Antibodies Raised against Bcvir15, an Extrachromosomal Double-Stranded RNA-Encoded Protein from Babesia canis, Inhibit the In Vitro Growth of the Parasite

As part of a search for homologous members of the Plasmodium falciparum Pf60 multigene family in the intraerythrocytic protozoan parasite Babesia canis, we report here the characterization of a cDNA of 1,115 bp, which was designated Bcvir for its potential viral origin. The Bcvir cDNA contained two overlapping open reading frames (ORFs) (ORF1 from nucleotide [nt] 61 to 486 and ORF2 from nt 417 to 919), where Bcvir15, the deduced ORF1 peptide (M(1) to I(141)), is the main expressed product. The Bcvir cDNA was derived from an extrachromosomal dsRNA element of 1.2 kbp that was always found associated with a double-stranded RNA (dsRNA) of 2.8 kbp by hybridization, and no copy of this cDNA sequence was found in B. canis genomic DNA. Biochemical characterization of Bcvir15, by using polyclonal rabbit sera directed against recombinant proteins, indicated that it is a soluble protein which remained associated with the cytoplasm of the B. canis merozoite. Interestingly, purified immunoglobulins from the anti-glutathione S-transferase-Bcvir15 (at a concentration of 160 μg/ml) induced 50% inhibition of the in vitro growth of B. canis, and the inhibitory effect was associated with morphological damage of the parasite. Our data suggest that the extrachromosomal dsRNA-encoded Bcvir15 protein might interfere with the intracellular growth of the parasite rather than with the process of invasion of the host cell by the merozoite. Epitope mapping of Bcvir15 identified three epitopes that might be essential for the function of the protein

Use of RNAlater (R) as a preservation method for parasitic coprology studies in wild-living chimpanzees

We evaluated the use of an RNA stabilisation buffer, RNAlater (R) (Ambion, Austin, Texas), as a preservation medium for parasitic coprology analysis of faecal samples collected from chimpanzees living in the wild (Pan troglodytes troglodytes). Thirty faecal samples collected in the forests of south-east Cameroon (Mambele area) from 2003 to 2011 were preserved in RNAlater (R) at -80 degrees C and analysed for their parasite content. We identified and counted parasitic elements and assessed their shape, size and morphology in relation to the storage time of the samples. We found that parasite elements were identifiable in RNAlater (R) preserved samples after as many as 7 years, showing that RNAlater (R) could be an effective and reliable preservation medium for coprology. Thus, its use could be an interesting way to optimise sample collection for several types of studies (parasitology and bacteriology/virology) at once, especially considering the logistically challenging and time-consuming field campaigns needed to obtain these faecal samples

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

African ST173 Cryptococcus deuterogattii strains are commonly less susceptible to fluconazole: an unclear mechanism of resistance

International audienceOBJECTIVES: Fluconazole, alone or in association, is often administered during cryptococcal meningitis treatment, especially in sub-Saharan Africa. Its extensive use has led to the emergence of resistant strains. The mechanisms underlying resistance are poorly documented for yeasts belonging to the Cryptococcus gattii species complex. The literature suggests that this resistance could be due to mutations and/or overexpression of the ERG11 gene (encoding the 14-α-demethylase) and efflux pumps, such as MDR (a sub-class of ABC transporters) or AFR (a second sub-class of ABC transporters). We highlight the presence of genotype VGII strains (C. deuterogattii) from the Ivory Coast with a rare sequence type 173, that are associated with high minimum inhibitory concentrations of FCZ compared to those for strains originating from the Pacific Northwest (USA).METHODS: We investigated the mechanisms of fluconazole resistance in 28 Ivorian clinical C. deuterogattii strains recovered from 3 patients during their follow-up and antifungal treatment.RESULTS: We demonstrated that (i) these strains exhibited no mutation in the ERG11 gene; (ii) some strains had increased ERG11 and MDR1 mRNA expression, while AFR1 and AFR2 were not overexpressed in strains with high MICs of FCZ compared with the expression levels for strains with low MICs of FCZ; and (iii) exposure to FCZ in strains with high MICs of FCZ induced AFR1 mRNA overexpression.CONCLUSION: We demonstrated that the FCZ-resistant mechanism commonly described in C. neoformans was not responsible for the resistance to FCZ in the rare subtype strains

Genotyping and antifungal susceptibility testing of Cryptococcus neoformans isolates from Cameroonian HIV-positive adult patients

Cryptococcus neoformans is the most common cause of meningitis amongst adult Africans with HIV/AIDS. The widespread use of fluconazole may lead to the emergence of isolates with reduced susceptibility. We studied C.neoformans isolates from HIV-infected patients with cryptococcal meningitis. Genotyping and antifungal testing were performed to assess the genetic diversity, occurrence of mixed infections and in vitro activity of antifungal agents. Isolates were recovered from cerebrospinal fluid prior to systemic antifungal treatment. Six isolates were studied for each sample (a total of 114 isolates from 19 patients). Serotyping was performed via LAC 1 and CAP 64 gene amplification and genotyping was performed using phage M13 core, (GACA)(4) and (GTG)(5) primers and restriction polymorphism analysis of the URA5 gene. Susceptibilities for amphotericin B, flucytosine, fluconazole, voriconazole and posaconazole were tested by the Sensititre YeastOne (R) method. All strains were identified as C.neoformans var. grubii serotype A. We identified nine major genotypes. Up to two genotypes were identified in the same sample. None of the isolates were resistant to the studied drugs. However, 13 of 114 strains exhibited a reduced susceptibility to fluconazole and 13 of 114 strains exhibited a reduced susceptibility to flucytosine. No correlation was found between the genotype and susceptibility. This study confirms the prevalence of C.neoformans serotype A in Cameroon. Two genotypes may be responsible for a single episode of cryptococcosis. The possibility of mixed infection and diminished susceptibility to fluconazole or flucytosine must be considered for the management of cryptococcosis