A Zn2+-triggered two-step mechanism of CLIC1 membrane insertion and activation into chloride channels.

The chloride intracellular channel (CLIC) protein family displays the unique feature of altering its structure from a soluble form to a membrane-bound chloride channel. CLIC1, a member of this family, is found in the cytoplasm or in internal and plasma membranes, with membrane relocalisation linked to endothelial disfunction, tumour proliferation and metastasis. The molecular switch promoting CLIC1 activation remains under investigation. Here, cellular Cl- efflux assays and immunofluorescence microscopy studies have identified intracellular Zn2+ release as the trigger for CLIC1 activation and membrane insertion. Biophysical assays confirmed specific binding to Zn2+, inducing membrane association and enhancing Cl- efflux in a pH-dependent manner. Together, our results identify a two-step mechanism with Zn2+ binding as the molecular switch promoting CLIC1 membrane insertion, followed by pH-mediated activation of Cl- efflux.

Immunological and senescence biomarker profiles in patients after spontaneous clearance of hepatitis C virus: gender implications for long-term health risk.

BACKGROUND: About 25% of patients with acute hepatitis C virus (HCV) infection show spontaneous clearance within the first six months of infection but may remain at risk of inflammaging, aging, and liver and non-liver disease complications. This study evaluated the differences in the plasma levels of immune checkpoints (ICs) and senescence-associated secretory phenotype (SASP) biomarkers between patients who had spontaneously eliminated HCV infection (SC group) and individuals without evidence of HCV infection (C group). METHODS: We performed a multicenter retrospective study of 56 individuals: 32 in the SC and 24 in the C groups. ICs and SASP proteins were analyzed using a Luminex 200TM analyzer. The statistical analysis used Generalized Linear Models with gamma distribution (log-link) adjusted by significant variables and sex. RESULTS: 13 ICs (BTLA, CD137(4-1BB), CD27, CD28, CD80, GITR, HVEM, IDO, LAG-3, PD-1, PD-L1, PD-L2, and TIM-3) and 13 SASP proteins (EGF, Eotaxin, IL-1alpha, IL-1RA, IL-8, IL-13, IL-18, IP-10, SDF-1alpha, HGF, beta-NGF, PLGF-1, and SCF) were significantly higher in SC group after approximately more than two years of HCV clearance. After stratifying by sex, differences remained significant for males, which showed higher levels for 13 ICs and 4 SASP proteins in SC. While only PD-L2 was significantly higher in SC women, and no differences in SASP were found. CONCLUSIONS: Higher plasma levels of different IC and SASP proteins were found in individuals after more than two years of HCV clearance, mainly in men. Alterations in these molecules might be associated with an increased risk of developing liver and non-hepatic diseases.

Resistance of Blastocystis to chlorine and hydrogen peroxide.

Blastocystis is a ubiquitous, widely distributed protist inhabiting the gastrointestinal tract of humans and other animals. The organism is genetically diverse, and so far, at least 28 subtypes (STs) have been identified with ST1-ST9 being the most common in humans. The pathogenicity of Blastocystis is controversial. Several routes of transmission have been proposed including fecal-oral (e.g., zoonotic, anthroponotic) and waterborne. Research on the latter has gained traction in the last few years with the organism having been identified in various bodies of water, tap water, and rainwater collection containers including water that has been previously filtered and/or chlorinated. Herein, we assessed the resistance of 11 strains maintained in culture, spanning ST1-ST9 to various chlorine and hydrogen peroxide concentrations for 24 h, and performed recovery assays along with re-exposure. Following the treatment with both compounds, all subtypes showed increased resistance, and viability could be visualized at the cellular level. These results are hinting at the presence of mechanism of resistance to both chlorine and hydrogen peroxide. As such, this pilot study can be the platform for developing guidelines for water treatment processes.

Localization and functional characterization of the alternative oxidase in Naegleria.

The alternative oxidase (AOX) is a protein involved in supporting enzymatic reactions of the Krebs cycle in instances when the canonical (cytochrome-mediated) respiratory chain has been inhibited, while allowing for the maintenance of cell growth and necessary metabolic processes for survival. Among eukaryotes, alternative oxidases have dispersed distribution and are found in plants, fungi, and protists, including Naegleria ssp. Naegleria species are free-living unicellular amoeboflagellates and include the pathogenic species of N. fowleri, the so-called "brain-eating amoeba." Using a multidisciplinary approach, we aimed to understand the evolution, localization, and function of AOX and the role that plays in Naegleria's biology. Our analyses suggest that AOX was present in last common ancestor of the genus and structure prediction showed that all functional residues are also present in Naegleria species. Using cellular and biochemical techniques, we also functionally characterize N. gruberi's AOX in its mitochondria, and we demonstrate that its inactivation affects its proliferation. Consequently, we discuss the benefits of the presence of this protein in Naegleria species, along with its potential pathogenicity role in N. fowleri. We predict that our findings will spearhead new explorations to understand the cell biology, metabolism, and evolution of Naegleria and other free-living relatives.

In vitro anti-Acanthamoeba activity of flavonoid glycosides isolated from Delphinium gracile, D. staphisagria, Consolida oliveriana and Aconitum napellus.

Acanthamoeba spp. are widely distributed in the environment and cause serious infections in humans. Treatment of Acanthamoeba infections is very challenging and not always effective which requires the development of more efficient drugs against Acanthamoeba spp. The purpose of the present study was to test medicinal plants that may be useful in the treatment of Acanthamoeba spp. Here we evaluated the trophozoital and cysticidal activity of 13 flavonoid glycosides isolated from Delphinium gracile, D. staphisagria, Consolida oliveriana and from Aconitum napellus subsp. Lusitanicum against the amoeba Acanthamoeba castellanii. AlamarBlue Assay Reagent® was used to determine the activity against trophozoites of A. castellanii, and cytotoxic using Vero cells. Cysticidal activity was assessed on treated cysts by light microscopy using a Neubauer chamber to quantify cysts and trophozoites. Flavonoids 1, 2, 3 and 4 showed higher trophozoital activity and selectivity indexes than the reference drug chlorhexidine digluconate. In addition, flavonoid 2 showed 100% cysticidal activity at a concentration of 50 µm, lower than those of the reference drug and flavonoid 3 (100 µm). These results suggest that flavonoids 2 and 3 might be used for the development of novel therapeutic approaches against Acanthamoeba infections after satisfactory in vivo evaluations.

Assessing the effectiveness of AS-48 in experimental mice models of Chagas' disease.

OBJECTIVES: We report the in vivo trypanocidal activity of the bacteriocin AS-48 (lacking toxicity), which is produced by Enterococcus faecalis, against the flagellated protozoan Trypanosoma cruzi, the aetiological agent of Chagas' disease. METHODS: We determined the in vivo activity of AS-48 against the T. cruzi Arequipa strain in BALB/c mice (in both acute and chronic phases of Chagas' disease). We evaluated the parasitaemia, the reactivation of parasitaemia after immunosuppression and the nested parasites in the chronic phase by PCR in target tissues. RESULTS: AS-48 reduced the parasitaemia profile in acute infection and showed a noteworthy reduction in the parasitic load in chronic infection after immunosuppression according to the results obtained by PCR (double-checking to demonstrate cure). CONCLUSIONS: AS-48 is a promising alternative that provides a step forward in the development of a new therapy against Chagas' disease.

In Vivo Biological Evaluation of a Synthetic Royleanone Derivative as a Promising Fast-Acting Trypanocidal Agent by Inducing Mitochondrial-Dependent Necrosis.

The life-long and life-threatening Chagas disease is one of the most neglected tropical diseases caused by the protozoan parasite Trypanosoma cruzi. It is a major public health problem in Latin America, as six to seven million people are infected, being the principal cause of mortality in many endemic regions. Moreover, Chagas disease has become widespread due to migrant populations. Additionally, there are no vaccines nor effective treatments to fight the disease because of its long-term nature and complex pathology. Therefore, these facts emphasize how crucial the international effort for the development of new treatments against Chagas disease is. Here, we present the in vitro and in vivo trypanocidal activity of some oxygenated abietane diterpenoids and related compounds. The 1,4-benzoquinone 15, not yet reported, was identified as a fast-acting trypanocidal drug with efficacy against different strains in vitro and higher activity and lower toxicity than benznidazole in both phases of murine Chagas disease. The mode of action was also evaluated, suggesting that quinone 15 kills T. cruzi by inducing mitochondrion-dependent necrosis through a bioenergetics collapse caused by a mitochondrial membrane depolarization and iron-containing superoxide dismutase inhibition. Therefore, the abietane 1,4-benzoquinone 15 can be considered as a new candidate molecule for the development of an appropriate and commercially accessible anti-Chagas drug.

Repositioning of leishmanicidal [1,2,3]Triazolo[1,5-a]pyridinium salts for Chagas disease treatment: Trypanosoma cruzi cell death involving mitochondrial membrane depolarisation and Fe-SOD inhibition.

Trypanosomatidae is a family of unicellular parasites belonging to the phylum Euglenozoa, which are causative agents in high impact human diseases such as Leishmaniasis, Chagas disease and African sleeping sickness. The impact on human health and local economies, together with a lack of satisfactory chemotherapeutic treatments and effective vaccines, justifies stringent research efforts to search for new disease therapies. Here, we present in vitro trypanocidal activity data and mode of action data, repositioning leishmanicidal [1,2,3]Triazolo[1,5-a]pyridinium salts against Trypanosoma cruzi, the aetiological agent of Chagas disease. This disease is one of the most neglected tropical diseases and is a major public health issue in Central and South America. The disease affects approximately 6-7 million people and is widespread due to increased migratory movements. We screened a suite of leishmanicidal [1,2,3]Triazolo[1,5-a]pyridinium salt compounds, of which compounds 13, 20 and 21 were identified as trypanocidal drugs. These compounds caused cell death in a mitochondrion-dependent manner through a bioenergetic collapse. Moreover, compounds 13 and 20 showed a remarkable inhibition of iron superoxide dismutase activity of T. cruzi, a key enzyme in the protection from the damage produced by oxidative stress.

Rational modification of Mannich base-type derivatives as novel antichagasic compounds: Synthesis, in vitro and in vivo evaluation.

The current chemotherapy against Chagas disease is inadequate and insufficient. A series of ten Mannich base-type derivatives have been synthesized to evaluate their in vitro antichagasic activity. After a preliminary screening, compounds 7 and 9 were subjected to in vivo assays in a murine model. Both compounds caused a substantial decrease in parasitemia in the chronic phase, which was an even better result than that of the reference drug benznidazole. In addition, compound 9 also showed better antichagasic activity during the acute phase. Moreover, metabolite excretion, effect on mitochondrial membrane potential and the inhibition of superoxide dismutase (SOD) studies were also performed to identify their possible mechanism of action. Finally, docking studies proposed a binding mode of the Fe-SOD enzyme similar to our previous series, which validated our design strategy. Therefore, the results suggest that these compounds should be considered for further preclinical evaluation as antichagasic agents.

Synthesis and biological evaluation of new long-chain squaramides as anti-chagasic agents in the BALB/c mouse model.

Chagas Disease is caused by infection with the insect-transmitted protozoan Trypanosoma cruzi and affects more than 10 million people. It is a paradigmatic example of a chronic disease without an effective treatment in Latin America where the current therapies, based on Benznidazole and Nifurtimox, are characterised by limited efficacy, toxic side-effects and frequent failures in the treatment. We present a series of new long-chain squaramides, identified based on their 1H and 13C NMR spectra, and their trypanocidal activity and cytotoxicity were tested in vitro through the determination of IC50 values. Compounds 4 and 7 were more active and less toxic than the reference drug Benznidazole, and these results were the basis of promoting in vivo assays, where parasitaemia levels, assignment of cure, reactivation of parasitaemia and others parameters were determined in mice treated in both the acute and chronic phases. Finally, the mechanisms of action were elucidated at metabolic and mitochondrial levels and superoxide dismutase inhibition. The experiments allowed us to select compound 7 as a promising candidate for treating Chagas Disease, where the activity, stability and low cost make long-chain squaramides appropriate molecules for the development of an affordable anti-chagasic agent versus current treatments.

In vitro assessment of 3-alkoxy-5-nitroindazole-derived ethylamines and related compounds as potential antileishmanial drugs.

Leishmaniasis is a widespread neglected tropical disease complex that is responsible of one million new cases per year. Current treatments are outdated and pose many problems that new drugs need to overcome. With the goal of developing new, safe, and affordable drugs, we have studied the in vitro activity of 12 different 5-nitroindazole derivatives that showed previous activity against different strains of Trypanosoma cruzi in a previous work. T. cruzi belongs to the same family as Leishmania spp., and treatments for the disease it produces also needs renewal. Among the derivatives tested, compounds 1, 2, 9, 10, 11, and 12 showed low J774.2 macrophage toxicity, while their effect against both intracellular and extracellular forms of the studied parasites was higher than the ones found for the reference drug Meglumine Antimoniate (Glucantime®). In addition, their Fe-SOD inhibitory effect, the infection rates, metabolite alteration, and mitochondrial membrane potential of the parasites treated with the selected drugs were studied in order to gain insights into the action mechanism, and the results of these tests were more promising than those found with glucantime, as the leishmanicidal effect of these new drug candidates was higher. The promising results are encouraging to test these derivatives in more complex studies, such as in vivo studies and other experiments that could find out the exact mechanism of action.

Effective Tetradentate Compound Complexes against Leishmania spp. that Act on Critical Enzymatic Pathways of These Parasites.

The spectrum and efficacy of available antileishmanial drugs is limited. In the present work we evaluated in vitro the antiproliferative activity of 11 compounds based on tetradentate polyamines compounds against three Leishmania species (L. braziliensis, L. donovani and L. infantum) and the possible mechanism of action. We identified six compounds (3, 5, 6, 7, 8 and 10) effective against all three Leishmania spp both on extracellular and intracellular forms. These six most active leishmanicidal compounds also prevent the infection of host cells. Nevertheless, only compound 7 is targeted against the Leishmania SOD. Meanwhile, on the glucose metabolism the tested compounds have a species-specific effect on Leishmania spp.: L. braziliensis was affected mainly by 10 and 8, L. donovani by 7, and L. infantum by 5 and 3. Finally, the cellular ultrastructure was mainly damaged by 11 in the three Leishmania spp. studied. These identified antileishmania candidates constitute a good alternative treatment and will be further studied.

Library of Seleno-Compounds as Novel Agents against Leishmania Species.

The in vitro leishmanicidal activities of a series of 48 recently synthesized selenium derivatives against Leishmania infantum and Leishmania braziliensis parasites were tested using promastigotes and intracellular amastigote forms. The cytotoxicity of the tested compounds for J774.2 macrophage cells was also measured in order to establish their selectivity. Six of the tested compounds (compounds 8, 10, 11, 15, 45, and 48) showed selectivity indexes higher than those of the reference drug, meglumine antimonate (Glucantime), for both Leishmania species; in the case of L. braziliensis, compound 20 was also remarkably selective. Moreover, data on infection rates and amastigote numbers per macrophage showed that compounds 8, 10, 11, 15, 45, and 48 were the most active against both Leishmania species studied. The observed changes in the excretion product profile of parasites treated with these six compounds were also consistent with substantial cytoplasmic alterations. On the other hand, the most active compounds were potent inhibitors of Fe superoxide dismutase (Fe-SOD) in the two parasite species considered, whereas their impact on human CuZn-SOD was low. The high activity, low toxicity, stability, low cost of the starting materials, and straightforward synthesis make these compounds appropriate molecules for the development of affordable antileishmanicidal agents.

In vitro antileishmanial activity and iron superoxide dismutase inhibition of arylamine Mannich base derivatives.

Leishmaniasis is one of the world's most neglected diseases, and it has a worldwide prevalence of 12 million. There are no effective human vaccines for its prevention, and treatment is hampered by outdated drugs. Therefore, research aiming at the development of new therapeutic tools to fight leishmaniasis remains a crucial goal today. With this purpose in mind, we present 20 arylaminoketone derivatives with a very interesting in vitro and in vivo efficacy against Trypanosoma cruzi that have now been studied against promastigote and amastigote forms of Leishmania infantum, Leishmania donovani and Leishmania braziliensis strains. Six out of the 20 Mannich base-type derivatives showed Selectivity Index between 39 and 2337 times higher in the amastigote form than the reference drug glucantime. These six derivatives affected the parasite infectivity rates; the result was lower parasite infectivity rates than glucantime tested at an IC25 dose. In addition, these derivatives were substantially more active against the three Leishmania species tested than glucantime. The mechanism of action of these compounds has been studied, showing a greater alteration in glucose catabolism and leading to greater levels of iron superoxide dismutase inhibition. These molecules could be potential candidates for leishmaniasis chemotherapy.

Effective anti-leishmanial activity of minimalist squaramide-based compounds.

In order to evaluate the in vitro leishmanicidal activity of N,N'-Squaramides derivatives, compounds that feature both hydrogen bond donor and acceptor groups and are capable of multiple interactions with complementary sites, against Leishmania infantum, Leishmania braziliensis and Leishmania donovani a series of 18compounds was prepared and assayed on extracellular and intracellular parasite forms. Infectivity and cytotoxicity tests were performed on J774.2 macrophage cells using meglumine antimoniate (Glucantime) as the reference drug. Changes in metabolite excretion by 1H-NMR and the ultrastructural alterations occurring in the parasites treated using transmission electron microscopy (TEM), was analyzed. Compounds 1, 7, 11, 14 and 17 were the more active and less toxic. Infection rates showed that the order of effectiveness was 17 > 11 > 14 > 7 for both L. infantum and L. braziliensis and in the same way, the compound 1 for L. donovani. All these compounds have altered the typical structure of the promastigotes, glycosomes and mitochondria. These severe modifications by the compounds are the ultimate reasons for the alterations observed in the excretion products. The Squaramide 17 (3-(butylamino)-4-((3-(dimetilamino)propyl)(methyl)amino)cyclobut-3-en-1,2-dione) was clearly the most efficient of all compounds. The data appear to confirm that the severe modifications generated in organelles such as glycosomes or mitochondria by the compounds are the ultimate reasons for the alterations observed in the excretion products of all species. The activity, stability, low cost of starting materials, and straightforward synthesis make amino squaramides appropriate molecules for the development of an affordable anti-leishmanial agent.

Plasma metabolomic profile is near-normal in people with HIV on long-term suppressive antiretroviral therapy.

Background: Combination antiretroviral therapy (ART) has transformed human immunodeficiency virus (HIV) infection in people with HIV (PWH). However, a chronic state of immune activation and inflammation is maintained despite achieving HIV suppression and satisfactory immunological recovery. We aimed to determine whether the plasma metabolomic profile of PWH on long-term suppressive ART and immunologically recovered approximates the normality by comparison with healthy controls with similar age and gender. Methods: We carried out a cross-sectional study in 17 PWH on long-term ART (HIV-RNA <50 copies/mL, CD4+ ≥500 cells/mm3, and CD4+/CD8+ ≥1) and 19 healthy controls with similar age and gender. Metabolomics analysis was performed by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). The statistical association analysis was performed by principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and Generalized Linear Models (GLM) with a gamma distribution (log-link). Significance levels (p-value) were corrected for multiple testing (q-value). Results: PCA and PLS-DA analyses found no relevant differences between groups. Adjusted GLM showed 14 significant features (q-value<0.20), of which only three could be identified: lysophosphatidylcholine (LysoPC) (22:6) (q-value=0.148), lysophosphatidylethanolamine (LysoPE) (22:6) (q-value=0.050) and hydroperoxy-octadecatrienoic acid (HpOTrE)/dihydroperoxy-octadecatrienoic acid (DiHOTrE)/epoxy-octadecadienoic acid (EpODE) (q-value=0.136). These significant identified metabolites were directly correlated to plasma inflammatory biomarkers in PWH and negatively correlated in healthy controls. Conclusion: PWH on long-term ART have a metabolomic profile that is almost normal compared to healthy controls. Nevertheless, residual metabolic alterations linked to inflammatory biomarkers persist, which could favor the development of age-related comorbidities among this population.

Predictive plasma biomarkers of long-term increase in hepatic steatosis index after HCV eradication in HIV/HCV-coinfected patients.

Hepatic steatosis is a common condition found in the liver of hepatitis C virus (HCV)-infected patients, contributing to more severe forms of liver disease. In addition, the human immunodeficiency virus (HIV) may accelerate this process. Alternatively, several immune checkpoint proteins have been reported to be upregulated and correlated with disease progression during HCV and HIV infections. In steatosis, a detrimental immune system activation has been established; however, the role of the immune checkpoints has not been addressed so far. Thus, this study aimed to evaluate the association between plasma immune checkpoint proteins at baseline (before antiviral therapy) with hepatic steatosis index (HSI) increase at the end of follow-up (∼ five years after sustained virologic response (SVR)). We performed a multicenter retrospective study in 62 patients coinfected with HIV/HCV who started antiviral therapy. Immune checkpoint proteins were analyzed at baseline using a Luminex 200TM analyzer. The statistical association analysis was carried out using Generalized Linear Models (GLM) and Partial Least Squares Discriminant Analysis (PLS-DA). Fifty-three percent of the patients showed HSI increase from baseline to the end of follow-up. Higher immune checkpoint protein levels of BTLA, CD137(4-1BB), CD80, GITR, LAG-3, and PD-L1 before HCV therapy were associated with a long-term increase in HSI after successful HCV therapy, suggesting a potential predictive role for early detection of progression towards steatosis in HIV/HCV-coinfected patients.

Biological characteristics of the Trypanosoma cruzi Arequipa strain make it a good model for Chagas disease drug discovery.

Trypanosoma cruzi, the causative agent of Chagas disease (CD), is a genuine parasite with tremendous genetic diversity and a complex life cycle. Scientists have studied this disease for more than 100 years, and CD drug discovery has been a mainstay due to the absence of an effective treatment. Technical advances in several areas have contributed to a better understanding of the complex biology and life cycle of this parasite, with the aim of designing the ideal profile of both drug and therapeutic options to treat CD. Here, we present the T. cruzi Arequipa strain (MHOM/Pe/2011/Arequipa) as an interesting model for CD drug discovery. We characterized acute-phase parasitaemia and chronic-phase tropism in BALB/c mice and determined the in vitro and in vivo benznidazole susceptibility profile of the different morphological forms of this strain. The tropism of this strain makes it an interesting model for the screening of new compounds with a potential anti-Chagas profile for the treatment of this disease.

An Updated View of the Trypanosoma cruzi Life Cycle: Intervention Points for an Effective Treatment.

Chagas disease (CD) is a parasitic, systemic, chronic, and often fatal illness caused by infection with the protozoan Trypanosoma cruzi. The World Health Organization classifies CD as the most prevalent of poverty-promoting neglected tropical diseases, the most important parasitic one, and the third most infectious disease in Latin America. Currently, CD is a global public health issue that affects 6-8 million people. However, the current approved treatments are limited to two nitroheterocyclic drugs developed more than 50 years ago. Many efforts have been made in recent decades to find new therapies, but our limited understanding of the infection process, pathology development, and long-term nature of this disease has made it impossible to develop new drugs, effective treatment, or vaccines. This Review aims to provide a comprehensive update on our understanding of the current life cycle, new morphological forms, and genetic diversity of T. cruzi, as well as identify intervention points in the life cycle where new drugs and treatments could achieve a parasitic cure.

5-Nitroindazole derivatives as potential therapeutic alternatives against Acanthamoeba castellanii.

Amoebas of the genus Acanthamoeba are distributed worldwide, including species with a high pathogenic capacity for humans. In a similar way to what occurs with other parasitic protozoa, the available treatments show variable effectiveness in addition to high toxicity, which demands the development of new treatments. Positive results of 5-nitroindazole derivatives against several protozoa parasites suggest that these compounds may be a promising tool for the development of efficient antiparasitic drugs. In the present work we have evaluated the in vitro activity of ten 5-nitroindazole derivatives against Acanthamoeba castellanii trophozoites and cysts. To that end, AlamarBlue Assay Reagent® was used to determine the activity against trophozoites compared to the reference drug chlorhexidine digluconate. Cytotoxicity of the compounds was evaluated using Vero cells. The activity on cysts was evaluated by light microscopy and using a Neubauer chamber to quantifying cysts and presence of trophozoites, as an indication of cyst. Our results showed the effectiveness of the 5-nitroindazole derivatives tested against both trophozoites and cysts of A. castellani highlighting 5-nitroindazole derivative 8 which showed a 80% activity on cysts, which is higher than that of the reference drug. Moreover, 5-nitroindazole derivatives 8, 9 and 10 were more effective on trophozoites than the reference drug showing IC50 values lower than 5 µM. Taking together these results, these 5-nitroindazole derivatives specially compound 8, might be a promising alternative for the development of more efficient treatments against A. castellani infection.