Etest ECVs/ECOFFs for Detection of Resistance in Prevalent and Three Nonprevalent Candida spp. to Triazoles and Amphotericin B and Aspergillus spp. to Caspofungin: Further Assessment of Modal Variability.

Susceptibility testing is an important tool in the clinical setting; its utility is based on the availability of categorical endpoints, breakpoints (BPs), or epidemiological cutoff values (ECVs/ECOFFs). CLSI and EUCAST have developed antifungal susceptibility testing, BPs, and ECVs for some fungal species. Although the concentration gradient strip bioMérieux Etest is useful for routine testing in the clinical laboratory, ECVs are not available for all agent/species; the lack of clinical data precludes development of BPs. We reevaluated and consolidated Etest data points from three previous studies and included new data. We defined ECOFFinder Etest ECVs for three sets of species-agent combinations: fluconazole, posaconazole, and voriconazole and 9 Candida spp.; amphotericin B and 3 nonprevalent Candida spp.; and caspofungin and 4 Aspergillus spp. The total of Etest MICs from 23 laboratories (Europe, the Americas, and South Africa) included (antifungal agent dependent): 17,242 Candida albicans, 244 C. dubliniensis, 5,129 C. glabrata species complex (SC), 275 C. guilliermondii (Meyerozyma guilliermondii), 1,133 C. krusei (Pichia kudriavzevii), 933 C. kefyr (Kluyveromyces marxianus), 519 C. lusitaniae (Clavispora lusitaniae), 2,947 C. parapsilosis SC, 2,214 C. tropicalis, 3,212 Aspergillus fumigatus, 232 A. flavus, 181 A. niger, and 267 A. terreus SC isolates. Triazole MICs for 66 confirmed non-wild-type (non-WT) Candida isolates were available (ERG11 point mutations). Distributions fulfilling CLSI ECV criteria were pooled, and ECOFFinder Etest ECVs were established for triazoles (9 Candida spp.), amphotericin B (3 less-prevalent Candida spp.), and caspofungin (4 Aspergillus spp.). Etest fluconazole ECVs could be good detectors of Candida non-WT isolates (59/61 non-WT, 4 of 6 species).

Antifungal susceptibility testing practices in mycology laboratories in France, 2018.

A survey of mycology laboratories for antifungal susceptibility testing (AFST) was undertaken in France in 2018, to better understand the difference in practices between the participating centers and to identify the difficulties they may encounter as well as eventual gaps with published standards and guidelines. The survey captured information from 45 mycology laboratories in France on how they perform AFST (number of strains tested, preferred method, technical and quality aspects, interpretation of the MIC values, reading and interpretation difficulties). Results indicated that 86% of respondents used Etest as AFST method, with a combination of one to seven antifungal agents tested. Most of the participating laboratories used similar technical parameters to perform their AFST method and a large majority used, as recommended, internal and external quality assessments. Almost all the participating mycology laboratories (98%) reported difficulties to interpret the MIC values, especially when no clinical breakpoints are available. The survey highlighted that the current AFST practices in France need homogenization, particularly for MIC reading and interpretation.

[About an autochtonous case of neurocysticercosis in Mali]. / A propos d'un cas autochtone de neurocysticercose au Mali (premier cas de la littérature?).

Cysticercosis has been reported in Muslim countries in workers coming from endemic regions for Taenia solium. For the first time in Mali, the authors report a case of autochtonous neurocysticercosis where Muslim religion is predominent. The patient was a woman student with fever, arthralgia, headaches, consciousness troubles followed by partial motor epilepsy. Diagnosis was confirmed by clinic, serology ELISA and Western blotting and cephalic tomodensitometry analysis. The medical treatment was successfully based on combination of albendazole and praziquantel. The origin of contamination remains unknown and further investigations are needed, particularly with veterinary research team and the National League against epilepsy recently founded in Mali.

Multicentre study to determine the Etest epidemiological cut-off values of antifungal drugs in Candida spp. and Aspergillus fumigatus species complex.

OBJECTIVES: To determine the Etest-based epidemiological cut-off values (ECVs) for antifungal agents against the most frequent yeast and Aspergillus fumigatus species isolated in 12 French hospitals. METHODS: For each antifungal agent, the Etest MICs in yeast and A. fumigatus isolates from 12 French laboratories were retrospectively collected from 2004 to 2018. The ECVs were then calculated using the iterative statistical method with a 97.5% cut-off. RESULTS: Forty-eight Etest ECVs were determined for amphotericin B, caspofungin, micafungin, anidulafungin, fluconazole, voriconazole, posaconazole and itraconazole, after pooling and analysing the MICs of 9654 Candida albicans, 2939 Candida glabrata SC, 1458 Candida parapsilosis SC, 1148 Candida tropicalis, 575 Candida krusei, 518 Candida kefyr, 241 Candida lusitaniae, 131 Candida guilliermondii and 1526 Aspergillus fumigatus species complex isolates. These ECVs were 100% concordant (identical or within one two-fold dilution) with the previously reported Etest-based ECVs (when available), and they were concordant in 76.1% of cases with the Clinical and Laboratory Standards Institute ECVs and in 81.6% of cases with the European Committee on Antimicrobial Susceptibility Testing ECVs. CONCLUSIONS: On the basis of these and other previous results, we recommend the determination of method-dependent ECVs. Etest ECVs should not be used instead of breakpoints, but may be useful to identify non-wild-type isolates with potential resistance to antifungal agents, and to indicate that an isolate may not respond as expected to the standard treatment.

[Criteria for diagnosis of the neurological stage of human African trypanosomiasis: update and perspectives]. / Marqueurs du stade neurologique de la trypanosomose humaine africaine: actualités et perspectives.

Sleeping sickness or human African trypanosomiasis (HAT) is due to parasite infection by a sanguicolous flagellate protozoan of the Trypanosoma brucei genus. The disease is classically divided into two stages, i.e., the hemolymphatic stage and the CNS stage. Disease staging is currently a major challenge for therapeutic decision-making. In the field, diagnosis is based solely on white blood cell (WBC) count and detection of the parasite in the patient's cerebrospinal fluid (CSF). This technique is unreliable and invasive. Numerous studies are now under way to adapt staging to field conditions and to develop a reliable, low-cost, non-invasive test. This article describes the mechanisms underlying CNS involvement during HAT and reviews the different techniques now being studied to simplify and improve diagnosis of the CNS stage.

Harbouring in the brain: A focus on immune evasion mechanisms and their deleterious effects in malaria and human African trypanosomiasis.

Malaria and human African trypanosomiasis represent the two major tropical vector-transmitted protozoan infections, displaying different prevalence and epidemiological patterns. Death occurs mainly due to neurological complications which are initiated at the blood-brain barrier level. Adapted host-immune responses present differences but also similarities in blood-brain barrier/parasite interactions for these diseases: these are the focus of this review. We describe and compare parasite evasion mechanisms, the initiating mechanisms of central nervous system pathology and major clinical and neuropathological features. Finally, we highlight the common immune mediated mechanisms leading to brain involvement. In both diseases neurological damage is caused mainly by cytokines (interferon-gamma, tumour necrosis factor-alpha and IL-10), nitric oxide and endothelial cell apoptosis. Such a comparative analysis is expected to be useful in the comprehension of disease mechanisms, which may in turn have implications for treatment strategies.

The vervet monkey (Chlorocebus aethiops) as an experimental model for Trypanosoma brucei gambiense human African trypanosomiasis: a clinical, biological and pathological study.

It has long been known that the vervet monkey, Chlorocebus (C.) aethiops, can be infected with Trypanosoma rhodesiense, but this model has not been described for T. gambiense. In this study, we report the development of such a model for human African trypanosomiasis. Twelve vervet monkeys infected with T. gambiense developed chronic disease. The duration of the disease ranged between 23 and 612 days (median 89 days) in five untreated animals. Trypanosomes were detected in the blood within the first 10 days post-infection and in the cerebrospinal fluid, with a median delay of 120 days (n = 4, range 28-348 days). Clinical changes included loss of weight, adenopathy, and in some cases eyelid oedema and lethargy. Haematological alterations included decreases in haemoglobin level and transitory decreases in platelet count. Biological modifications included increased gamma globulins and total proteins and decreased albumin. Pathological features of the infection were presence of Mott's cells, inflammatory infiltration of either mononuclear cells or lymphocytes and plasma cells in the brain parenchyma, and astrocytosis. These observations indicate that the development of the disease in vervet monkeys is similar to human T. gambiense infection. We conclude that C. aethiops is a promising experimental primate model for the study of T. gambiense trypanosomiasis.

[Electroencephalograms (EEG) in 250 patients with epilepsy in a cysticercosis endemic area in Burundi]. / Electroencéphalogrammes réalisés chez 250 patients épileptiques dans une zone d'endémie cysticerquienne au Burundi.

OBJECTIVES: This work aimed at describing EEG abnormalities in epileptic patients living in areas endemic for cysticercosis, underlining the electroclinical correlations and discussing the interest of EEG examination in this context. METHODS: During a case-control study, 250 EEGs from patients with epilepsy were recorded with a portable system. Types of seizures were assessed clinically and from information obtained through a standardised questionnaire, and along with EEG were related to the results of cysticercosis serological tests. RESULTS: Among the 249 EEGs, 48% were normal, 5.2% had epileptic abnormalities, 6.8% showed an association between epileptic abnormalities and slow alterations. Slow theta and delta abnormalities were found in 21.8% of cases, and isolated deterioration of basic rhythms was observed in 17.3% of cases. Most seizures were generalized, and 61% of the patients had positive serology. One EEG was uninterpretable and another showed isolated spikes. Electroclinical agreement was considered to be satisfactory in 33 patients, and was better with the epileptic than with slow abnormalities. The existence of epileptiform EEG abnormalities confirmed clinically diagnosed epilepsy, but did not allow etiological diagnosis. Electroserological agreement was good in 24 patients. A significant association (Chi2, p = 0.03) existed between slow focal abnormalities and positive cysticercosis serology. Conversely, no significant association was detected between epileptic patterns and serology results. CONCLUSION: While the EEG alone clearly does not allow aetiological diagnosis, its joint use with clinical and biological results was a key element of the etiological and therapeutic discussion. When it shows focal abnormalities in a patient with epilepsy living in a high prevalence cysticercosis area, it confirms the clinical suspicion of neurocysticercosis. Morphological imagery alone can provide etiological information on the seizures by showing the nature and localization of the parenchymal lesions.

Isoenzymic characterization of seven strains of Toxoplasma gondii by isoelectrofocusing in polyacrylamide gels.

Toxoplasma gondii strains are usually defined by biological parameters such as pathogenicity in mice. A characterization of toxoplasma strains by biochemical techniques has not been reported. In this study, extracts of tachyzoites of 7 toxoplasma strains were compared on the basis of their isoenzyme patterns for 39 enzymes by means of isoelectrofocusing in polyacrylamide gels. Eighteen enzymes gave clear and reproducible bands. Of these, 14 had identical electrophoretic patterns for all strains. Two different isoenzyme types were found for the enzymes aspartate aminotransferase, glutathione reductase, glucose phosphate isomerase, and amylase. This allowed the description of 3 isoenzyme pattern groups among the 7 toxoplasma strains. The possible relationship between biological behavior and isoenzyme pattern groups is discussed.

Human African trypanosomiasis: presence of antibodies to galactocerebrosides.

Improvements were made in the immunodetection of anti-galactocerebroside (anti-GalC) antibody in sera of patients with human African trypanosomiasis by thin-layer chromatography, enzyme-linked immunosorbent assay, and immunoadsorption. Rabbit anti-GalC antibodies were used to standardize these techniques and demonstrate their specificity. Anti-GalC antibodies were found in the sera of 42.8% of 63 patients with human African trypanosomiasis. Thirty-four control subjects living in the same endemic area were also tested. Anti-GalC levels were higher in human African trypanosomiasis patients with neurologic disturbances compared with patients without such disturbances. These antibodies were distributed mainly between the IgG and IgM classes, but 28% of the patients with human African trypanosomiasis had increased IgA levels without anti-GalC antibody activity.

Detection and characterization of autoantibodies directed against neurofilament proteins in human African trypanosomiasis.

In serum and in cerebrospinal fluid (CSF) from patients with human African trypanosomiasis (HAT) with central nervous system involvement, we detected autoantibodies directed to some proteins from these tissues. The characterization of antigenic proteins by Western blotting showed that the antibodies recognized the 200-kD and 160-kD proteins of neurofilament (NF). Serum anti-NF antibodies were more frequent in HAT patients than in control subjects (86% versus 24%; P < 10[-9]) and they belonged predominantly to the IgM class (anti-NF IgM = 86% versus anti-NF IgG = 4%; P < 10[-9]) in the patients with stage II (central nervous system involvement) HAT. The CSF antibodies to NF were IgM in 88% (22 of 25) of the cases and IgG in 32% (8 of 25) of the cases. Epitopes shared by NF and trypanosomes were detected by indirect immunofluorescence and this was confirmed by the disappearance of anti-NF reactivity after adsorption with trypanosome antigens (Trypanosoma brucei brucei or T. b. gambiense). Anti-NF antibodies were undetectable in the CSF from stage I HAT patients.

Galactocerebrosides are antigens for immunoglobulins in sera of an experimental model of trypanosomiasis in sheep.

In an experimental model of human African trypanosomiasis in sheep inoculated with Trypanosoma brucei brucei, we report an immunoglobulin reactivity towards central nervous system (CNS) glycolipids. Immunocharacterization of the glycolipid antigens was performed on thin-layer chromatography using peroxidase-labelled second antibody. An immunoreactivity against galactocerebroside antigens was observed in inoculated animals up to a dilution of 1:600 and was suppressed after immunoadsorption of sera on pure galactocerebrosides. As galactocerebroside antigen is responsible for demyelination in several experimental models, the relation between the important glycolipid immunoreactivity in inoculated sheep sera and the pathogenesis of CNS demyelination in African trypanosomiasis is suggested.

Blood-cerebrospinal fluid barrier and intrathecal immunoglobulins compared to field diagnosis of central nervous system involvement in sleeping sickness.

Diagnosis of central nervous system (CNS) involvement in sleeping sickness is crucial in order to give an appropriate treatment regimen. Neurological symptoms occur late, therefore field diagnosis is based on white blood cell count, total protein concentration and presence of trypanosomes in cerebrospinal fluid (CSF). More sensitive and specific parameters are now available. Blood-CSF barrier (B-CSFB) dysfunction, intrathecal total and specific immunoglobulin synthesis were evaluated in 95 patients with and without obvious meningoencephalitis, and compared to field criteria.B-CSFB dysfunction is a rather late event in the course of CNS involvement and correlates with the presence of trypanosomes, neurological signs and intrathecal polyspecific and specific immune response. IgM intrathecal response and particularly IgM antibody index are early markers of CNS invasion. We showed that 29% of patients with CSF abnormalities but without trypanosome detection in the field had no neuro-immunological response. In contrast, patients with normal CSF according to field diagnosis showed an intrathecal immune response in 31% of the cases.Field diagnosis can therefore fail to determine neurological involvement but can also provide false positive results. Improved criteria including B-CSFB dysfunction and IgM detection are needed in order to provide an adapted treatment regimen.

Clinical follow-up in the rat experimental model of African trypanosomiasis.

Animal models of Human African Trypanosomiasis (HAT) have been developed to understand the pathogenic mechanisms leading to the passage into the neurological phase, most of them referring to histological aspects but not clinical or behavioral data. Our study aimed at defining simple clinical and/or behavioral markers of the passage between the hemolymphatic phase and the meningo-encephalitic stage of the disease. Sprague-Dawley rats (n=24) were infected with Trypanosoma brucei brucei AnTat 1.1E. Food intake and body weight were measured daily from the day of infection until death. Hematocrit was measured twice a week. Behavioral disturbances were evaluated through an Open-field test. A sudden weight loss occurred on the twelfth day after infection, due to a significant drop of food intake starting two days before. The rats developed an anemic state shown by the hematocrit measurements. The Open-field test showed them to be less active and reactive as soon as the second week after infestation. A complementary histological study observed trypanosomes and inflammatory cells in the choroid plexus at the same period. These results are in favor of central nervous system functional disturbances. The observed weight loss is discussed as being a parameter of the entry in the meningo-encephalitic phase. The rat model reproduces neurological symptoms observed in the human disease and may prove to be useful for further neurohistological and therapeutic studies.

Seroprevalence of cysticercosis in Bénin.

We report the results of a seroepidemiological study on the prevalence of cysticercosis in Bénin. Cluster sampling at 3 levels was performed in the 6 départements (Atacora, Borgou, Zou, Mono, Atlantique and Oueme) and 2625 serum samples, from 1329 adult females and 1296 adult males, were collected. Antibodies against Taenia solium cysticerci were first searched for by enzyme-linked immunosorbent assay and the 41 seropositive samples were then examined by enzyme-linked electroimmunotransfer blot assay (EITB). Thirty-five samples gave positive results in the EITB. The overall seroprevalence of cysticercosis was therefore 1.3% (95% confidence interval [95% CI] 0.9-1.9). The seroprevalence was 1.9% in males (95% CI 1.2-2.7) and 0.8% (95% CI 0.4-1.5) in females (P < 0.05). A progressive increase in seroprevalence with increasing age was found. The highest seroprevalences were observed in Atacora and Atlantique, 2 non-Muslim départements (3.3% and 3.0%, respectively). This study demonstrated the public health importance of cysticercosis in Bénin.

Effect of megazol on Trypanosoma brucei brucei acute and subacute infections in Swiss mice.

Human African trypanosomiasis (HAT) or sleeping sickness is a major public health problem in 36 sub-Saharan African countries and is caused by Trypanosoma brucei gambiense and T. b. rhodesiense. About 25,000 new cases of the disease are reported annually, and around 50 million people are classed as at risk of contracting the disease. Until now; the only effective drug available for treatment of advanced HAT was the trypanocide melarsoprol. The mortality rate of melarsoprol treated patients is 1-5%. Megazol is a nitroimidazole derivative shown to be effective in vitro against T. b. brucei with an EC50 of 0.01 micrograms.ml-1. When this compound was tested for its in vivo activity in T. b. brucei infected Swiss mice, it was shown to cure the acute disease. However, megazol alone did not cause cure of mice carrying a subacute infection with involvement of the central nervous system (CNS). Combined suramin and megazol treatment did prove effective and the mice were shown to have remission without further relapse from the CNS. The study of three megazol derivatives is also described here. Substitution of a bromine, methyl or trifluoromethyl moiety at the 4 position of the imidazole ring abolished trypanocidal activity both in vivo and in vitro. Intermediates of megazol synthesis (imidazole sulfoxide and imidazole sulfone) were also tested, but were shown not to be active. It is thought that megazol trypanocidal effect may be due to the triggering of radical production by the compound, which have toxic effects on the trypanosomes metabolism. In depth study of megazol is needed to fully elucidate its pharmacokinetics and to precisely pin down its mode of action.

Sedimentation field-flow fractionation application to Toxoplasma gondii separation and purification.

Toxoplasmosis is a worldwide disease caused by Toxoplasma gondii, an intracellular protozoa of micronic size range (4-10 microm). Its classical purification processes are complex and often associated with low recovery. All investigation procedures concerning this parasite require its isolation and purification from at least the mouse ascitic fluid. For this purpose, a recently developed laboratory technology was used, i.e. sedimentation field-flow fractionation. This chromatographic-like separation technology was demonstrated to be particularly selective for isolation and separation of micron-sized biological particles. Sedimentation field-flow fractionation operated on the steric-hyperlayer mode was used to isolate the parasite from the remanent ascitic contaminants of different origins and from red blood cells. With this technology, 86% recovery with 97% viability was obtained in less than 30 min.

Isoenzyme analysis of 35 Toxoplasma gondii isolates and the biological and epidemiological implications.

Isoenzyme analysis was conducted on the tachyzoite stage of 35 Toxoplasma gondii isolates. Fifteen enzyme systems were studied after isoelectrofocusing of tachyzoite extracts in polyacrylamide or agarose gels. Six enzyme systems showed variable electrophoretic patterns: aspartate aminotransferase (EC 2.6.1.1), glutathione reductase (EC 1.6.4.2), glucose phosphate isomerase (EC 5.3.1.9), amylase (EC 3.2.1.1), acid phosphatase (EC 3.1.3.2), and propionyl esterase. Their combination allows the description of 5 zymodemes among the 35 T. gondii isolates. Zymodeme 1 involves 6 isolates that are highly pathogenic to mice and for which oocysts could not be obtained. Isolates belonging to zymodemes 2, 3, and 4 are less pathogenic to mice and produced oocysts. Zymodeme 5 involves only 1 isolate, which was highly pathogenic to mice.

Antigenic similarity between Hammondia hammondi and Toxoplasma gondii tachyzoites.

Hammondia hammondi is an obligate heteroxenous intestinal coccidian of cats, sharing many characteristics with Toxoplasma gondii. The tachyzoite stage antigens of T. gondii and H. hammondi were studied by immunofluorescence assays (IFA) and western blotting (WB) techniques to demonstrate antigenic similarities. Five monoclonal antibodies (MAbs), anti-T. gondii antigens, P22, P23, P30, P35, and P43, and mice polyclonal anti-H. hammondi serum were investigated. Antigens of H. hammondi were recognized by anti-P30 MAb both in IFA and in WB and by anti-P22 and anti-P35 MAbs only in IFA. Polyclonal anti-H. hammondi serum revealed many common antigens between the 2 parasites (30, 32, 35, 66, and 90 kDa). The differences of host parasite relationship between these 2 coccidians lead us to suggest that many of these antigens with similar molecular weights are not the same, but homologous, molecules or that they are not the only factors involved in these differences.

Isoenzyme analysis of Hammondia hammondi and Toxoplasma gondii sporozoites.

Isoenzyme analysis using isoelectrofocusing in polyacrylamide gels was used to distinguish Hammondia hammondi and Toxoplasma gondii sporozoites. Five enzyme systems were studied: aconitase (EC 4.2.1.3), aspartate aminotransferase (EC 2.6.1.1), glucose phosphate isomerase (EC 5.3.1.9), lactate dehydrogenase (EC 1.1.1.27), and phosphoglucomutase (EC 2.7.5.1). Three stocks of T. gondii belonging to 3 zymodemes were compared to 1 stock of H. hammondi. Hammondia hammondi differed from T. gondii at all 5 loci analyzed. This was observed for all 3 zymodemes of T. gondii. These results indicated clear genetic differences between the 2 species.