In vitro and in vivo activity of iclaprim, a diaminopyrimidine compound and potential therapeutic alternative against Pneumocystis pneumonia.

Pneumocystis pneumonia is a serious complication that may affect immunosuppressed patients. The absence of reliable and safe therapeutic alternatives to trimethoprim-sulfamethoxazole (TMP/SMX) justifies the search for more effective and less toxic agents. In this study, the in vitro and in vivo anti-Pneumocystis jirovecii activity of iclaprim, a diaminopyrimidine compound that exerts its antimicrobial activity through the inhibition of dihydrofolate reductase (DHFR), as does TMP, was evaluated alone or in combination with SMX. The antimicrobial activity of iclaprim was tested in vitro using an efficient axenic culture system, and in vivo using P. carinii endotracheally inoculated corticosteroid-treated rats. Animals were orally administered iclaprim (5, 25, 50 mg/kg/day), iclaprim/SMX (5/25, 25/125, 50/250 mg/kg/day), TMP (50 mg/kg/day), or TMP/SMX (50/250 mg/kg/day) once a day for ten consecutive days. The in vitro maximum effect (Emax) and the drug concentrations needed to reach 50% of Emax (EC50) were determined, and the slope of the dose-response curve was estimated by the Hill equation (Emax sigmoid model). The iclaprim EC50 value was 20.3 µg/mL. This effect was enhanced when iclaprim was combined with SMX (EC50: 13.2/66 µg/mL) (p = 0.002). The TMP/SMX EC50 value was 51.4/257 µg/mL. In vivo, the iclaprim/SMX combination resulted in 98.1% of inhibition compared to TMP/SMX, which resulted in 86.6% of inhibition (p = 0.048). Thus, overall, the iclaprim/SMX combination was more effective than TMP/SMX both in vitro and in vivo, suggesting that it could be an alternative therapy to the TMP/SMX combination for the treatment of Pneumocystis pneumonia.

Comparison of different blood compartments for the detection of circulating DNA using a rat model of Pneumocystis pneumonia.

Pneumocystis is mostly found in the alveolar spaces, but circulation of viable organisms also occurs and suggests that the detection of DNA in blood could be used as a noninvasive procedure to improve the diagnosis of Pneumocystis pneumonia (PcP). In order to determine the optimal compartment for Pneumocystis DNA detection, we used a rat model of PcP and tested the presence of Pneumocystis with a quantitative mtLSU targeting real-time PCR in four blood compartments: whole blood, clot, serum and Platelet-Rich-Plasma (PRP). All samples from 4 Pneumocystis-free control rats were negative. Pneumocystis was detected in 79, 64, 57, and 57% of samples from 14 PcP rats, respectively, but DNA release was not related to pulmonary loads. These data confirm the potential usefulness of Pneumocystis DNA detection in the blood for PcP diagnosis and suggest that whole blood could be the most appropriate compartment for Pneumocystis detection.

An Hcp100 gene fragment reveals Histoplasma capsulatum presence in lungs of Tadarida brasiliensis migratory bats.

Histoplasma capsulatum was sampled in lungs from 87 migratory Tadarida brasiliensis bats captured in Mexico (n=66) and Argentina (n=21). The fungus was screened by nested-PCR using a sensitive and specific Hcp100 gene fragment. This molecular marker was detected in 81·6% [95% confidence interval (CI) 73·4-89·7] of all bats, representing 71 amplified bat lung DNA samples. Data showed a T. brasiliensis infection rate of 78·8% (95% CI 68·9-88·7) in bats captured in Mexico and of 90·4% (95% CI 75·2-100) in those captured in Argentina. Similarity with the H. capsulatum sequence of a reference strain (G-217B) was observed in 71 Hcp100 sequences, which supports the fungal findings. Based on the neighbour-joining and maximum parsimony Hcp100 sequence analyses, a high level of similarity was found in most Mexican and all Argentinean bat lung samples. Despite the fact that 81·6% of the infections were molecularly evidenced, only three H. capsulatum isolates were cultured from all samples tested, suggesting a low fungal burden in lung tissues that did not favour fungal isolation. This study also highlighted the importance of using different tools for the understanding of histoplasmosis epidemiology, since it supports the presence of H. capsulatum in T. brasiliensis migratory bats from Mexico and Argentina, thus contributing new evidence to the knowledge of the environmental distribution of this fungus in the Americas.

Pneumocystis diversity as a phylogeographic tool.

Parasites are increasingly used to complement the evolutionary and ecological adaptation history of their hosts. Pneumocystis pathogenic fungi, which are transmitted from host-to-host via an airborne route, have been shown to constitute genuine host markers of evolution. These parasites can also provide valuable information about their host ecology. Here, we suggest that parasites can be used as phylogeographic markers to understand the geographical distribution of intra-specific host genetic variants. To test our hypothesis, we characterised Pneumocystis isolates from wild bats living in different areas. Bats comprise a wide variety of species; some of them are able to migrate. Thus, bat chorology and migration behaviour can be approached using Pneumocystis as phylogeographic markers. In the present work, we find that the genetic polymorphisms of bat-derived Pneumocystis are structured by host chorology. Therefore, Pneumocystis intra-specific genetic diversity may constitute a useful and relevant phylogeographic tool.

Hypobaric hypoxia-related impairment of pulmonary surfactant proteins A and D did not favour Pneumocystis carinii Frenkel 1999 growth in non-immunocompromised rats.

It has been suggested that patients with pulmonary surfactant impairment are more susceptible to Pneumocystis infection than healthy controls. Owing the fact that most patients with pulmonary surfactant impairment also suffer from hypoxia, we explored the effect of intermittent hypobaric hypoxia conditions on the ability of non-immunocompromised rats infected by endotracheal route with P. carinii to clear the infection from their lungs. Control rats, inoculated or not with P. carinii, were maintained in normobaric normoxic conditions, and were submitted or not to dexamethasone administration. It was found that even if hypobaric hypoxia weakened host immune mechanisms and impaired significantly the surfactant composition, mainly of surfactant proteins A and D, these changes were not enough to favour the Pneumocystis growth or to inhibit the clearing of Pneumocystis organisms from the lungs of non-immunocompromised rats. The potential influence of surfactant protein changes on Pneumocystis infection is discussed.

First data on Pneumocystis jirovecii colonization in patients with respiratory diseases in North Lebanon.

Pneumocystis colonization may play a role in transmission and local inflammatory response. It was explored in patients with respiratory diseases in North Lebanon. Overall prevalence reached only 5.2% (95% CI 2.13-10.47) but it was higher (17.3%) in the subpopulation of patients with chronic obstructive pulmonary disease (COPD). COPD was the only factor associated with a significantly increased risk of colonization. mtLSU genotyping revealed predominance of genotype 2, identified in five patients (71.4%), including one patient who had co-infection with genotype 3. These first data in North Lebanon confirm Pneumocystis circulation among patients with respiratory diseases and the potential for transmission to immunocompromised patients.

Different ultrastructural morphology of Pneumocystis carinii derived from mice, rats, and rabbits.

Pneumocystis carinii (PC) is a fungus present in the lungs of many mammal species. Even though studies of the genome, the isoenzymes, and the antigens have proved some host-species-linked heterogeneity, the existence of distinct Pneumocystis species or subspecies has still not been accepted. Comparative studies of the ultrastructural morphology of pneumocysts derived from several host species may support evidence of host-species-linked heterogeneity. We have compared the ultrastructural morphology of pneumocysts derived from mice, rats, and rabbits. The density of membrane-limited electron-dense cytoplasmic granules was found to be higher in mouse-derived pneumocysts than in rabbit-derived pneumocysts, and furthermore the average diameter of the granules from mouse pneumocysts was larger than that of granules from rabbit-derived pneumocysts. The average diameter of the filopodia of mouse-derived pneumocysts was smaller than that of filopodia from rat-derived pneumocysts, which was smaller than that of filopodia from rabbit-derived pneumocysts. Globular electron-dense bulbous dilatations at the tip of the filopodia were described for the first time and they were only found on filopodia of mouse-derived pneumocysts. These distinct host-species-linked morphological differences of pneumocysts from mouse, rat, and rabbit may support previous biochemical data indicating the existence of different Pneumocystis species or subspecies.

Evaluation of drug efficacy by using animal models or in vitro systems.

The efficacy of most therapeutic and prophylactic protocols against Pneumocystis carinii pneumonia used in human patients has been tested in animal models, especially in the corticosteroid-treated rat. The advantages and drawbacks of this model have been examined in brief in Chapter 1 of this section. More recently, the nude rat, intratracheally inoculated with Pneumocystis, was used to test new anti-microbian molecules for their anti-Pneumocystis activity. In vitro systems, co-cultures of Pneumocystis with feeder cells as well as axenic cultures, were also used many times for drug screening. In this paper, the most used in vivo or in vitro drug screening systems are described. Moreover, as immunocompromised individuals, AIDS patients, especially, are often infected simultaneously by several infectious agents, a recent co-infection model is described.

Animal models of pneumocystosis.

As in vitro culture systems allowing to isolate Pneumocystis samples from patients or other mammal hosts are still not available, animal models have critical importance in Pneumocystis research. The parasite was reported in numerous mammals but P. carinii pneumonia (PCP) experimental models were essentially developed by using rats, mice, rabbits and ferrets. The rat treated with corticosteroids for 9-12 weeks is a useful PCP model. Like laboratory rats, conventional mice develop PCP after prolonged corticosteroid administration. The ferret (Mustela putorius furo) also develop PCP under corticosteroid regime. Whilst bronchoalveolar lavage (BAL) is really difficult to perform on live laboratory rodents, serial BAL sampling can be performed on live ferrets. Rabbits currently develop spontaneous PCP at weaning without corticosteroid administration. For this reason this model has been used for studying the host immune response as well as Pneumocystis-surfactant interactions. Pigs and horses also develop spontaneous PCP. Treated with corticosteroids, piglets develop extensive PCP and could be used as a non-rodent model. Pneumocystis was detected in many non-human primates. Primates could represent a source of parasites taxonomically related to P. carinii sp. f. hominis. Moreover, primates might be used as experimental hosts to human Pneumocystis. A marked variability of parasite levels among corticosteroid-treated animals and the fact that the origin of the parasite strain remains unknown, are important drawbacks of the corticosteroid-treated models. For these reasons, inoculated animal models of PCP were developed. The intratracheal inoculation of lung homogenates containing viable parasites in corticosteroid-treated non-latently infected rats resulted in extensive, reproducible Pneumocystis infections. Extensive PCP can be obtained within 5-7 weeks, whilst 9-12 weeks are needed in the classical model. The severe combined immunodeficiency (SCID) mouse inoculated by nasal route and the athymic nude rats intratracheally inoculated were used to test the infectivity of Pneumocystis samples coming from cultures or from different hosts. They were also used to test the anti-Pneumocystis activity of antimicrobial molecules.

Current in vitro culture systems for Pneumocystis.

Although Pneumocystis continuous culture systems have not yet been developed, efficient short-term in vitro methods allowing the production of infectious forms of Pneumocystis can now be employed. The quality of the inoculum will influence the in vitro development of P. carinii. For this reason, efficient extraction and cryopreservation techniques are considered in this section. In vitro growth and limited passage were obtained by inoculating freshly extracted parasites onto fibroblast- or epithelial-like cell monolayers cultivated in ordinary tissue culture flasks, culture plates, microcarrier beads or other culture devices. Cultures were usually maintained in an atmosphere of 5% CO2 at 35-37 degrees C. The results obtained in these different systems were surprisingly similar: the number of parasites increased about 6-10 times within the first 3-4 days post-inoculation, then remained stationary until day 7-14 and decreased rapidly. If passages were attempted, the growth decreased gradually and no growth was recorded after 2-3 passages. Proof of the in vitro Pneumocystis attachment to feeder cells has been furnished by electron microscopy. Two currently used feeder cell culture systems were selected in this subchapter. The first system is a co-culture of monolayer lung epithelial-like cells with Pneumocystis. After trypsin treatment and passage of cells with attached parasites to culture bottles containing fresh medium, 3 or more new culture bottles can be plated. A 2-4-fold increase in parasite number can be obtained but, interestingly, cultured parasites were more infectious to the nude rat than freshly extracted lung parasites. In the second system, the spinner flask culture method, Pneumocystis is cultivated on cell coated microbeads in slow stirring vessels, in order to exploit the beads' huge surface where microorganisms can transiently adhere and grow and from where they can be easily detached by simply leaving the beads to settle down. This culture system has ensured 10(8)-10(9) viable trophozoites in each harvest after 7-10 days of slow stirring incubation.

Morphological and ultrastructural methods for Pneumocystis.

Pneumocystis is a eukaryotic unicellular microorganism with marked fungal affinities. All known life cycle stages of this parasite were observed in the lung of mammals. The cystic forms of this microorganism may be observed microscopically by using stains with affinity for the components of their relatively thick cell wall. However, about 100 years ago they were observed for the first time thanks to panoptic stains which do not stain their cell wall. Methanol-Giemsa technique as well as Giemsa-like rapid stainings are often used to reveal vegetative or cystic forms of this parasite on air dried smears of clinical or experimental samples. For many years, hypotheses on its life cycle, which remains unknown, were based on transmission electron microscopy (TEM) studies. However, only for the last years progresses in the quality of fixation for TEM led to a better understanding of the Pneumocystis cell structure. In this chapter, strategies to reveal Pneumocystis organisms in clinical or experimental specimens by using light microscopy, as well as techniques allowing a good preparation of parasitic samples for TEM, are given and shortly discussed.

The yeast killer phenomenon: a hypothetical way to control Pneumocystis carinii pneumonia.

Pneumocystis carinii is an important agent of pneumonia in immunocompromised individuals, especially in acquired immunodeficiency syndrome AIDS patients P. carinii attaches specifically to type 1 pneumocytes. Although this phenomenon must play a marked role in pneumocystosis pathophysiology, no therapeutic molecules able to inhibit specifically the parasite attachment were found. A killer toxin, secreted by the yeast Pichia anomala, induced a significant decrease in P. carinii in vitro attachment and inhibited the parasite infectivity in SCID mice. Killer toxins cannot be used as systemic antibiotics. However, it was possible to produce antiidiotypic antibodies against a monoclonal antibody specific of the toxin active site. These antilds were shown to mimic the in vitro killer effect for the toxin and were called 'antibiobodies'. The susceptibility of P. carinii to the antimicrobial activity of the killer toxin made it possible to hypothesize that the killer phenomenon could constitute a new way for the treatment and prophylaxis of P. carinii infections.

Three optimized and validated (using accuracy profiles) LC methods for the determination of pentamidine and new analogs in rat plasma.

Three novel LC-UV methods for the determination of pentamidine (PTMD) and two of its new analogs in rat plasma are described. The chromatographic conditions (wavelength, acetonitrile percentage in the mobile phase, internal standard) were optimized to have an efficient selectivity. A pre-step of extraction was simultaneously developed for each compound. For PTMD, a solid phase extraction (SPE) with Oasis(®) HLB cartridges was selected, while for the analogs we used protein precipitation with acetonitrile. SPE for PTMD gave excellent results in terms of extraction yield (99.7 ± 2.8) whereas the recoveries for the analogs were not so high but were reproducible as well (64.6 ± 2.6 and 36.8 ± 1.6 for analog 1 and 2, respectively). By means of a recent strategy based on accuracy profiles (ß-expectation tolerance interval), the methods were successfully validated. ß was fixed at 95% and the acceptability limits at ± 15% as recommended by the FDA. The method was successfully validated for PTMD (29.6-586.54 ng/mL), analog 1 (74.23-742.3 ng/mL) and analog 2 (178.12-890.6 ng/mL). The first concentration level tested was considered as the LLOQ (lower limit of quantification) for PTMD and analog 1 whereas for analog 2, the LLOQ was not the first level tested and was raised to 178.12 ng/mL.

Pneumocystitis carinii organisms from in vitro culture are highly infectious to the nude rat.

Many in vitro systems have been used to cultivate Pneumocystis, but only limited parasite growth has been obtained by different authors. A reliable in vitro system enabling a sustained propagation of Pneumocystis appears to be an important condition for a better definition of the transmission of P. carinii pneumonia. In this work, Pneumocystis in vitro culture was performed on monolayers of L2 rat lung epithelial-like cells. Ultrastructural assessment revealed that culture parasites were structurally intact. Pneumocystis culture samples were intratracheally inoculated into corticosteroid-treated nude rats (nonlatently infected by P. carinii), which developed P. carinii pneumonia at 40 days postinoculation. The infectious power of parasites obtained in vitro was 7-10 times higher than that of parasites freshly extracted from parasitized rat lung. In summary, the present results show that it is possible to obtain in vitro highly infectious Pneumocystis forms, and this study provides a promising infectivity test for use by investigators working on Pneumocystis in vitro systems.

In vitro attachment of Pneumocystis carinii from mouse and rat origin.

The attachment of Pneumocystis carinii to lung cells could play a role in the pathophysiology of P carinii pneumonia. The trophozoite attaches to type I alveolar epithelial cells. Physical, chemical, and extracellular matrix factors, involved in the mouse-or rat-derived P carinii attachment to fibroblastic cells in culture, were examined using a new model of in vitro adherence. The development of parasite filopodia penetrating deeply the host cell cytoplasm was observed using transmission electronic microscopy. Killed P carinii organisms were unable to attach to cultured cells. Also, parasites were unable to attach to killed target cells. The P carinii in vitro attachment was partially inhibited by cytochalasin B. In contrast, the parasite attachment was not affected when the target cell cytoskeleton was altered. In our work conditions, sialic acids were not involved in the attachment process. Present results showed that fibronectin (Fn) plays a role in the parasite attachment, and suggest that a specific Fn-binding receptor is present at the surface of mouse-derived P carinii organisms.

Production of a monoclonal antibody using lymphocytes from Pneumocystis infected mice.

A colony of BALB/c mice maintained in a protected area of our laboratory was not infected with Pneumocystis carinii. During corticosteroid treatment, animals became infected by exposure to infected mice. After four months of corticosteroid treatment, BALB/c mice developed severe pneumocystosis. Stopping of treatment was associated with: (i) high mortality of mice, (ii) decreased lung parasite level and (iii) appearance of anti-P. carinii antibodies in survivors. A monoclonal antibody (MAb) 4F2 was obtained by immortalisation of spleen lymphocytes of a female BALB/c mouse 3 months after the cessation of corticosteroid treatment. The MAb 4F2 recognized a 210-220 kDa mouse P. carinii antigen, but did not react with rat-, rabbit- or human-derived P. carinii. This MAb reacted with all stages of mouse P. carinii.

In vitro pharmacodynamic parameters of sordarin derivatives in comparison with those of marketed compounds against Pneumocystis carinii isolated from rats.

Pneumocystis carinii pneumonia remains one of the most serious complications of immunosuppressed patients. In this study, the in vitro pharmacodynamic parameters of four sordarin derivatives (GM 191519, GM 237354, GM 193663, and GM 219771) have been evaluated by a new quantitative approach and compared with the commercially available drugs pentamidine, atovaquone, and trimethoprim-sulfamethoxazole (TMP-SMX). In vitro activities and in vivo therapeutic efficacies of sordarin derivatives against P. carinii were also evaluated. In vitro activity was determined by the broth microdilution technique, comparing the total number of microorganisms in treated and drug-free cultures by using Giemsa staining. The in vitro maximum effect (E(max)), the drug concentrations to reach 50% of E(max) (EC(50)), and the slope of the dose-response curve were then estimated by the Hill equation (E(max) sigmoid model). Sordarin derivatives were the most potent agents against P. carinii, with EC(50)s of 0.00025, 0.0007, 0.0043, and 0. 025 microg/ml for GM 191519, GM 237354, GM 193663, and GM 219771, respectively. The EC(50)s of pentamidine, atovaquone, and TMP-SMX were 0.025, 0.16, and 26.7/133.5 microg/ml, respectively. The results obtained with this approach showed GM 237354 and GM 191519 to be approximately 35- and 100-fold more active in vitro than pentamidine, the most active marketed compound. All sordarin derivatives tested were at least 5,000-fold more active in vitro than TMP-SMX. The three sordarin derivatives tested in vivo-GM 191519, GM 237354, and GM 219771-showed a marked therapeutic efficacy, defined as reduction of cyst forms per gram of lung. GM 191519 was the most potent (daily dose reducing 50% of the P. carinii burden in the lungs [ED(50)], 0.05 mg/kg/day) followed by GM 237354 and GM 219771 (ED(50)s, 0.30 and 0.49 mg/kg/day, respectively). Good agreement between in vitro parameters and in vivo outcome was obtained when P. carinii pneumonia in rats was treated with sordarin derivatives.

Surfactant changes during experimental pneumocystosis are related to Pneumocystis development.

Pneumocystosis-related surfactant changes have been reported in both humans and corticosteroid-treated experimental hosts. As corticosteroids induce an increase in pulmonary surfactant, some findings could be considered as controversial. The aim of this study was to investigate whether the surfactant composition changes during experimental pneumocystosis were related to the Pneumocystis development. In this work two corticosteroid-untreated animal models were used: rabbits, which develop spontaneous pneumocystosis at weaning; and severe combined immunodeficiency mice, which were intranasally inoculated with Pneumocystis carinii. Surfactant phospholipid and protein content was explored by bronchoalveolar lavage. The in vitro effect of surfactant on P. carinii growth was also explored. In the two models, the surfactant phospholipid/protein ratio was significantly increased, whereas parasite rates were low. This ratio decreases with the slope increase of the parasite growth curve. These early surfactant changes suggested that Pneumocystis proliferation requires alveolar lining fluid changes, and that normal surfactant is not suitable for parasite development. In this way, in vitro experiments presented here have revealed an inhibitory effect of synthetic or seminatural surfactants on the P. carinii growth. Further studies are needed to determine how Pneumocystis induces the reported early modifications of the surfactant, and why the parasite development is inhibited by pulmonary surfactant.

Ultrastructural observations on the attachment of Pneumocystis carinii in vitro.

Pneumocystis carinii trophozoites grow in vivo in close contact with host cells. The attachment of Pneumocystis to the lung cells seems to be a critical step in the parasite's development. Up to now, the contact of Pneumocystis with mammalian tissue culture cells was shown using light and scanning electron microscopy. The methods are not sufficient to observed in detail the parasite-feeder cell area of contact. In this work, the attachment of Pneumocystis trophozoites to feeder cells was examined in serial sections using transmission electron microscopy. When the contact of a trophozoite with a feeder cell took place, the development of filopodia penetrating deeply into invaginations of the feeder cell plasma membrane was observed. Then, the apical tips of filopodia become bulged anchoring the parasite to the feeder cell. The behaviour of Pneumocystis in feeder cell cultures is compared to that of the parasite in other in vitro or in vivo experimental models.