Genetic diversity of the Hwp1 gene and HIS3, EF3, CDC3 microsatellites and antifungal susceptibility profiles of Candida albicans isolates from Yaoundé HIV-infected patients.

The molecular epidemiology and the antifungal susceptibility profiles of Candida albicans are scarce in Cameroon. Authors studied the genetic diversity and the antifungal susceptibility of C. albicans isolates from Yaoundé HIV-infected patients. Clinical isolates were obtained by mycological diagnosis of oropharyngeal swabs, stools, urine, and vaginal swabs from patients. C. albicans isolates were confirmed by the Light cycler real-time PCR of the ITS1 region of the 5.8s ribosomal DNA. The ABC genotypes and the Hwp1 gene amplification were carried out with specific primers. Microsatellite length polymorphism of HIS3, CDC3, and EF3 microsatellites was analysed. The antifungal susceptibility testing was carried out by the CLSI broth microdilution M27-A3 and M27-S4 protocols. The minimal inhibitory concentration (MIC) results were interpreted according to updated clinical breakpoints (CBPs) recommended by the CLSI or epidemiological cut-off values (ECVs). One hundred and thirteen (113) isolates were obtained from the analysis of 1218 samples. The ABC genotyping showed 79 (69.91%) genotype A, 24 (21.23%) genotype B, and 10 (8.84%) genotype C. The Hwp1 gene amplification provided a newly observed genetic polymorphism, named H and 5 genotypes described (H1-H5). The microsatellite analysis generated 65 molecular types. All the isolates were susceptible to amphotericin B (MIC ≤ 1 µg/ml); 79.64% of isolates were wild type to itraconazole (MIC ≤ 0.12 µg/ml); and 86.72% of isolates were susceptible to fluconazole (MIC ≤ 2 µg/ml). These results highlight the important genetic diversity of C. albicans isolates among Yaoundé HIV-infected patients and bring clues for the comprehension of the molecular epidemiology of the yeast in Cameroon.

Molecular epidemiology reveals genetic diversity among 363 isolates of the Cryptococcus neoformans and Cryptococcus gattii species complex in 61 Ivorian HIV-positive patients.

Cryptococcal meningitis is a severe opportunistic infection in HIV-infected patients. In Ivory Coast, despite the availability of antiretroviral treatment (ART), this infection is still prevalent. The study investigates the genetic diversity of 363 clinical isolates of Cryptococcus from 61 Ivorian HIV-positive patients, the occurrence of mixed infections and the in vitro antifungal susceptibility of the isolates. Serotyping was performed via LAC1 and CAP64 gene amplification. Genotyping was performed using the phage M13 core (GACA)4 and (GTG)5 primers and restriction fragment length polymorphism analysis of the URA5 gene. By PCR fingerprinting, the presence of the three serotypes were demonstrated among the 363 isolates in the population studied: A (n=318; 87.6%), AD (n=40; 11%) and B (n=4; 1.1%). Using PCR fingerprinting with primers M13 (GACA)4 and (GTG)5 , we grouped the isolates into 56 molecular subtypes. We observed a high frequency (39.3%) of mixed infections, with up to two different genotypes per sample. None of the isolates were resistant to amphotericin B. Only 0.3% and 1.1% of the isolates were resistant to fluconazole and flucytosine respectively. This study revealed the high genetic diversity among Cryptococcus isolates, the occurrence of mixed infections and a high antifungal susceptibility for the majority of Ivorian cryptococcal isolates.

Environmental distribution of Cryptococcus neoformans and C. gattii around the Mediterranean basin.

In order to elucidate the distribution of Cryptococcus neoformans and C. gattii in the Mediterranean basin, an extensive environmental survey was carried out during 2012-2015. A total of 302 sites located in 12 countries were sampled, 6436 samples from 3765 trees were collected and 5% of trees were found to be colonized by cryptococcal yeasts. Cryptococcus neoformans was isolated from 177 trees and C. gattii from 13. Cryptococcus neoformans colonized 27% of Ceratonia, 10% of Olea, Platanus and Prunus trees and a lower percentage of other tree genera. The 13 C. gattii isolates were collected from five Eucalyptus, four Ceratonia, two Pinus and two Olea trees. Cryptococcus neoformans was distributed all around the Mediterranean basin, whereas C. gattii was isolated in Greece, Southern Italy and Spain, in agreement with previous findings from both clinical and environmental sources. Among C. neoformans isolates, VNI was the prevalent molecular type but VNII, VNIV and VNIII hybrid strains were also isolated. With the exception of a single VGIV isolate, all C. gattii isolates were VGI. The results confirmed the presence of both Cryptococcus species in the Mediterranean environment, and showed that both carob and olive trees represent an important niche for these yeasts.

Cryptococcus neoformans isolates from Yaoundé human immunodeficiency virus-infected patients exhibited intra-individual genetic diversity and variation in antifungal susceptibility profiles between isolates from the same patient.

Cryptococcal meningitis is a dreadful opportunistic fungal infection amongst human immunodeficiency virus (HIV)-infected patients. One complication in the management of the disease is the possible infection of a patient by two or more different strains of Cryptococcus neoformans. This study investigated the intra-individual genetic diversity and antifungal susceptibility of C. neoformans isolates from Yaoundé (Cameroon) HIV-infected patients with cryptococcal meningitis. Twenty-five clinical isolates were obtained during a prospective study. Five colonies were randomly collected from each initial sample. The 150 isolates obtained (125 colonies and 25 initial samples) were submitted to serotyping by multiplex PCR. Genotyping analyses were achieved using RFLP, and minisatellite- and microsatellite-length polymorphism. The antifungal susceptibility testing was carried out using a Sensititre YeastOne kit. Seven antifungals were tested: itraconazole, fluconazole, amphotericin B, ketoconazole, 5-fluorocytosine, posaconazole and voriconazole. The 150 isolates were identified as C. neoformans serotype A and genotype VNI. The microsatellite and minisatellite sequence analyses generated 15 genotypes. Six out of 25 (24 %) patients were found to be infected by two different genotypes. Antifungal susceptibility showed several profiles: posaconazole (0.015-0.25 µg ml-1), amphotericin B (0.06-1 µg ml-1), fluconazole (0.5-16 µg ml-1), itraconazole (0.008-0.12 µg ml-1), ketoconazole (0.008-0.12 µg ml-1), 5-fluorocytosine (0.25-16 µg ml-1) and voriconazole (0.008-0.12 µg ml-1). It was noted that isolates from the same patient might present different susceptibility profiles to an antifungal drug with differences of more than four dilutions. The results achieved highlighted the possible presence of isolates with different genotypes in a patient with dissimilar antifungal susceptibility profiles during a single episode of cryptococcal meningitis.

First case of mixed infection with Cryptococcus deuterogattii and Cryptococcus neoformans VNI in an Ivorian HIV-positive patient.

INTRODUCTION: Cryptococcal meningitis (CM) may be caused by several species of Cryptococcus. CASE PRESENTATION: We describe a fatal case of CM in a HIV-positive patient from Ivory Coast infected by Cryptococcus neoformans VNI and Cryptococcusdeuterogattii. Isolates were recovered from cerebrospinal fluid (CSF) prior to systemic antifungal treatment. Six isolates were studied (the entire culture plus five isolated colonies from it). Serotyping was performed via LAC 1 and CAP 64 gene amplification. Genotyping was performed using restriction fragment length polymorphism (RFLP) analysis of the URA5 gene, (GACA)4, (GTG)5 and M13 PCR fingerprinting. URA5-RFLP analysis identified the original culture with two different molecular type combinations. However, URA5-RFLP profiles of the five colonies isolated from the original sample revealed two different species. Four colonies were identified as C.deuterogattii and the last isolate as C.neoformans VNI. The in vitro susceptibility profile was determined using the standard method according to the CLSI M27-A3 protocol. The isolates were susceptible to the tested antifungals (fluconazole, flucytosine and amphotericin B). Treatment with fluconazole (1200 mg day-1) was initiated; however, the patient died 17 days after the onset of antifungal therapy. CONCLUSION: This is the first reported case of mixed infection with C. neoformans and C.deuterogattii in a HIV-positive patient.

Potent and Synergistic Extract Combinations from Terminalia Catappa, Terminalia Mantaly and Monodora tenuifolia Against Pathogenic Yeasts.

Mycoses caused by Candida and Cryptococcus species, associated with the advent of antifungal drug resistance have emerged as major health problems. Improved control measures and innovative therapies are needed. This paper describes results from the screening of bio-guided fractionated extracts alone and combinations of Terminalia catappa, Terminalia mantaly and Monodora tenuifolia harvested in Cameroon. Crude ethanolic, hydro-ethanolic and aqueous extracts and bio-guided fractions were screened for antifungal activity against isolates of C. albicans, C. glabrata, C. parapsilosis and Cr. neoformans and the reference strain C. albicans NR-29450. Minimal inhibitory concentrations (MIC) were determined using a broth micro dilution method according to the Clinical & Laboratory Standards Institute (CLSI). Time kill kinetics of extracts alone and in combination were also evaluated. Extracts from T. mantaly stem bark were the most active with the best MIC values ranging from 0.04 mg/mL to 0.16 mg/mL. Synergistic interactions were observed with combinations of sub-fractions from M. tenuifolia, T. mantaly and T. catappa. Combination of sub-fractions from M. tenuifolia and T. mantaly (C36/C12) showed synergistic interaction and fungicidal effect against four out of five tested yeasts. These results support further investigation of medicinal plant extracts alone and in combination as starting points for the development of alternative antifungal therapy.

Investigation of minor species Candida africana, Candida stellatoidea and Candida dubliniensis in the Candida albicans complex among Yaoundé (Cameroon) HIV-infected patients.

Minor species of the Candida albicans complex may cause overestimation of the epidemiology of C. albicans, and misidentifications could mask their implication in human pathology. Authors determined the occurrence of minor species of the C. albicans complex (C. africana, C. dubliniensis and C. stellatoidea) among Yaoundé HIV-infected patients, Cameroon. Stool, vaginal discharge, urine and oropharyngeal samples were analysed by mycological diagnosis. Isolates were identified by conventional methods and mass spectrometry (MS; carried out by the matrix-assisted laser desorption-ionisation time-of-flight MS protocol). Candida albicans isolates were thereafter submitted to the PCR amplification of the Hwp1 gene. The susceptibility of isolates to antifungal drugs was tested using the Clinical and Laboratory Standards Institute M27-A3 protocol. From 115 C. albicans obtained isolates, neither C. dubliniensis nor C. stellatoidea was observed; two strains of C. africana (422PV and 448PV) were identified by PCR electrophoretic profiles at 700 bp. These two C. africana strains were vaginal isolates. The isolate 448PV was resistant to ketoconazole at the minimal inhibitory concentration of 2 µg ml(-1), and showed reduced susceptibility to amphotericin B at 1 µg ml(-1). This first report on C. africana occurrence in Cameroon brings clues for the understanding of the global epidemiology of this yeast as well as that of minor species of the C. albicans complex.

Assessment of gastrointestinal parasites in wild chimpanzees (Pan troglodytes troglodytes) in southeast Cameroon.

We tested 114 faecal samples from wild simian immunodeficiency virus (SIV)-positive (n = 43) and SIV-negative (n = 71) chimpanzees (Pan troglodytes troglodytes) in southeast Cameroon for the presence of gastrointestinal parasites by direct smear. We observed cysts from different protozoa (Entamoeba coli and Entamoeba histolytica / Entamoeba dispar, Endolimax nana, Iodamoeba butschlii, Chilomastix mesnili, Balantidium coli and Blastocystis cells) and trophozoites from Troglodytella abrassarti and Balantidium coli. Eggs from different helminths (strongylids, Ascaris lumbricoides, Abbreviata caucasica, Trichuris sp., Capillaria sp., Enterobius anthropopeci, Bertiella sp., Hymenolepis diminuta and an undetermined fluke) were also observed. Finally, we observed eggs that could not be properly identified and classified. We did not observe any differences between the SIV+ and SIV- samples except for the unidentified eggs. The studied chimpanzees were highly parasitised by strongylid (85.1% of prevalence), Troglodytella (43.8%) and Blastocystis (2.9%), and the frequency of the other parasites ranged from 0.9 to 8.8%. These high levels of parasite infections could represent an additional burden in a population where there is a high rate of the SIV virus in circulation.

Fungicidal activity and morphological alterations of Candida albicans induced by echinocandins: study of strains with reduced caspofungin susceptibility.

Caspofungin is a member of the echinocandin class of antifungal compounds that inhibit 1,3-ß-d-Glucan synthase. As patient exposure to caspofungin (CAS) broadens, the number of infecting strains with reduced susceptibility to this drug is expected to rise. In the present study, the in vitro effects of varying concentrations of CAS against Candida albicans isolates presenting reduced susceptibility to CAS were studied in comparison with a reference strain. Two C. albicans isolates presenting high minimal inhibitory concentrations (MIC = 8 µg ml(-1) ) were selected: one isolate obtained in the laboratory under continuous antifungal selection pressure (CaIn-R) and one clinical isolate (CaClin-R) from a patient with a therapeutic failure. Results showed that after 24 h of CAS exposure, CaIn-R and CaClin-R presented a partial growth inhibition in comparison with the reference strain. Moreover, scanning electron microscopy and transmission electron microscopy studies showed that the cell walls of CaIn-R and CaClin-R were less altered than that of the reference strain. These observations suggested that although CaIn-R and CaClin-R cells were misshapen after CAS exposure, cell lysis was limited after 24 h of treatment indicating higher survival ability for CaIn-R and CaClin-R in the presence of CAS.

Geographic variation in the frequency of isolation and fluconazole and voriconazole susceptibilities of Candida glabrata: an assessment from the ARTEMIS DISK Global Antifungal Surveillance Program.

Geographic differences in frequency and azole resistance among Candida glabrata may impact empiric antifungal therapy choice. We examined geographic variation in isolation and azole susceptibility of C. glabrata. We examined 23 305 clinical isolates of C. glabrata during ARTEMIS DISK global surveillance. Susceptibility testing to fluconazole and voriconazole was assessed by disk diffusion, and the results were grouped by geographic location: North America (NA) (2470 isolates), Latin America (LA) (2039), Europe (EU) (12 439), Africa and the Middle East (AME) (728), and Asia-Pacific (AP) (5629). Overall, C. glabrata accounted for 11.6% of 201 653 isolates of Candida and varied as a proportion of all Candida isolated from 7.4% in LA to 21.1% in NA. Decreased susceptibility (S) to fluconazole was observed in all geographic regions and ranged from 62.8% in AME to 76.7% in LA. Variation in fluconazole susceptibility was observed within each region: AP (range, 50-100% S), AME (48-86.9%), EU (44.8-88%), LA (43-92%), and NA (74.5-91.6%). Voriconazole was more active than fluconazole (range, 82.3-84.2% S) with similar regional variation. Among 22 sentinel sites participating in ARTEMIS from 2001 through 2007 (84 140 total isolates, 8163 C. glabrata), the frequency of C. glabrata isolation increased in 14 sites and the frequency of fluconazole resistance (R) increased in 11 sites over the 7-year period of study. The sites with the highest cumulative rates of fluconazole R were in Poland (22% R), the Czech Republic (27% R), Venezuela (27% R), and Greece (33% R). C. glabrata was most often isolated from blood, normally sterile body fluids and urine. There is substantial geographic and institutional variation in both frequency of isolation and azole resistance among C. glabrata. Prompt species identification and fluconazole susceptibility testing are necessary to optimize therapy for invasive candidiasis.

Detection and identification of Candida spp. in human serum by LightCycler real-time polymerase chain reaction.

The aim of this work was to develop LightCycler real-time polymerase chain reaction method to allow rapid detection and identification of Candida spp. in human serum with panfungal primers (internal transcribed spacer [ITS] and L18). Melting-curve analysis of the ITS sequences showed that each amplicon presented a specific melting point and enabled identification of 5 Candida spp. After parameters optimization, 58 sera were preliminary analyzed from 23 patients. For L18 primers, the LightCycler system enabled detection of DNA in 92% of patients with positive blood culture. These primers were not able to differentiate between species of Candida. By using ITS primers, the LightCycler system enabled detection of DNA in sera from 76.9% of patients with positive blood culture. With ITS primers, the species responsible for the infection was identified for 11 patients. These data revealed the LightCycler as a potential tool for early detection and identification of Candida.

Clonal population structure and genetic diversity of Candida albicans in AIDS patients from Abidjan (Côte d'Ivoire).

We have investigated the genotype at 14 enzyme-encoding loci in 275 isolates of the pathogenic yeast Candida albicans sampled from 42 HIV-positive patients (all but one with AIDS) from Abidjan (Côte d'Ivoire). We separately analyzed the following variables: patient, residence, age, gender, T cell count, hospitalization (yes or no), drug treatment, date of sampling, multilocus genotype, and serotype. The most important factors contributing to the genetic variability of C. albicans are individual patient and gender. Our data manifest that the population size of the parasite is relatively small within each patient, although larger in women than in men, and that, at least for the patients involved in the study, the transmission rate of C. albicans between human adults is very low. Most important is the inference that the prevailing mode of reproduction of C. albicans in natural populations is clonal, so that sexual reproduction is extremely rare, if it occurs at all.

Antiplasmodial activity and cytotoxicity of plants used in West African traditional medicine for the treatment of malaria.

Eighteen plants originating from Ivory Coast were selected by ethnobotanical survey as plants commonly used by traditional healers for the treatment of malaria. Extracts of these plants were tested on two strains of Plasmodium falciparum: FcM29-Cameroon (chloroquine-resistant strain) and a Nigerian chloroquine-sensitive strain. The powdered plants were used to prepare three kinds of extracts: by decoction in water, in ethanol (95%) and in pentane. A radioactive micromethod allowed the evaluation of the antiplasmodial in vitro activity of the extracts on P. falciparum. Concentrations inhibiting 50% of the parasite growth (IC50) ranged from 18 microg/ml to more than 500 microg/ml for aqueous and ethanol extracts and from 4.3 microg/ml to more than 500 microg/ml for pentane extracts. Cytotoxicity was estimated on A375 melanoma cells and a cytotoxicity/antiplasmodial index (CAR) was calculated for each extract, ranging from 1 to 10. The pentane extracts of Cola caricaefolia and Uvaria afzelii, which revealed the strongest antiplasmodial activity had CAR values of about 10.

Spore diffusate isolated from some strains of Aspergillus fumigatus inhibits phagocytosis by murine alveolar macrophages.

Aspergillus fumigatus is a ubiquitous fungus that grows in decaying organic matter. It can cause disease in both immunodeficient and immunocompetent patients by using virulence factors to escape the host defenses. Some of these factors, such as a diffusate, released from the spores of A. fumigatus, have previously been described. This diffusate was demonstrated to inhibit oxidative burst and phagocytosis of coated red blood cells. The present study has shown that this substance can inhibit the phagocytosis of A. fumigatus spores by murine alveolar macrophages (MALU) and evaluated the action of this substance. We quantified phagocytosis by MALU cells with and without diffusate and evaluated the inhibition of phagocytosis by testing diffusates from different strains. We conclude that the spore diffusate of some strains of A. fumigatus can reversibly decrease the ability of alveolar macrophages to ingest A. fumigatus spores.

Antiplasmodial and cytotoxic activity of galipinine and other tetrahydroquinolines from Galipea officinalis.

The antimalarial and toxicological properties of four tetrahydroquinoline alkaloids from Galipea officinalis trunk bark were studied. Crude extracts and pure alkaloids were tested for in vitro antimalarial activity on Plasmodium falciparum. The IC50 were evaluated after 24 and 72 h contact between compounds and the parasite culture, and ranged from 1.8 to 40 microg/ml for the chloroquine-sensitive strain (CQS) and from 0.09 to 38 microg/ml for the chloroquine-resistant strains (CQR). Galipinine yielded the best antimalarial effect (IC50: 0.09 - 0.9 microg/ml on CQR strain) and this compound interacted particularly between the 32(nd) and the 40(th) hour of the P. falciparum erythrocytic cycle. The cytotoxicity of the extracts and pure tetrahydroquinoline alkaloids was assessed on the HeLa cell line and showed IC50 values ranging from 5.8 to above 50 microg/ml.