Genomic and Phenotypic Characterization of Experimentally Selected Resistant Leishmania donovani Reveals a Role for Dynamin-1-Like Protein in the Mechanism of Resistance to a Novel Antileishmanial Compound.

The implementation of prospective drug resistance (DR) studies in the research-and-development (R&D) pipeline is a common practice for many infectious diseases but not for neglected tropical diseases (NTDs). Here, we explored and demonstrated the importance of this approach using as paradigms Leishmania donovani, the etiological agent of visceral leishmaniasis (VL), and TCMDC-143345, a promising compound of the GlaxoSmithKline (GSK) "Leishbox" to treat VL. We experimentally selected resistance to TCMDC-143345 in vitro and characterized resistant parasites at the genomic and phenotypic levels. We found that it took more time to develop resistance to TCMDC-143345 than to other drugs in clinical use and that there was no cross-resistance to these drugs, suggesting a new and unique mechanism. By whole-genome sequencing, we found two mutations in the gene encoding the L. donovani dynamin-1-like protein (LdoDLP1) that were fixed at the highest drug pressure. Through phylogenetic analysis, we identified LdoDLP1 as a family member of the dynamin-related proteins, a group of proteins that impacts the shapes of biological membranes by mediating fusion and fission events, with a putative role in mitochondrial fission. We found that L. donovani lines genetically engineered to harbor the two identified LdoDLP1 mutations were resistant to TCMDC-143345 and displayed altered mitochondrial properties. By homology modeling, we showed how the two LdoDLP1 mutations may influence protein structure and function. Taken together, our data reveal a clear involvement of LdoDLP1 in the adaptation/reduced susceptibility of L. donovani to TCMDC-143345. IMPORTANCE Humans and their pathogens are continuously locked in a molecular arms race during which the eventual emergence of pathogen drug resistance (DR) seems inevitable. For neglected tropical diseases (NTDs), DR is generally studied retrospectively once it has already been established in clinical settings. We previously recommended to keep one step ahead in the host-pathogen arms race and implement prospective DR studies in the R&D pipeline, a common practice for many infectious diseases but not for NTDs. Here, using Leishmania donovani, the etiological agent of visceral leishmaniasis (VL), and TCMDC-143345, a promising compound of the GSK Leishbox to treat VL, as paradigms, we experimentally selected resistance to the compound and proceeded to genomic and phenotypic characterization of DR parasites. The results gathered in the present study suggest a new DR mechanism involving the L. donovani dynamin-1-like protein (LdoDLP1) and demonstrate the practical relevance of prospective DR studies.

The potential role of nicotinamide on Leishmania tropica: An assessment of inhibitory effect, cytokines gene expression and arginase profiling.

Leishmaniasis represents a major health concern worldwide which has no effective treatment modality. Nicotinamide (NAm) has been used for a wide range of applications from anticancer to antimicrobial usage. This study aimed to assess the effect of NAm combination on Leishmania tropica Inhibition, as well as on cytokines gene expression and arginase (ARG) activity in L. tropica-infected macrophages in an in vitro model. The leishmanicidal effects of NAm and Glucantime (meglumine antimoniate, MA) alone and in combination (NAm/MA) were evaluated using a colorimetric assay and macrophage model. Additionally, immunomodulatory effects and enzymatic activity were assessed by analyzing Th1 and Th2 cytokines gene expression and ARG level, respectively, in infected macrophages treated with NAm and MA, alone and in combination. Findings indicated that the NAm/MA combination demonstrated greater inhibitory effects on L. tropica promastigotes and amastigotes compared with each drug individually. Docking results proved the affinity of NAm to IFN-γ, which can affirm the increased levels of IFN-γ, IL-12p40 and TNF-α as well as reductions in IL-10 secretion with a dose-response effect, especially in the combination group. The NAm/MA combination also showed a significant reduction in the level of ARG activity at all concentrations used compared to each drug individually. These findings indicate higher effectiveness of NAm plus MA in reducing parasite growth, promoting immune response and inhibiting ARG level. This combination should be considered as a potential therapeutic regimen for treatment of volunteer patients with anthroponotic cutaneous leishmaniasis (ACL) in future control programs.

Targeting Parasite-Produced Macrophage Migration Inhibitory Factor as an Antivirulence Strategy With Antibiotic-Antibody Combination to Reduce Tissue Damage.

Targeting virulence factors represents a promising alternative approach to antimicrobial therapy, through the inhibition of pathogenic pathways that result in host tissue damage. Yet, virulence inhibition remains an understudied area in parasitology. Several medically important protozoan parasites such as Plasmodium, Entamoeba, Toxoplasma, and Leishmania secrete an inflammatory macrophage migration inhibitory factor (MIF) cytokine homolog, a virulence factor linked to severe disease. The aim of this study was to investigate the effectiveness of targeting parasite-produced MIF as combination therapy with standard antibiotics to reduce disease severity. Here, we used Entamoeba histolytica as the model MIF-secreting protozoan, and a mouse model that mirrors severe human infection. We found that intestinal inflammation and tissue damage were significantly reduced in mice treated with metronidazole when combined with anti-E. histolytica MIF antibodies, compared to metronidazole alone. Thus, this preclinical study provides proof-of-concept that combining antiparasite MIF-blocking antibodies with current standard-of-care antibiotics might improve outcomes in severe protozoan infections.

A Pluronic® F127-based polymeric micelle system containing an antileishmanial molecule is immunotherapeutic and effective in the treatment against Leishmania amazonensis infection.

Clioquinol (5-chloro-7-iodoquinolin-8-ol or ICHQ) was recently showed to presents an in vitro effective antileishmanial action, causing changes in membrane permeability, mitochondrial functionality, and parasite morphology. In the present study, ICHQ was incorporated into a Poloxamer 407-based polymeric micelles system (ICHQ/M), and its antileishmanial activity was in vivo evaluated in L. amazonensis-infected BALB/c mice. Amphotericin B (AmpB) and its liposomal formulation (Ambisome®) were used as controls. Parasitological and immunological evaluations were performed 30 days after the treatment. Results indicated more significant reductions in the average lesion diameter and parasite burden in ICHQ or ICHQ/M-treated mice, which were associated with the development of a polarized Th1 immune response, based on production of high levels of IFN-γ, IL-12, TNF-α, GM-CSF, and antileishmanial IgG2a antibody. Control groups´ mice produced high levels of IL-4, IL-10, and IgG1 isotype antibody. No organic toxicity was found by using ICHQ or ICHQ/M to treat the animals, although those receiving AmpB and Ambisome® have presented higher levels of renal and hepatic damage markers. In conclusion, results suggested that the ICHQ/M composition can be considered as an antileishmanial candidate to be tested against human leishmaniasis.

Prospects for antimicrobial peptide-based immunotherapy approaches in Leishmania control.

INTRODUCTION: Leishmaniasis is one of the neglected tropical diseases and is highly endemic in many countries. Currently, there is no adequate human vaccine and treatment to control the disease. Areas covered: As a result of the failure of chemotherapy and toxicity, it is necessary to find another approach for the treatment of leishmaniasis. Recently, antimicrobial peptides (AMPs), originating from natural resources, have attracted much attention for their use as a new antibiotics for many infectious and noninfectious diseases. Natural AMPs are named interchangeably as host defense peptides. They are naturally active in the innate immune system as a primary defense mechanism in most species all over the world. Several AMPs have been tested in in vitro and in vivo experiments against leishmaniasis. Expert commentary: Most AMPs require proper conformation to be active. Leishmania (L.) tarentolae as a nonpathogenic strain, is an effective tool not only for vaccine development but also for therapy. Recombinant L. tarentolae expressing selective or combined AMPs is a suggestive approach for leishmaniasis or any other infectious disease treatment.

Leishmania Antigenuria to Predict Initial Treatment Failure and Relapse in Visceral Leishmaniasis/HIV Coinfected Patients: An Exploratory Study Nested Within a Clinical Trial in Ethiopia.

Background: Biomarkers predicting the risk of VL treatment failure and relapse in VL/HIV coinfected patients are needed. Nested within a two-site clinical trial in Ethiopia (2011-2015), we conducted an exploratory study to assess whether (1) levels of Leishmania antigenuria measured at VL diagnosis were associated with initial treatment failure and (2) levels of Leishmania antigenuria at the end of treatment (parasitologically-confirmed cure) were associated with subsequent relapse. Methods:Leishmania antigenuria at VL diagnosis and cure was determined using KAtex urine antigen test and graded as negative (0), weak/moderate (grade 1+/2+) or strongly-positive (3+). Logistic regression and Kaplan-Meier methods were used to assess the association between antigenuria and (1) initial treatment failure, and (2) relapse over the 12 months after cure, respectively. Results: The analysis to predict initial treatment failure included sixty-three coinfected adults [median age: 30 years interquartile range (IQR) 27-35], median CD4 count: 56 cells/µL (IQR 38-113). KAtex results at VL diagnosis were negative in 11 (17%), weak/moderate in 17 (27%) and strongly-positive in 35 (36%). Twenty (32%) patients had parasitologically-confirmed treatment failure, with a risk of failure of 9% (1/11) with KAtex-negative results, 0% (0/17) for KAtex 1+/2+ and 54% (19/35) for KAtex 3+ results. Compared to KAtex-negative patients, KAtex 3+ patients were at increased risk of treatment failure [odds ratio 11.9 (95% CI 1.4-103.0); P: 0.025]. Forty-four patients were included in the analysis to predict relapse [median age: 31 years (IQR 28-35), median CD4 count: 116 cells/µL (IQR 95-181)]. When achieving VL cure, KAtex results were negative in 19 (43%), weak/moderate (1+/2+) in 10 (23%), and strongly positive (3+) in 15 patients (34%). Over the subsequent 12 months, eight out of 44 patients (18%) relapsed. The predicted 1-year relapse risk was 6% for KAtex-negative results, 14% for KAtex 1+/2+ and 42% for KAtex 3+ results [hazard ratio of 2.2 (95% CI 0.1-34.9) for KAtex 1+/2+ and 9.8 (95% CI 1.8-82.1) for KAtex 3+, compared to KAtex negative patients; P: 0.03]. Conclusion: A simple field-deployable Leishmania urine antigen test can be used for risk stratification of initial treatment failure and VL relapse in HIV-patients. A dipstick-format would facilitate field implementation.

Building confidence in quantitative systems pharmacology models: An engineer's guide to exploring the rationale in model design and development.

This tutorial promotes good practice for exploring the rationale of systems pharmacology models. A safety systems engineering inspired notation approach provides much needed rigor and transparency in development and application of models for therapeutic discovery and design of intervention strategies. Structured arguments over a model's development, underpinning biological knowledge, and analyses of model behaviors are constructed to determine the confidence that a model is fit for the purpose for which it will be applied.

Elevated TGF-beta levels in drug-resistant visceral leishmaniasis.

BACKGROUND: Poor and neglected populations in Africa are particularly affected with visceral leishmaniasis. The widespread emergence of resistance to pentavalent antimonials occurs globally and the unavailability of a vaccine in clinical use constitutes a major obstacle in disease control. OBJECTIVE: To investigate the cytokine profile in human visceral leishmaniasis. DESIGN: A cross-sectional laboratory-based study. SETTING: Single center study carried out at the Institute of Endemic Diseases, University of Khartoum, Sudan. PATIENTS AND METHODS: Soluble lysates of L major and L donovani were used to stimulate the lymphocytes of two groups of confirmed VL patients (group 1 [n=20] had respond to pentostam treatment and group 2 [n=5] were recorded as drug resistant after follow up) in a cellular proliferation assay and the levels of IFNg, IL-10, TNFa and TGFb were detected by cytokine ELISA. MAIN OUTCOME MEASURES: Levels of IFNg, TNFa, IL-10 and TGFb. RESULTS: A significant increase of IFNg and TNFa levels were reported in stimulated cells of drug susceptible and drug resistant groups, but no significant difference in IL-10 production was observed between the different antigens or between the patients groups. TGFb from stimulated lymphocytes was secreted in statistically significant amounts in patients reported as drug resistant in response to both L major and L donovani antigens (P < .001). CONCLUSIONS: In VL patients, IFNg and TNFa are extremely produced in response to in vitro re-stimulation which means that the parasitic infection, although virulent and chronic, does not render patients as immunocompromised. However, TGFb is mostly associated with treatment failure. LIMITATIONS: This study assessed secretory TGFb. A study with a larger sample size to assess TGFb gene expression and to follow its intracytoplasmic synthesis in drug resistant VL patients is recommended.

Successful Therapy of Murine Visceral Leishmaniasis with Astrakurkurone, a Triterpene Isolated from the Mushroom Astraeus hygrometricus, Involves the Induction of Protective Cell-Mediated Immunity and TLR9.

In our previous report, we showed that astrakurkurone, a triterpene isolated from the Indian mushroom Astraeus hygrometricus (Pers.) Morgan, induced reactive oxygen species, leading to apoptosis in Leishmania donovani promastigotes, and also was effective in inhibiting intracellular amastigotes at the 50% inhibitory concentration of 2.5 µg/ml. The aim of the present study is to characterize the associated immunomodulatory potentials and cellular activation provided by astrakurkurone, leading to effective antileishmanial activity in vitro and in vivo Astrakurkurone-mediated antileishmanial activity was evaluated by real-time PCR and flow cytometry. The involvement of Toll-like receptor 9 (TLR9) was studied by in vitro assay in the presence of a TLR9 agonist and antagonist and by in silico modeling of a three-dimensional structure of the ectodomain of TLR9 and its interaction with astrakurkurone. Astrakurkurone caused a significant increase in TLR9 expression of L. donovani-infected macrophages along with the activation of proinflammatory responses. The involvement of TLR9 in astrakurkurone-mediated amastigote killing has been evidenced from the fact that a TLR9 agonist (CpG, ODN 1826) in combination with astrakurkurone enhanced the amastigote killing, while a TLR9 antagonist (bafilomycin A1) alone or in combination with astrakurkurone curbed the amastigote killing, which could be further justified by in silico evidence of docking between mouse TLR9 and astrakurkurone. Astrakurkurone was found to reduce the parasite burden in vivo by inducing protective cytokines, gamma interferon and interleukin 17. Moreover, astrakurkurone was nontoxic toward peripheral blood mononuclear cells of immunocompromised patients with visceral leishmaniasis. Astrakurkurone, a nontoxic antileishmanial, enhances the immune efficiency of host cells, leading to parasite clearance in vitro and in vivo.

2-Amino-thiophene derivatives present antileishmanial activity mediated by apoptosis and immunomodulation in vitro.

This study evaluated the effects of 2-amino-thiophene derivatives on the promastigote and amastigote forms of Leishmania (Leishmania) amazonensis and their possible mechanisms of action. Initially, we evaluated the antileishmanial activity of ten 2-amino-thiophene derivatives on promastigote and axenic amastigote forms of Leishmania amazonensis and their cytotoxicity against murine macrophages and human red blood cells. Three promising compounds were selected for studies of the cell death process using flow cytometry analysis and a DNA fragmentation assay. The effects of the compounds were assessed on intramacrophagic amastigotes, and the modulation of cytokine and NO production was investigated. All thiophene derivatives showed antileishmanial activity against promastigotes and axenic amastigotes with less toxicity for murine macrophages and human red blood cells. The best values were obtained for compounds containing a lateral indole ring. Docking studies suggested that these compounds played an important role in inhibiting trypanothione reductase (TryR) activity. The selected compounds SB-200, SB-44, and SB-83 induced apoptosis in promastigotes involving phosphatidylserine externalization and DNA fragmentation in a pattern similar to that observed for the positive control. Additionally, SB-200, SB-44, and SB-83 significantly reduced the infection index of macrophages by the parasites; for compounds SB-200 and SB-83 this reduction was associated with increased TNF-α, IL-12, and NO levels. This study demonstrated the effective and selective action of 2-amino-thiophene derivatives against L. amazonensis, resulting in apoptosis-like cell death and immunomodulation in vitro. The results suggest that they are promising compounds for the development of new leishmanicidal drugs.

Th-1 biased immunomodulation and synergistic antileishmanial activity of stable cationic lipid-polymer hybrid nanoparticle: biodistribution and toxicity assessment of encapsulated amphotericin B.

To address issues related to Amphotericin B (AmpB) clinical applications, we developed macrophage targeted cationic stearylamine lipid-polymer hybrid nanoparticles (LPNPs) with complementary characteristics of both polymeric nanoparticles and liposomes, for enhancement of therapeutic efficacy and diminishing toxic effect of encapsulated AmpB. The LPNPs (size 198.3 ± 3.52 nm, PDI 0.135 ± 0.03, zeta potential +31.6 ± 1.91 mV) provide core-shell type structure which has the ability to encapsulate amphiphilic AmpB in higher amount (Encapsulation efficiency 96.1 ± 2.01%), sustain drug release and stabilize formulation tremendously. Attenuated erythrocytes and J774A.1 toxicity of LPNPs demonstrated safe applicability for parenteral administration. Elevated macrophage uptake of LPNPs, rapid plasma clearance and higher drug allocation in macrophage abundant liver and spleen illustrated admirable antileishmanial efficacy of AmpB-LPNPs in vitro (IC50, 0.16 ± 0.04 µg AmpB/ml) and in vivo (89.41 ± 3.58% parasite inhibition) against visceral leishmaniasis models. Augmentation in antileishmanial activity due to Th-1 biased immune-alteration mediated by drug-free LPNPs which elevated microbicidal mediators of macrophages. Moreover, minimal distribution to kidney tissues and low level of nephrotoxicity markers (creatinine and BUN) demonstrated the safety profile of AmpB-LPNPs. Conclusively, reliable safety and macrophage directed therapeutic performance of AmpB-LPNPs suggest it as promising alternative to commercial AmpB-formulations for the eradication of intra-macrophage diseases.

Identification and characterization of a human IL-10 receptor antagonist.

Interleukin-10 (IL-10) is an anti-inflammatory cytokine that works through IL-10 receptor alpha subunit- and suppresses immune responses in many infectious diseases such as leishmaniasis as well as in cancer. Therefore, in order to restore the host-protective immune responses in such diseases, an antagonist to this cytokine is a pressing need. Herein, using phage peptide library display, we have identified a dodecameric peptide that functions as an antagonist to human IL-10 receptor in an IL-10-induced STAT3 phosphorylation assay. The peptide antagonist's ability to restore anti-leishmanial function in CD40-activated macrophages was also tested. We observed that the peptide reduced IL-10-induced STAT-3 phosphorylation and enhanced CD40-activated leishmanial functions in macrophages.

Targeting caspases in intracellular protozoan infections.

Caspases are cysteine aspartases acting either as initiators (caspases 8, 9, and 10) or executioners (caspases 3, 6, and 7) to induce programmed cell death by apoptosis. Parasite infections by certain intracellular protozoans increase host cell life span by targeting caspase activation. Conversely, caspase activation, followed by apoptosis of lymphocytes and other cells, prevents effective immune responses to chronic parasite infection. Here we discuss how pharmacological inhibition of caspases might affect the immunity to protozoan infections, by either blocking or delaying apoptosis.

Management of trichomonas vaginalis in women with suspected metronidazole hypersensitivity.

BACKGROUND/OBJECTIVE: Standard treatment for Trichomonas vaginalis is metronidazole or tinidazole. Hypersensitivity to these drugs has been documented but is poorly understood. Desensitization is an option described in limited reports of women with hypersensitivity to nitroimidazoles. The purpose of this analysis is to improve documentation of management for trichomonas infections among women with metronidazole hypersensitivity. STUDY DESIGN: Clinicians who consulted Centers for Disease Control and Prevention concerning patients with suspected hypersensitivity to metronidazole were provided with treatment options and asked to report outcomes. RESULTS: From September 2003-September 2006, complete information was obtained for 59 women. The most common reactions were urticaria (47%) and facial edema (11%). Fifteen of these women (25.4%) were treated with metronidazole desensitization and all had eradication of their infection. Seventeen women (28.8%) were treated with alternative intravaginal drugs, which were less successful; 5 of 17 infections (29.4%) were eradicated. CONCLUSION: Metronidazole desensitization was effective in the management of women with nitroimidazole hypersensitivity.

The mechanism behind the antileishmanial effect of zinc sulphate. I. An in-vitro study.

In an attempt to determine the possible mechanism(s) behind the antileishmanial activity of zinc sulphate, promastigotes, axenic amastigotes and intracellular amastigotes of both Leishmania major and L. tropica were incubated with different concentrations of the compound. For each of the two Leishmania species, all three forms were found to be inhibited by the zinc sulphate, in a dose-dependent manner, the promastigotes being the most resistant form, followed by the axenic amastigotes. These results indicate that zinc sulphate has a direct antileishmanial effect. Compared with macrophages from starch-treated mice, the macrophages recovered from mice that had been injected intraperitoneally with zinc sulphate (daily for the 4 days prior to the macrophage collection) or BCG (once, 4 days before the cell collection) showed increased phagocytosis and increased killing of L. major and L. tropica. As the effects of the zinc sulphate were not statistically different from those of the known immunomodulating agent BCG, zinc sulphate appears to have an immunomodulating effect, in addition to its direct antileishmanial effect.

Fusion of two malaria vaccine candidate antigens enhances product yield, immunogenicity, and antibody-mediated inhibition of parasite growth in vitro.

A Plasmodium falciparum chimeric protein 2.9 (PfCP-2.9) was constructed consisting of the C-terminal regions of two leading malaria vaccine candidates, domain III of apical membrane ag-1 (AMA-1) and 19-kDa C-terminal fragment of the merozoite surface protein 1 (MSP1). The PfCP-2.9 was produced by Pichia pastoris in secreted form with a yield of 2600 mg/L and approximately 1 g/L of final product was obtained from a three-step purification process. Analysis of conformational properties of the chimeric protein showed that all six conformational mAbs interacted with the recombinant protein were reduction-sensitive, indicating that fusion of the two cysteine-rich proteins retains critical conformational epitopes. PfCP-2.9 was found to be highly immunogenic in rabbits as well as in rhesus monkeys (Macaca mulatta). The chimeric protein induced both anti-MSP1-19 and anti-AMA-1(III) Abs at levels 11- and 18-fold higher, respectively, than individual components did. Anti-PfCP-2.9 sera from both rabbits and rhesus monkeys almost completely inhibited in vitro growth of the P. falciparum FCC1/HN and 3D7 lines when tested at a 6.7-fold dilution. It was shown that the inhibition is dependent on the presence of Abs to the chimeric protein and their disulfide bond-dependent conformations. Moreover, the activity was mediated by a combination of growth-inhibitory Abs generated by the individual MSP1-19 and AMA-1(III) of PfCP-2.9. The combination of the extremely high yield of the protein and enhancement of its immune response provides a basis to develop an effective and affordable malaria vaccine.

Haemolysin A and listeriolysin--two vaccine delivery tools for the induction of cell-mediated immunity.

Haemolysin A of Escherichia coli and listeriolysin of Listeria monocytogenes represent important bacterial virulence factors. While such cytolysins are usually the reason for morbidity and even mortality, vaccine researchers have turned haemolysin A and listeriolysin into tools for vaccine delivery. Both cytolysins have found widespread application in vaccine research and are highly suitable for the elicitation of cell-mediated immunity. In this paper, we will review vaccine delivery mediated by the haemolysin A secretion system and listeriolysin and will highlight their use in vaccination approaches against protozoan parasites.

Prophylaxis for human immunodeficiency virus-related Pneumocystis carinii pneumonia: using simulation modeling to inform clinical guidelines.

BACKGROUND: Human immunodeficiency virus (HIV)-infected patients receiving highly active antiretroviral therapy (HAART) have experienced a dramatic decrease in Pneumocystis carinii pneumonia (PCP), necessitating reassessment of clinical guidelines for prophylaxis. METHODS: A simulation model of HIV infection was used to estimate the lifetime costs and quality-adjusted life expectancy (QALE) for alternative CD4 cell count criteria for stopping primary PCP prophylaxis in patients with CD4 cell count increases receiving HAART and alternative agents for second-line PCP prophylaxis in those intolerant of trimethoprim-sulfamethoxazole (TMP/SMX). The target population was a cohort of HIV-infected patients in the United States with initial CD4 cell counts of 350/microL who began PCP prophylaxis after their first measured CD4 lymphocyte count less than 200/microL. Data were from randomized controlled trials and other published literature. RESULTS: For patients with CD4 cell count increases during HAART, waiting to stop prophylaxis until the first observed CD4 cell count was greater than 300/microL prevented 9 additional cases per 1000 patients and cost $9400 per quality-adjusted life year (QALY) gained compared with stopping prophylaxis at 200/microL. For patients intolerant of TMP/SMX, using dapsone increased QALE by 2.7 months and cost $4500 per QALY compared with no prophylaxis. Using atovaquone rather than dapsone provided only 3 days of additional QALE and cost more than $1.5 million per QALY. CONCLUSIONS: Delaying discontinuation of PCP prophylaxis until the first observed CD4 cell count greater than 300/microL is cost-effective and provides an explicit "PCP prophylaxis stopping criterion." In TMP/SMX-intolerant patients, dapsone is more cost-effective than atovaquone.

The binding and distribution of albendazole and its principal metabolites in Giardia duodenalis.

Trophozoites of the protozoan parasite Giardia duodenalis were exposed to various albendazole concentrations for 4 h, washed, fixed and incubated with antibodies raised against albendazole and its two major metabolites albendazole sulphoxide and albendazole sulphone. Tubulin antibodies were also used. A peroxidase- or FITC-conjugated secondary antibody was used to detect the primary antibody with transmission electron microscopy or confocal laser scanning microscopy, respectively. Albendazole, a benzimidazole compound, was detected in the mid-dorsal region of trophozoites, albendazole sulphoxide in the posterior-dorsal region and albendazole sulphone in clusters above the median bodies. Tubulin was recognised in the ventral disk. This is the first indication that G. duodenalis may be capable of metabolising albendazole and the potential path of the metabolised drug traced within the trophozoite. Fluorescence measurements revealed that albendazole sulphoxide binding decreased and albendazole sulphone binding increased with exposure of the trophozoites to increasing albendazole concentration. This indicates that if albendazole was being metabolised by trophozoites, it occurred to a greater extent following exposure to higher albendazole concentrations.