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.

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.

[Epidemiology of cysticercosis and neurocysticercosis]. / Épidémiologie de la cysticercose et de la neurocysticercose.

Within the genus Taenia, three species are human parasites: T. solium, T. saginata and a new uncommon species, T. asiatica, described recently in Asia. T. saginata and T. solium live as adult tapeworms in human intestines, where they cause taeniasis. T. saginata is widely present worldwide, in all regions where cattle are bred. T. solium is endemic in many countries where livestock and consumption of pigs are common. Cattle and pigs become infected by ingesting eggs emitted by humans into the environment and serve as the respective intermediate hosts of these helminths and host larval forms, or metacestodes or cysticerci. Cysticerci develop into adult worms in the human intestines after a person has eaten contaminated raw or undercooked meat. In the T. solium, eggs are also human contaminants. Humans, like swine, can develop cysticercosis after ingesting eggs with water or contaminated food, or via dirty hands. The clinical manifestations of cysticercosis are highly variable both in kind and in severity. The period between initial infection and the onset of symptoms can also vary. The clinical expression of cysticercosis is generally dependent on the number, size and location of the cysts, as well as the host immune response to the parasite. The preferred locations are the muscles, subcutaneous tissues, central nervous system (CNS), and eyes. Subcutaneous and muscular forms are often asymptomatic. Severe cysticercosis is due to larvae located in human CNS - neurocysticercosis. The World Health Organization (WHO) lists neurocysticercosis as a neglected tropical disease. It estimates that about 50 million people worldwide have neurocysticercosis in the world and that it causes about 50,000 deaths each year. Its most frequent clinical manifestations are seizures, intracranial hypertension, neurological deficits, and sometimes psychiatric manifestations. It is also responsible for more than 50% of the cases of late-onset epilepsy in developing countries. The T. solium taeniasis/cysticercosis complex is endemic in many developing countries in sub-Saharan Africa, Latin America, and Asia. Although T. solium had virtually disappeared in developed countries due to industrialization, improved methods of husbandry, and health checks, cysticercosis and neurocysticercosis are diagnosed anew in North America, Europe and Australia due to increased immigration from endemic areas. Cysticercosis is considered an eradicable disease. Although theoretically feasible, this concept has been replaced by projects to control and reduce the impact of cysticercosis on human health (through mass treatment of people, veterinary control of pigs, improved farming techniques, and health education).

[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.

[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.

Review of epidemiological studies searching for a relationship between onchocerciasis and epilepsy.

A review and a meta-analysis of the available epidemiological literature for evidence of an association between onchocerciasis infection and epilepsy were carried out. We used EMBASE (1974-2002), MEDLINE (1966-2002), and PASCAL (1987-2002) databases and relevant journals and bibliographies. We limited our analysis to the epidemiological studies, where the status regarding onchocerciasis infection and epilepsy was available for each subject. Nine African studies were included. The common relative risk estimated by the random-effects model was 1.21 (95% CI 0.99-1.47; p = 0.06). The meta-analysis did not show any difference according to the onchocerciasis endemicity level and the African areas. Our results do not allow to conclude for an association between Onchocerca volvulus infection and epilepsy. However, the results are nearly significant. Further research is needed in this neglected subject, in particular for the better understanding of the neurological pathogenicity in onchocerciasis.

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.

[Sleeping sickness: forgotten research?]. / Maladie du sommeil: la recherche oubliée?

Has research on sleeping sickness, i.e., human African trypanosomiasis (HAT), been forgotten? To get an idea on funding, we consulted the Medline bibliographic database for the last 14 years. The number of publications on HAT was stagnant over the study period. By comparison there was a steady increase in the number of publications dealing with malaria. These findings suggest that interest in HAT research waned in favor of other endemics even though government or other funding agencies continued to finance research networks. To illustrate this situation, we present the funding and findings of our multidisciplinary working group in a wide range of domains including sleep, endocrine rhythms, identification of biological markers, research on physiopathologic mechanisms of the host-pathogen relationship, and development on new medications. Over the last 14 years, a total of 1 million Euros was spent to produce 68 publications on Medline, i.e., roughly 15000 [symbol: see text] per publication.

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.

[Human African trypanosomiasis: present and future treatment ]. / La trypanosomose humaine africaine: propos sur le traitement actuel et les perspectives.

During his life General Lapeyssonnie coped with the hazards linked to the therapeutics of the human African trypanosomiasis (HAT), sometimes with passion and disappointment, sometimes with revolt and hope. Because of a lack of political and financial concern during the past decades, a real global policy against the disease and a drug research against HAT didn't emerge. Today, some changes seem to take place. They are the result of the frightening spread of the disease and of the moral obligation that forces pharmaceutical companies to intervene. Drug research needs to be increased. New drugs should present no toxicity and should be able to cross through the blood-brain barrier with efficient cerebrospinal fluid concentrations. Moreover, new drugs should be easy to synthesize, easy to use and at a low cost. Today, megazol is the only one product in preclinical development, which seems to reach each of these goals.

Neurocysticercosis: validity of ELISA after storage of whole blood and cerebrospinal fluid on paper.

Cysticercosis is an infestation of Cysticercus cellulosae. When it occurs in the brain, chronic neurological complications can ensue, most commonly seizures. Neurocysticercosis is usually diagnosed by neuroimaging, a technique not available in most endemic countries. Hence immunological tests are valuable for diagnosis and epidemiological surveys. We evaluated the suitability of paper for storing blood and cerebrospinal fluid (CSF) until subsequent testing by enzyme-linked immunosorbent assay (ELISA), by testing whole blood samples on filter paper from 305 patients and CSF samples from 117 patients stored on ordinary white typing paper and on filter paper. Optimal preservation of biological samples is achieved when whole blood is stored on filter paper, CSF on white paper, and when samples are frozen within 1 week after collection. Our results could improve diagnostic capabilities and facilitate epidemiological surveys in endemic countries where immunodiagnostic tests cannot be rapidly performed because of inadequate laboratory infrastructure.

Human macrophage tumor necrosis factor (TNF)-alpha production induced by Trypanosoma brucei gambiense and the role of TNF-alpha in parasite control.

Trypanosoma brucei gambiense, a causative agent of sleeping sickness, induced a dose-dependent production of tumor necrosis factor (TNF)-alpha by human macrophages in vitro. TNF-alpha was also induced in the Mono Mac 6 cell line, which indicates a direct effect of parasite components on macrophages. Parasite-soluble factors were also potent inducers of TNF-alpha. The addition of anti-TNF-alpha to cocultures of macrophages and parasites increased the number of trypanosomes and their life span, whereas irrelevant antibodies had no effect. TNF-alpha may have a direct role (i.e., direct trypanolytic activity) and/or an indirect one, such as TNF-alpha-mediated induction of cytotoxic molecules. A direct dose-dependent lytic effect of TNF-alpha on purified parasites was observed. This lytic effect was inhibited by anti-TNF-alpha. These data suggest that, as in experimental trypanosomiasis, TNF-alpha is involved in parasite growth control in human African trypanosomiasis.

The duality of sleeping sickness: focusing on sleep.

Sleeping sickness, once under control, is a re-emergent endemic parasitic disease in intertropical Africa. Its originality resides in its duality. Two trypanosome groups (Trypanososma brucei gambiense vs.rhodesiense ) are transmitted to humans by tsetse flies from two geographical areas (Western and Central Africa humid forest vs. Eastern Africa arboreous savannah), provoking a slowly or a rapidly evolutive disease. The two stage (haemolymphatic vs. neurological invasion) pathogenic evolution leads to the duality of the immune response, depending on the host-parasite inter-relation differences in the blood and the brain. In the blood, the immune processes involved are both specific (anti-variant surface glycoprotein (VSG) antibodies) and non-specific (complement-mediated lysis, opsonification-facilitated phagocytosis and antibody dependent cell-mediated cytotoxicity). Although macrophages are activated in the blood and infiltrate the brain, nitric oxide decreases in the blood and increases in the brain, with a breakage in the blood-brain barrier, leading to brain lesions through the production of deleterious molecules. Prophylactic means are affected by the duality of pathogenic processes. This finally leads to a two stage disease (haemolymphatic vs. neurological) with two different therapeutic strategies. The sleep-wake cycle and other biological rhythms are also marked by the disappearance of circadian rhythmicity demasking basic ultradian activities and relationships, such as the interdependence of endocrine profiles and the sleep-wake alternation. 2001 Harcourt Publishers Ltd

Changes in human fasciolosis in a temperate area: about some observations over a 28-year period in central France.

A retrospective study of 616 patients affected by fasciolosis was carried out to determine the numerical fluctuations of this disease over time, the recruitment of patients, and the modes of infection in a cattle-rearing area under a temperate climate (region of Limousin, central France). The annual number of patients showed irregular fluctuations between 1955 and 1987, after which it showed subsequent decreases until 1998. Before 1980, 66.3% of the persons under study were living in villages, whereas the others inhabited larger towns. An inverse relationship was noted after 1981: 80.8% of the individuals lived in towns consisting of > 1,000 inhabitants, whereas only 19.2% resided in smaller villages. Watercress was the infection mode in 98% of persons, with infections being attributed to four watercress species before 1980 versus only two species of Nasturtium sp. after 1981. The 235 watercress beds investigated in this study were found to contain 1 or 2 Lymnaea species as follows: L. truncatula only (84% of watering places), L. glabra only (6.3%), and both species (6.3%). In the populations of L. truncatula, natural infections of snails with Fasciola hepatica were irregular and occurred up to six times over the 28-year period in the region of Limousin. According to the authors, the decrease in human cases and the changes in the recruitment of patients might be explained by the demographic movements that occurred over several decades in the region of Limousin, with the moving of younger age groups into towns.

L-Arginine availability modulates local nitric oxide production and parasite killing in experimental trypanosomiasis.

Nitric oxide (NO) is an important effector molecule of the immune system in eliminating numerous pathogens. Peritoneal macrophages from Trypanosoma brucei brucei-infected mice express type II NO synthase (NOS-II), produce NO, and kill parasites in the presence of L-arginine in vitro. Nevertheless, parasites proliferate in the vicinity of these macrophages in vivo. The present study shows that L-arginine availability modulates NO production. Trypanosomes use L-arginine for polyamine synthesis, required for DNA and trypanothione synthesis. Moreover, arginase activity is up-regulated in macrophages from infected mice from the first days of infection. Arginase competes with NOS-II for their common substrate, L-arginine. In vitro, arginase inhibitors decreased urea production, increased macrophage nitrite production, and restored trypanosome killing. In vivo, a dramatic decrease in L-arginine concentration was observed in plasma from infected mice. In situ restoration of NO production and trypanosome killing were observed when excess L-arginine, but not D-arginine or L-arginine plus N(omega)-nitro-L-arginine (a NOS inhibitor), was injected into the peritoneum of infected mice. These data indicate the role of L-arginine depletion, induced by arginase and parasites, in modulating the L-arginine-NO pathway under pathophysiological conditions.