in Mice
et al. (2013) Therapeutic Potential of Caspofungin Combined with Trimethoprim-
Sulfamethoxazole for Pneumocystis Pneumonia: A Pilot Study in Mice. PLoS ONE 8(8): e70619. doi:10.1371/journal.pone.0070619
Therapeutic Potential of Caspofungin Combined with Trimethoprim-Sulfamethoxazole for Pneumocystis Pneumonia: A Pilot Study in Mice
Maria Lusa Lobo 0
Francisco Esteves 0
Bruno de Sousa 0
Fernando Cardoso 0
Melanie T. Cushion 0
Francisco Antunes 0
Olga Matos 0
Herbert B. Tanowitz, Albert Einstein College of Medicine, United States of America
0 1 Unidade de Parasitologia Me dica, Grupo de Protozoa rios Oportunistas/VIH e Outros Protozoa rios, CMDT, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa , Lisboa , Portugal , 2 Faculdade de Psicologia e Ciencias da Educac a o, Universidade de Coimbra , CMDT, Coimbra , Portugal , 3 University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America, 4 Faculdade de Medicina, Hospital de Santa Maria, Universidade de Lisboa , Lisboa , Portugal
Pneumocystis pneumonia (PcP) is a major cause of mortality and morbidity in immunocompromised patients. There are limited alternative therapeutic choices to trimethoprim-sulfamethoxazole (TMP-SMX) which is the standard first line therapy/prophylaxis for PcP. The efficacy of low doses of caspofungin and caspofungin in association with TMP-SMX standard-prophylactic dose was evaluated in an experimental model of Pneumocystis. Susceptibility of Pneumocystis spp. to low doses of caspofungin and caspofungin/TMP-SMX was evaluated in Balb/c immunosuppressed mice, infected intranasally with P. murina. Caspofungin was administered once daily at 0.1 mg/kg, 0.05 mg/kg, and 0.001 mg/kg and TMPSMX was administered by oral gavage (12.25 mg/62.5 mg/day), for 21 days. Efficacy was calculated based on the reduction in organism burden determined through quantitative fluorescent-based real-time PCR (qPCR). Serum b-1,3-D-glucan was measured as an additional marker of infection. The present data showed that caspofungin demonstrated antiPneumomocystis effect. However, the doses administrated were too low to achieve Pneumocystis eradication, which suggests that echinocandin treatment should not be administrated as mono-therapy. After 21 days of treatment, P. murina was not detected in the lungs of mice with either TMP-SMX or caspofungin/TMP-SMX. The results showed that, even at the lowest concentrations tested, the efficacy of caspofungin in association with TMP-SMX was higher than the efficacy of either drug used alone. The administration of caspofungin/TMP-SMX was at least 1.4 times more effective against P. murina infection than TMP-SMX used alone. The most promising result was achieved with the combination of caspofungin 0.05 mg/kg/day with TMP-SMX 12.5 mg-62.5 mg/day, which reduced the parasite burden to undetectable levels immediately at the 14th day of treatment, showing a highly marked anti-Pneumomocystis effect. These data suggest that the administration of low doses of caspofungin in combination with low doses of TMP-SMX may provide an improved treatment protocol for Pneumocystis infection clearance.
Funding: The present study was supported by the scientific project Ref. 38903 financed by Merck Sharp & Dohme Corp. The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: Merck Sharp & Dohme Corp. funded this study and manufactures the product Caspofungin used in this study. There are no further
patents, products in development or marketed products to declare. This does not alter the authors adherence to all the PLOS ONE policies on sharing data and
Invasive fungal infections are an important cause of morbidity
and mortality, especially in immunocompromised and/or
hospitalized patients with serious underlying diseases [1,2]. Pneumocystis
pneumonia (PcP) is a common and serious life-threatening
opportunistic disease in hosts with impaired/debilitated immune
systems, especially HIV-positive persons, but also in patients who
are undergoing immunosuppressive treatments related to
malignancies, connective tissue diseases or organ transplantation. These
organisms are also emerging as a co-morbidity factor associated
with chronic diseases such as chronic obstructive pulmonary
disorder (COPD) .
Pneumocystis spp. are atypical fungi with bi-phasic life cycle in the
mammalian lung, consisting of an asexual phase (binary fission of
trophic forms) and a sexual cycle (conjugation of trophic forms
resulting in formation of cysts). As the organism cannot be cultured
reliably in vitro, there is a poor understanding of the life cycle, and
the development of alternative therapeutic choices is limited.
Therefore, drug efficacy studies have been carried out directly on
rodent models . PcP transmission is thought to be via
airborne route. Pneumocystis spp. attaches to the host alveolar
epithelium through Type I pneumocytes and fill the alveolar sacs.
Without effective treatment, the host succumbs to respiratory
failure and related organ dysfunction [6,10].
Due to the lack of ergosterol in the cell membrane,
amphotericin B and the azoles are ineffective against Pneumocystis spp.
infections. The standard first line therapy/prophylaxis for PcP is
the combination therapy, trimethoprim-sulfamethoxazole
(TMPSMX). Despite being effective, there are significant prophylactic
and treatment failures as well as intolerance or side effects
associated with this anti-folate inhibitors combination that may
hamper the clinical outcome of the disease. Relapse and
recurrence of infection are high when using secondary therapies
(pentamidine, clindamycin-primaquine or atovaquone). New
therapeutic approaches are needed [6,7,11,1315].
In the early 2000s, echinocandins were licensed for the
treatment and prevention of fungal infections. Caspofungin
(caspofungin acetate) is a semi-synthetic water soluble lipopeptide
(fermentation product of the fungus Glorea lozoyensis) and a
noncompetitive inhibitor of the b(1,3)D-glucan (BG) synthase
(not present in mammalian cells) that presents excellent safety
profiles [1,12,16,17]. This echinocandin is effective against
Pneumocystis spp. cysts that strongly express BG synthase, but is
less effective against the trophic forms, which are poor in BG.
Studies suggest that treatment of PcP with caspofungin alone may
not likely result in the effective eradication of the infection, which
could result in relapse [3,13,16,1821].
There is a scarcity of data about the efficacy of the addition of
caspofungin to TMP-SMX for treatment of PcP patients. A few
reports suggest that the use of caspofungin with TMP-SMX
regimen may lead to a faster improvement of the patient followed
by complete cure of pneumonia [20,22], however, the use of low
doses of these anti-PcP molecules in association was not evaluated.
In order to improve PcP treatment and to overcome the
potential intolerance and adverse effects of the standard
antiPneumocystis spp. drugs, the aim of the present study was to evaluate
the susceptibility of Pneumocystis spp. organisms to low doses of
caspofungin alone and caspofungin in association with
TMPSMX, in the rodent model.
Materials and Methods
Specific pathogen-free BALB/c male mice were obtained from
the Instituto de Higiene e Medicina Tropical, Universidade Nova
de Lisboa Breeding Laboratory and were housed in filter-topped
cages and fed autoclaved chow and water ad libitum.
Caspofungin (25 mg) was supplied by Merck Sharp & Dohme
Corp. TMP-SMX (BactrimH, Roche) pediatric suspension (TMP
8 mg/ml+SMX 40 mg/ml) was purchased.
P. murina (26107 nuclei/ml) for mice inoculation were purified
from the lungs of C3H/HeN wild type mice. The techniques
adopted have been described in earlier reports [3,23].
Evaluation of anti-Pneumocystis activity was conducted with an
immunosuppressed mouse model of PcP. The experimental design
of the study is adapted from Cushion et al., 2010  and
summarized in Figure 1. The immunosuppressed state was
induced by administration of 4 mg/ml dexamethasone (Sigma)
in the drinking water. Tetracycline (1 g/L, Sigma) was added to
the water to avoid bacterial infection. Animals were anesthetized
with a drug cocktail (ketamine hydrochloride 2 mg and Xylazine
HCl 2%) and injected intranasally with P. murina at a dose of 106
nuclei per mouse. Four P. murina-free mice were housed separately
(sentinel controls, Group 1 Pm uninfected). When the infection
reached a moderate intensity (after 7.5 weeks), mice were
randomly divided into treatment and control groups of four
animals each. A total of eight groups were generated (see Table 1):
Group 2 Non-medicated (immunosuppressed mice without
antiP. murina therapy), Group 3 Caspofungin (three subgroups: 3.1,
3.2 and 3.3), Group 4 TMP-SMX (4), Group 5 Caspofungin/
TMP-SMX (three subgroups: 5.1, 5.2 and 5.3). Four mice were
sacrificed at the start of the study to confirm the presence of acute
P. murina infection. Caspofungin (dissolved in 0.9% NaCl) was
administered intravenously (i.v.) during the first 8 days and by
intraperitoneal (i.p,) injection during the remaining 13 days of
treatment on a mg/kg basis once daily for 21 days, at varying dose
levels (0.1 mg/kg, 0.05 mg/Kg and 0.001 mg/Kg). The change in
the treatment regimen was a consequence of the overly frequent
(daily) administration of caspofungin in the mice tail vein that
started to induce vein collapse, signs of necrosis in their tails and
high levels of animal stress. In previous case reports of caspofungin
treatment of PcP cases, standard-doses of 5070 mg/day were
usually administrated [16,20,21]. Also, Cushion and co-workers
2010, tested 1 mg/kg-0.1 mg/kg doses i.p. one to three times a
week in the rodent model, suggesting its effectiveness at reducing
cyst burdens. In the present study, lower concentrations of
caspofungin were selected to test the efficacy of the concomitant
use of caspofungin in association with TMP-SMX. TMP-SMX
pediatric suspension was administered by oral gavage at the
standard-prophylactic dose (12.5 mg62.5 mg daily) [3,23]. The
drug treatment continued for 21 days during which time the mice
remained under the immunosuppressive regimen. Control animals
receiving steroids (Group 2, P. murina infected mice without
therapy) received no treatment. At the time points 0, 14, and 21
days, mice were anesthetized and euthanized (Group 1: one mouse
at day 0 and 14, respectively and two mice at day 21; four mice
randomly selected from the four large cages harboring
immunosuppressed P.murina infected mice prior grouping them into 8 small
boxes according the distinct groups were euthanized at day 0;
Group 2, Group 3 subgroups 3.1, 3.2 and 3.3, Group 4 and
Group 5 subgroups 5.1, 5.2 and 5.3: one mouse at day 14, and
three mice at day 21). Mice were exsanguinated, and their lungs
were aseptically removed and homogenized for further histological
and molecular analysis. All caging procedures and surgical
manipulations were done in a laminar flow hood. The
experimental protocols were approved by the Sociedade Portuguesa de
Ciencias em Animais de Laborato rio/Direcc ao Geral de
Veterinaria (SPCAL/DGV), Institutional Animal Care, in strict
accordance with the recommendations in the Guide for the Care
and Use of Laboratory Animals.
Evaluation of P. murina Infection Levels
Extraction of DNA from mice lungs was performed using a
Mini-BeadBeater/guanidinium thiocyanate-silica method, as
described previously [24,25]. Infection levels were determined by a
quantitative fluorescence-based real-time PCR (qPCR) for P.
murina mitochondrial large-subunit rRNA gene (mtLSU rRNA)
quantification. The qPCR assays (TaqmanH MGB probes,
FAMTM dye-labelled, Applied Biosystems) were performed in
the 7300 Real-Time PCR System (Applied Biosystems, Foster
City, CA), based on data reported previously [4,26]. The baseline
was taken from cycles three to 20 and the threshold was set at 0.3.
The maximum Cq value was determined at dilution 1:300 from
the initial P. murina 26107 nuclei/ml suspension. To convert the
threshold cycle data into P. murina nuclei, a standard curve was
generated using DNA isolated from a purified sample with 107 P.
Microscopic Evaluation of P. murina Infection
Smears from the homogenized lungs were stained with Giemsa
and Grocotts methenamine silver (GMS) to confirm the presence
of P. murina organims. Slides were examined in a blinded manner
using an Olympus BX51 microscope [3,27].
Serum b(1,3)D-glucan Content
The BG serum levels from mice were measured using the
FUNGITELLTM assay (Associates of Cape Cod, Inc.,East
Falmouth, MA) and performed in the NanoQuant Infinite M200 Pro
The efficacy of the anti-Pneunocystis therapy regimens was
evaluated calculating the decrease of organism burden by qPCR in
treated mice in comparison with untreated mice (fold reduction).
Statistical tests were applied to investigate associations at a
significance level of 0.05 using SPSS v.20.0 software (SPSS Inc.,
Chicago, IL, USA). The non-parametric Kruskal-Wallis test was
used to investigate differences between the median P. murina
burden distribution variations across the groups of mice treated
with different anti-Pneumocystis regimens.
Evaluation of P. murina Infection Levels
The results obtained for qPCR quantification of P. murina mtLSU
RNA gene in the nine distinct mice groups enrolled in this study
are summarized in Table 1. Mice from negative control group
remained uninfected during the time of the experiment. The P.
murina infected untreated control had a 1.1-fold increase of
organisms per lung during the 21 days. The caspofungin treatment
lowered the average organisms count per lung: in subgroup 3.1
(0.1 mg/Kg/day) a 2.1-fold reduction (day 14), and a 3.1-fold
reduction at day 21; in subgroup 3.2 (0.05 mg/Kg/day) a 3.8-fold
reduction (day 14) and a 2.0-fold reduction (day 21); in subgroup
3.3 (0.001 mg/Kg/day) was observed an initial increase of
272fold (day 14) and a reduction of 1.2-fold (day 21) during the 21
days of the experiment. The TMP-SMX treatment decreased
65.5-fold the average of organisms count per lung in Group 4
(12.5 mg62.5 mg/day) at day 14 and to undetectable levels at the
end of the study (day 21).
The addition of low doses of caspofungin to TMP-SMX
enhanced the efficacy of the treatment, dropping the average
organisms count per lung (day 14): in subgroup 5.1 (0.1 mg/Kg/
day caspofungin; 12.5 mg62.5 mg/day TMP-SMX) a 130.5-fold
reduction; in subgroup 5.2 (0.05 mg/Kg/day caspofungin;
12.5 mg62.5 mg/day TMP-SMX) a marked reduction, from
7.176108 to undetectable levels; and in subgroup 5.3 (0.001 mg/
Kg/day caspofungin; 12.5 mg62.5 mg/day TMP-SMX) a
92.7fold reduction. All caspofungin/TMP-SMX regimens exhibited
very effective anti-P. murina activity, reducing organisms counts to
undetectable levels at the 21st day.
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Therefore, at the end of the 21st day, both regimens TMP-SMX
alone and Caspofungin in association with TMP-SMX reduced P.
murina counts to undetectable levels. However, at the 14th day, the
combination caspofungin/TMP-SMX demonstrated higher
effectiveness against P. murina infection than TMP-SMX alone. In
comparison with the TMP-SMX regimen, the combination
0.05 mg/Kg/day caspofungin with 12.5 mg62.5 mg/day
TMP-SMX was more effective, reducing the average P. murina
burden to undetectable levels immediately at day 14. Also, the
concentrations 0.01 mg/Kg/day caspofungin with 12.5 mg
62.5 mg/day TMP-SMX and 0.001 mg/Kg/day caspofungin
with 12.5 mg62.5 mg/day TMP-SMX suggested a 2.0 times
and a 1.4 times more effective fold reduction than TMP-SMX
Microscopic Evaluation of P. murina Infection
The presence of P. murina organisms was confirmed in the lung
smears of infected animals by the staining procedures adopted.
Samples with undetectable levels by qPCR showed no P. murina
organisms. As expected, no P. murina organisms were observed in
the lungs of the sentinel control mice. All samples were analyzed
using this quality control.
Serum b(1,3)D-glucan Content
The levels of BG measured in the serum of the rodents, at days
0 and 21, are presented in Table 2. As expected, the decrease of P.
murina organisms in all the treated mice correlated with a reduction
of BG content. However, the reduction in BG levels was not
proportional to the decrease of P. murina organisms. At day 21, the
lowest levels of BG (68.94 pg/ml) were observed in mice treated
with 0.05 mg/Kg/day caspofungin, followed by the TMP-SMX
regimen (75.53 pg/ml). With the exception of group 5.2
(0.05 mg/Kg/day caspofungin; 12.5 mg62.5 mg/day
TMPSMX), the BG content in the mice treated with the combination
caspofungin/TMP-SMX was lower than in the ones treated with
caspofungin in mono-therapy.
The Kruskal-Wallis test showed no statistical significances
between the median P. murina loads distribution variation across
the distinct groups (TMP-SMX or caspofungin/TMP-SMX
treatment regimens) studied by qPCRs, mainly due to the small
number of mice in each combination of treatment and doses.
Nevertheless, if mice are grouped according to treatment regimen,
regardless of the concentrations of drug administered (group 2 - P.
murina infected untreated mice; group 3 - P. murina infected mice
treated with caspofungin; group 4 - P. murina infected mice treated
with TMP-SMX; group 5 - P. murina infected mice treated with
caspofungin/TMP-SMX), there are some relevant statistical
associations. Kruskal-Wallis test rejected the null hypothesis that
the median P. murina burden are the same in treated (caspofungin,
TMP-SMX, or caspofungin/TMP-SMX) and untreated mice,
rejecting the equality of the median values at the 21st day between
group 5 and group 2 (caspofungin/TMP-SMX versus untreated,
P,0.001; Std. error 4.877), group 4 and group 2 (TMP-SMX
versus untreated, P = 0.016; Std. error 6.678) and group 5 and
group 3 (caspofungin/TMP-SMX versus TMP-SMX, P,0.023;
Std. error 4.702).
There is a lack of clinical experience with using caspofungin to
treat PcP. Nevertheless, few studies raised the importance of the
potential clinical use of this BG synthase inhibitor [1,13,2022]. In
the present study Pneumocystis spp. susceptibility to low doses of
caspofungin and caspofungin in association with TMP-SMX, in
the rodent model was evaluated. The obtained data demonstrated
that addition of caspofungin (active against cysts) in low doses to
TMP-SMX (active against trophic forms and cysts) in prophylactic
doses may provide an additive anti-Pneumomocystis effect,
completely inhibiting Pneumocystis spp. life cycle. Our results suggest
that the administration of low doses of caspofungin in combination
with low doses of TMP-SMX provides an improved hastening
clearance of the infection, as they are faster acting over Pneumocystis
spp. organisms than either TMP-SMX alone, or caspofungin in
In the course of the 21 days of PcP treatment with caspofungin
mono-therapy, the highest concentration (0.1 mg/Kg/day) tested
showed the most effective anti-Pneumocystis activity, followed by the
second highest concentration (0.05 mg/Kg/day) and by the lowest
concentration (0.001 mg/Kg/day) (Table 1), as expected.
Nevertheless, on the 21st day, P. murina was still detectable in the lungs of
mice, adding evidence that the doses administrated were too low
to achieve an effective treatment, or that this drug alone was
Groups/subgroups of mice
Pm uninfected control
Pm infected, untreated control
Pm infected, Caspofungin
Pm infected, TMP-SMX
Pm infected, Caspofungin/TMP-SMX
(12.25 mg62.5 mg/day)
(0.1 mg/kg/day+12.5 mg62.5 mg/day)
(0.05 mg/kg/day+12.5 mg62.5 mg/day)
(0.001 mg/kg/day+12.5 mg62.5 mg/day)
Note: The reference values established for fungal infections and the baseline limits of the method are optimized to measure the b-1,3-D-glucan serum/plasma levels
from humans. However the same values were adopted for this study: Negative: ,60 pg/ml; Indeterminate: 6079 pg/ml; Positive: .80 pg/ml; Upper limit: #500 pg/ml;
Lower limit $31 pg/ml.
unable to eradicate Pneumocystis spp. infection. These results
confirmed the evidence that echinocandin treatment should not
be administrated as mono-therapy. Since these compounds chiefly
target the cysts and are less effective against the trophic forms, it
should be taken into account that cessation of echinocandin
treatment while the host remains immunosuppressed could result
in inadvertent cyst repopulation and subsequent relapse as
reported previously [3,12,16,20]. Unlike the other groups under
treatment, in which effective decreases of organisms were detected
in days 14 and 21, it was observed that in the group of mice
treated with 0.001 mg/Kg/day caspofungin there was an initial
272-fold (day 14) increase of P. murina organisms, followed by a
1.2-fold (day 21) reduction. These results may be a consequence of
the particularly low dose of caspofungin administrated in this
group, which probably has a weaker anti-P. murina effect, delaying
the clearance of infection. A higher fungal load was observed at
14th day in mice treated with caspofungin (0.001 mg/Kg/day)
than in untreated controls. We hypothesize that such changes may
be associated to intrinsic features of each animal, namely to mice
inter-individual dissimilarities, reported in rodent models by other
The present data showed that the concomitant use of
caspofungin in low doses and TMP-SMX in lower doses was
effective against Pneumocystis spp. infection, reducing the parasite
burden to undetectable levels in the lungs of mice at the end of the
21 days of treatment. The qPCR data clearly showed that even at
the lowest concentrations administrated (caspofungin 0.001 mg/
Kg/day; TMP-SMX 12.5 mg - 62.5 mg/day), the efficacy of the
association was higher than the one observed for the drugs used
alone, whatever the concentrations tested (Table 1). The most
interesting and promising result was achieved by using the
combination of caspofungin 0.05 mg/kg/day with TMP-SMX
12.5 mg62.5 mg/day, which reduced the parasite burden to
undetectable levels immediately at the 14th day of treatment.
Although additional studies are needed to confirm the trend
observed the results evidenced that the use of caspofungin in
association with TMP-SMX may provide an additive effect against
Pneumocystis spp. organisms [3,20,22], even when low
concentrations are administrated. Taking into account the concentrations
used, the concomitant administration of caspofungin in association
with TMP-SMX was at least 1.4 times more effective against P.
murina infection than TMP-SMX mono-therapy, suggesting a
highly marked anti-Pneumomocystis effect.
The lack of statistical significance of the difference between the
median P. murina burden distribution variation across the distinct
groups resulted from the small number of mice enrolled in each
group (four mice), making it meaningless to test the assumptions of
normality and homogeneity of variance necessary to apply the
ttest. In order to overcome this limitation, the results of the median
P. murina burden were pooled according to the treatment regimens.
Taking into consideration the significance levels, this approach
strongly supports the hypotheses that at the end of the treatment:
1) caspofungin/TMP-SMX combination was statistically more
effective than caspofungin in mono-therapy, 2) both TMP-SMX
and caspofungin/TMP-SMX were statistically effective treatments
to deplete P. murina infection, in the rodent model. The treatment
with caspofungin/TMP-SMX showed the highest significance
level (P,0.001), which suggests this therapeutic association as a
potential candidate regimen to treat PcP that should be studied in
The potential additive effect of caspofungin/TMP-SMX
combined therapy against Pneumocystis spp. infection may be
advantageous in the treatment of PcP patients, as it appears to fully
inhibit the organisms life cycle and promote a faster recovery and
a better outcome when compared with the therapy standards
normally applied. The use of these molecules in combination
produces a double effect: (1) fungistatic effect that results from (a)
the blockade of the cell wall synthesis by caspofungin, and (b)
nucleic acids and protein breakdown by TMP-SMX, reducing the
trophic forms and consequently the cysts formation; (2) fungicidal
effect that results from (a) a change in the integrity of the cell wall,
which loses its mechanical strength and becomes unable to resist
the intracellular osmotic pressure through the action of
caspofungin, and from (b) the action of TMP-SMX that impairs the
function of folic acids, promotes an inability to translate and
synthesize proteins, which conducts a metabolic knock-out, both
leading ultimately to destruction of the Pneumocystis spp. cells
[3,15,16,30]. Additionally, the use of caspofungin in combination
with TMP-SMX may decrease the harsh inflammatory responses
linked to BG, a well-known pro-inflammatory factor implicated in
detrimental inflammatory responses in PcP, as reported previously
As expected the BG serum levels decreased throughout the
treatment period for all the drugs tested. Although the reduction in
BG levels was not proportional to the decrease in P. murina
organisms, the present data confirmed the efficacy of the
compounds used. 0.05 mg/Kg/day caspofungin mono-therapy
and 12.5 mg62.5 mg/day TMP-SMX have shown the highest
BG serum levels decreases. Overall, the BG results suggest that the
combination caspofungin/TMP-SMX was more efficient than
caspofungin in mono-therapy to treat PcP. This data may be
related to the fact that: a) TMP-SMX targets trophic forms
resulting mostly in anti-Pneumomocystis activity without fluctuations
in the BG basal levels; b) caspofungin destroys the cystic forms of
Pneumocystis followed by elevation of serum BG. Additionally, other
authors proposed the possibility that immunocompromised hosts
clear BG slowly, remaining longer in the bloodstream [3,33].
In conclusion, the results of this study suggest but do not prove
the potential benefit of the combined PcP treatment caspofungin/
TMP-SMX in lower doses in comparison with those tested in
previous studies . Further studies involving a larger number of
mice and time points over the treatment period are required, to
confirm the efficacy of this drug combination and to determine the
shortest period of drugs administration. This new approach
provided a significant contribution to the development and
enrichment of anti-Pneumomocystis armamentarium, contributing
for further selection of drugs that lead to a reliably, minimally
toxic, better tolerated, highly effective and time/cost saving
treatment for PcP.
Conceived and designed the experiments: MLL FA OM. Performed the
experiments: MLL FC FE. Analyzed the data: MLL FE BDS. Contributed
reagents/materials/analysis tools: MTC. Wrote the paper: MLL FE OM.
Critical revision of the manuscript: FA MTC.
1. Espinel-Ingroff A ( 2009 ) Novel antifungal agents, targets or therapeutic strategies for the treatment of invasive fungal diseases: a review of the literature (2005- 2009) . Rev Iberoam Micol 26 : 15 - 22 .
2. Enoch DA , Ludlam HA , Brown NM ( 2006 ) Invasive fungal infections: a review of epidemiology and management options . J Med Microbiol 55 : 809 - 18 .
3. Cushion MT , Linke MJ , Ashbaugh A , Sesterhenn T , Collins MS , et al. ( 2010 ) Echinocandin treatment of Pneumocystis pneumonia in rodent models depletes cysts leaving trophic burdens that cannot transmit the infection . PLoS ONE 5 : e8524 .
4. Esteves F , Gaspar J , de Sousa B , Antunes F , Mansinho K , et al. ( 2012 ) Pneumocystis jirovecii multilocus genotyping in pooled DNA samples: a new approach for clinical and epidemiological studies . Clin Microbiol Infect 18 : E177 - E184 .
5. Morris A , Sciurba FC , Norris KA ( 2008 ) Pneumocystis: a novel pathogen in chronic obstructive pulmonary disease ? COPD 5: 43 - 51 .
6. Calderon EJ , Gutierrez-Rivero S , Durand-Joly I , Dei-Cas E ( 2010 ) Pneumocystis infection in humans: diagnosis and treatment . Expert Rev Anti Infect Ther 8 : 683 - 701 .
7. Huang L , Cattamanchi A , Davis JL , den Boon S , Kovacs J , et al. ( 2011 ) HIVassociated Pneumocystis pneumonia . Proc Am Thorac Soc 8 : 294 - 300 .
8. Aliouat-Denis CM , Chabe M , Demanche C , Aliouat eM , Viscogliosi E , et al. ( 2008 ) Pneumocystis species, co-evolution and pathogenic power . Infect Genet Evol 8 : 708 - 26 .
9. Redhead SA , Cushion MT , Frenkel JK , Stringer JR ( 2006 ) Pneumocystis and Trypanosoma cruzi: nomenclature and typifications . J Eukaryot Microbiol 53 : 2 - 11 .
10. Thomas CF Jr, Limper AH ( 2007 ) Current insights into the biology and pathogenesis of Pneumocystis pneumonia . Nat Rev Microbiol 5 : 298 - 308 .
11. Stringer JR ( 1996 ) Pneumocystis carinii: what is it , exactly? Clin Microbiol Rev 9 : 489 - 98 .
12. Powles MA , Liberator P , Anderson J , Karkhanis Y , Dropinski JF , et al. ( 1998 ) Efficacy of MK-991 (L-743 ,872), a semisynthetic pneumocandin, in murine models of Pneumocystis carinii . Antimicrob Agents Chemother 42 : 1985 - 9 .
13. Schmatz DM , Romancheck MA , Pittarelli LA , Schwartz RE , Fromtling RA , et al. ( 1990 ) Treatment of Pneumocystis carinii pneumonia with 1,3-beta-glucan synthesis inhibitors . Proc Natl Acad Sci U S A 87 : 5950 - 4 .
14. Barry SM , Johnson MA ( 2001 ) Pneumocystis carinii pneumonia: a review of current issues in diagnosis and management . HIV Med 2 : 123 - 32 .
15. Matos O , Esteves F ( 2010 ) Epidemiology and clinical relevance of Pneumocystis jirovecii Frenkel, 1976 dihydropteroate synthase gene mutations . Parasite 17 : 219 - 32 .
16. Letscher-Bru V , Herbrecht R ( 2003 ) Caspofungin: the first representative of a new antifungal class . J Antimicrob Chemother 51 : 513 - 21 .
17. Finkelman MA ( 2010 ) Pneumocystis jirovecii infection: cell wall (1-3)-B-D-glucan biology and diagnostic utility . Crit Rev Microbiol 36 : 271 - 81 .
18. Vazquez JA , Sobel JD ( 2006 ) Anidulafungin: a novel echinocandin . Clin Infect Dis 43 : 215 - 22 .
19. Matsumoto Y , Yamada M , Amagai T ( 1991 ) Yeast glucan of Pneumocystis carinii cyst wall: an excellent target for chemotherapy . J Protozool 38 : 6S - 7S .
20. Utili R , Durante-Mangoni E , Basilico C , Mattei A , Ragone E , et al ( 2007 ) Efficacy of caspofungin addition to trimethoprim-sulfamethoxazole treatment for severe Pneumocystis pneumonia in solid organ transplant recipients . Transplantation 84 : 685 - 8 .
21. Annaloro C , Della VA , Usardi P , Lambertenghi DG ( 2006 ) Caspofungin treatment of Pneumocystis pneumonia during conditioning for bone marrow transplantation . Eur J Clin Microbiol Infect Dis 25 : 52 - 4 .
22. Beltz K , Kramm CM , Laws HJ , Schroten H , Wessalowski R , et al. ( 2006 ) Combined trimethoprim and caspofungin treatment for severe Pneumocystis jiroveci pneumonia in a five year old boy with acute lymphoblastic leukemia . Klin Padiatr 218 : 177 - 9 .
23. Walzer PD , Foy J , Steele P , Kim CK , White M , et al. ( 1992 ) Activities of antifolate, antiviral, and other drugs in an immunosuppressed rat model of Pneumocystis carinii pneumonia . Antimicrob Agents Chemother 36 : 1935 - 42 .
24. Costa MC , Gaspar J , Mansinho K , Esteves F , Antunes F , et al. ( 2005 ) Detection of Pneumocystis jirovecii dihydropteroate synthase polymorphisms in patients with Pneumocystis pneumonia . Scand J Infect Dis 37 : 766 - 71 .
25. Esgalhado R , Esteves F , Antunes F , Matos O ( 2013 ) Study of the epidemiology of Pneumocystis carinii f . sp. suis in abattoir swine in Portugal . Med Mycol 51 ( 1 ): 66 - 71 .
26. Ruan S , Tate C , Lee JJ , Ritter T , Kolls JK , et al. ( 2002 ) Local delivery of the viral interleukin-10 gene suppresses tissue inflammation in murine Pneumocystis carinii infection . Infect Immun 70 : 6107 - 13 .
27. Holten-Andersen W , Kolmos HJ ( 1989 ) Comparison of methenamine silver nitrate and Giemsa stain for detection of Pneumocystis carinii in bronchoalveolar lavage specimens from HIV infected patients . APMIS 97 : 745 - 7 .
28. Persat F , Ranque S , Derouin F , Michel-Nguyen A , Picot S , et al. ( 2008 ) Contribution of the (1-.3)-beta-D-glucan assay for diagnosis of invasive fungal infections . J Clin Microbiol 46 : 1009 - 13 .
29. Hufeldt MR , Nielsen DS , Vogensen FK , Midtvedt T , Hansen AK ( 2010 ) Family relationship of female breeders reduce the systematic inter-individual variation in the gut microbiota of inbred laboratory mice . Lab Anim 44 ( 4 ): 283 - 289 .
30. Esteves F , Gaspar J , Marques T , Leite R , Antunes F , et al. ( 2010 ) Identification of relevant single-nucleotide polymorphisms in Pneumocystis jirovecii: relationship with clinical data . Clin Microbiol Infect 16 : 878 - 84 .
31. Limper AH , Lebron F , Evans SE , Hahn RY ( 2003 ) Pneumocystis carinii: cell wall beta-glucan-mediated pulmonary inflammation . J Eukaryot Microbiol 50 Suppl: 646.
32. Vassallo R , Standing JE , Limper AH ( 2000 ) Isolated Pneumocystis carinii cell wall glucan provokes lower respiratory tract inflammatory responses . J Immunol 164 : 3755 - 63 .
33. Yoshida M , Roth RI , Grunfeld C , Feingold KR , Levin J ( 1996 ) Soluble (1-.3)- beta-D-glucan purified from Candida albicans: biologic effects and distribution in blood and organs in rabbits . J Lab Clin Med 128 : 103 - 114 .
34. Linke MJ , Ashbaugh A , Collins MS , Lynch K , Cushion MT ( 2013 ) Characterization of a distinct host response profile to Pneumocystis murina asci during clearance of Pneumocystis pneumonia . Infect Immu 81 : 984 - 95 .