Use of a serum-free medium to produce in vitro Neospora caninum and Toxoplasma gondii tachyzoites on Vero cells

Neospora caninum and Toxoplasma gondii are cyst-forming coccidian parasites of human and veterinary clinical relevance. In vitro cultivation of the protozoans using Vero cells is usually performed in order to produce antigenic materials. Quantitative and qualitative comparisons of Vero cells grown in RPMI medium supplemented either with foetal calf serum (FCS), horse serum (HS) or a specific serum-free additive (DefCell) were performed. A serum-free cell culture system used to propagate N. caninum (NC-1 isolate) and T. gondii tachyzoites (Rh stain) were compared with the other two cell culture systems. FCS supplemented media was found to be more effective than the others in promoting Vero cells and N. caninum tachyzoites. However, it was found unable to support adequate T. gondii tachyzoite proliferation. Vero cells, T. gondii and N. caninum tachyzoite production gave similar growth patterns with either HS or DefCell supplemented media. Defcell was considered as a good alternative to supplement culture medium.Utilisation d'un milieu sans sérum pour la production in vitro de tachyzoites de Neospora caninum et Toxoplasma gondii sur cellules Vero. Neospora caninum et Toxoplasma gondii sont deux sporozoaires présentant un intérêt en médecine humaine et vétérinaire. La culture in vitro utilisant, entre autres, les cellules Vero comme support de la multiplication des deux parasites, est généralement employée en vue de l'obtention de tachyzoïtes. Au cours de cette étude, une évaluation quantitative et qualitative de la production de cellules Vero dans un milieu complémenté en sérum de veau foetal, de cheval ou bien avec un additif (DefCell) exempt de sérum, a été réalisée. Les trois types de milieu de culture ont également été employés et comparés dans le cadre de la production sur cellules Vero de tachyzoïtes de T. gondii (souche RH) et de N. caninum (isolat NC-1). Le milieu complémenté en sérum de veau fœtal s'est révélé être le plus adéquat pour la croissance de cellules Vero et la production de tachyzoïtes de N. caninum. Cependant, ce milieu s'est avéré incapable d'assurer une production optimale de tachyzoïtes de T. gondii. La production de cellules Vero, ainsi que de tachyzoïtes de T. gondii et de N. caninum, a présenté des caractéristiques communes en milieu complémenté en sérum de cheval et en DefCell. Ce dernier s'est révélé être une bonne alternative au sérum de veau foetal et de cheval pour complémenter les milieux de culture. L'absence de protéines animales dans ce milieu présente un certain nombre d'avantages qui sont discutés

A Recurrent Mutation at Position 26340 of SARS-CoV-2 Is Associated with Failure of the E Gene Quantitative Reverse Transcription-PCR Utilized in a Commercial Dual-Target Diagnostic Assay

Control of the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic requires accurate laboratory testing to identify infected individuals while also clearing essential staff to continue to work. At the current time, a number of quantitative real-time PCR (qRT-PCR) assays have been developed to identify SARS-CoV-2, targeting multiple positions in the viral genome. While the mutation rate of SARS-CoV-2 is moderate, given the large number of transmission chains, it is prudent to monitor circulating viruses for variants that might compromise these assays. Here, we report the identification of a C-to-U transition at position 26340 of the SARS-CoV-2 genome that is associated with failure of the cobas SARS-CoV-2 E gene qRT-PCR in eight patients. As the cobas SARS-CoV-2 assay targets two positions in the genome, the individuals carrying this variant were still called SARS-CoV-2 positive. Whole-genome sequencing of SARS-CoV-2 showed all to carry closely related viruses. Examination of viral genomes deposited on GISAID showed this mutation has arisen independently at least four times. This work highlights the necessity of monitoring SARS-CoV-2 for the emergence of single-nucleotide polymorphisms that might adversely affect RT-PCRs used in diagnostics. Additionally, it argues that two regions in SARS-CoV-2 should be targeted to avoid false negatives.status: publishe

Absence de mutation K13 associée à une résistance de l'artémisine chez Plasmodium falciparum en RDC

Artemisinin-based combination therapies (ACTs) have been recommended by the World Health Organization (WHO) as first-line treatment of uncomplicated Plasmodium falciparum (P. falciparum) malaria since 2005 in Democratic Republic of Congo (DRC) and a regular surveillance of the ACT efficacy is required to ensure the treatment effectiveness. Mutations in the propeller domain of the pfk13 gene were identified as molecular markers of artemisinin resistance (ART-R). This study investigated the pfk13-propeller gene polymorphism in clinical isolates of P. falciparum collected in the DRC. In 2017, ten geographical sites across DRC were selected for a cross-sectional study that was conducted first in Kinshasa from January to March, then in the nine other sites from September to December. Dried blood samples were collected from patients attending health centers for fever where diagnosis of Malaria was first made by rapid diagnostic test (RDT) available on site (SD Bioline malaria Ag Pf or CareStart Malaria Pf) or by thick blood smear and then confirmed by a P. falciparum real-time PCR assay. A pfk13-propeller segment containing a fragment that codes for amino acids at positions 427-595 was amplified by conventional PCR before sequencing. In total, 1070 patients were enrolled in the study. Real-time PCR performed confirmed the initial diagnosis of P. falciparum infection in 806 samples (75.3%; 95% CI: 72.6%- 77.9%). Of the 717 successfully sequenced P. falciparum isolates, 710 (99.0%; 95% CI: 97.9% - 99.6) were wild-type genotypes and 7 (1.0%; 95% CI: 0.4% - 2.1%) carried non-synonymous (NS) mutations in pfk13-propeller including 2 mutations (A578S and V534A) previously detected and 2 other (M472I and A569T) not yet detected in the DRC. Mutations associated with ART-R in Southeast Asia were not observed in DRC. However, the presence of other mutations in pfk13-propeller gene calls for further investigations to assess their implication in drug resistance

Assessment of Plasmodium falciparum anti-malarial drug resistance markers in pfk13-propeller, pfcrt and pfmdr1 genes in isolates from treatment failure patients in Democratic Republic of Congo, 2018–2019

Background: The national policy for malaria treatment of the Democratic Republic of Congo recommends two first-line artemisinin-based combinations for the treatment of uncomplicated malaria: artesunate-amodiaquine and artemether-lumefantrine. This study investigated the presence of markers associated with resistance to the current first-line artemisinin-based combination therapy (ACT) in isolates of Plasmodium falciparum from treatment failure patients in the Democratic Republic of Congo. Methods: From November 2018 to November 2019, dried blood spots were taken from patients returning to health centres for fever within 28 days after an initial malaria treatment in six sentinel sites of the National Malaria Control Programme across Democratic Republic of Congo. The new episode of malaria was first detected by a rapid diagnostic test and then confirmed by a real-time PCR assay to define treatment failure. Fragments of interest in pfk13 and pfcrt genes were amplified by conventional PCR before sequencing and the Pfmdr1 gene copy number was determined by a TaqMan real-time PCR assay. Results: Out of 474 enrolled patients, 364 (76.8%) were confirmed positive by PCR for a new episode of P. falciparum malaria, thus considered as treatment failure. Of the 325 P. falciparum isolates obtained from 364 P. falciparum-positive patients and successfully sequenced in the pfk13-propeller gene, 7 (2.2%) isolates carried non-synonymous mutations, among which 3 have been previously reported (N498I, N554K and A557S) and 4 had not yet been reported (F506L, E507V, D516E and G538S). Of the 335 isolates successfully sequenced in the pfcrt gene, 139 (41.5%) harboured the K76T mutation known to be associated with chloroquine resistance. The SVMNT haplotype associated with resistance to amodiaquine was not found. None of the isolates carried an increased copy number of the pfmdr1 gene among the 322 P. falciparum isolates successfully analysed

Biennial surveillance of Plasmodium falciparum anti-malarial drug resistance markers in Democratic Republic of Congo, 2017 and 2019

Background: Because of the loss of chloroquine (CQ) effectiveness, the Democratic Republic of Congo (DRC)’s malaria treatment policy replaced CQ by sulfadoxine–pyrimethamine (SP) as first‑line treatment of uncomplicated malaria in 2003, which in turn was replaced by artemisinin‑based combination therapies (ACT) in 2005. The World Health Organization (WHO) recommends monitoring of anti‑malarial drug resistance every 2 years. The study aimed to provide baseline data for biennial molecular surveillance of anti‑malarial drug resistance by comparing data from a study conducted in 2019 to previously published data from a similar study conducted in 2017 in the DRC. Methods: From July to November 2019, a cross‑sectional study was conducted in ten sites which were previously selected for a similar study conducted in 2017 across the DRC. P. falciparum malaria was diagnosed by a rapid diagnostic test (RDT) or by microscopy and dried blood samples (DBS) were taken from patients who had a positive test. Segments of interest in pfcrt and pfk13 genes were amplified by conventional PCR before sequencing. Results: Out of 1087 enrolled patients, 906 (83.3%) were PCR‑confirmed for P. falciparum. Like in the 2017‑study, none of the mutations known to be associated with Artemisinine (ART) resistance in Southeast Asia was detected. How‑ ever, non‑synonymous (NS) mutations with unknown functions were observed among which, A578S was detected in both 2017 and 2019‑studies. The overall prevalence of pfcrt‑K76T mutation that confers CQ‑resistance was 22.7% in 2019‑study compared to 28.5% in 2017‑study (p‑value = 0.069), but there was high variability between sites in the two studies. Like in 2017‑study, the pfcrt 72–76 SVMNT haplotype associated with resistance to amodiaquine was not detected. Conclusion: The study reported, within 2 years, the non‑presence of molecular markers currently known to be associated with resistance to ART and to AQ in P. falciparum isolated in the DRC. However, the presence of polymorphisms with as‑yet unknown functions was observed, requiring further characterization. Moreover, an overall decrease in the prevalence of CQ‑resistance marker was observed in the DRC, but this prevalence remained highly variable from region to regio