The protozoan Toxoplasma gondii is a ubiquitous and highly prevalent parasite that can theoretically infect all warm-blooded vertebrates. In humans, toxoplasmosis causes infections in both immunodeficient and immunocompetent patients, congenital toxoplasmosis, and ocular lesions. These manifestations have different degrees of severity. Clinical severity is determined by multiple factors, including the genotype of the T. gondii strain involved in the infection. T. gondii exhibits remarkable genetic diversity, which varies according to geography and ecotype (domestic or wild). Previous studies have demonstrated that wild strains of T. gondii are of particular epidemiological interest, as they have been associated with more severe forms of toxoplasmosis in different regions of the world. However, no data on wild strains of T. gondii are available from Africa. In this study, we describe for the first time a wild T. gondii strain from Africa. Wild animals from the forest environment of Gabon, Central Africa, were screened for chronic infection with T. gondii using quantitative PCR. The infecting T. gondii strains were genotyped whenever possible by the analysis of 15 microsatellite markers and by whole-genome sequencing. A new T. gondii genotype was identified in the DNA extract from a heart sample of a duiker (Cephalophus sp.) and was found to be highly divergent from previously described T. gondii populations worldwide, including those from domestic environments in Gabon. This discovery suggests the existence of a wild T. gondii population in Africa. The role of wild T. gondii strains in the incidence of severe toxoplasmosis in Africa remains unclear and requires further investigation.

IntroductionThere have only been a few molecular studies conducted on the detection of T. gondii in tissues of carnivores in South Africa, with no data on the genetic diversity of this parasite. That is why the aim of this study was to detect and genotype T. gondii DNA in tissues of selected wild and domestic carnivores in South Africa.MethodsSamples were collected from 80 animals of 20 species (mainly road-killed) in the four provinces of Limpopo (n=57), Mpumalanga (n=21), Gauteng (n=1) and Free State (n=1) during the period 2014–2018. Samples of brain (n=31), heart (n=4), liver (n=40), spleen (n=2) and lung (n=3) were used to detect T. gondii by real-time PCR targeting a 529 bp repeating fragment of T. gondii DNA. Samples that were positive in real-time PCR were genotyped using 15 microsatellite markers.ResultsT. gondii DNA was detected in 4 (5 %) samples: in the brain from a Black-backed Jackal (Canis mesomelas), in the liver from a African Wildcat (Felis silvestris lybica) and in the liver and heart of two Rusty-spotted Genets (Genetta maculata) respectively. The DNA sample from Black-backed Jackal was genotyped and characterized as belonging to the type Africa 4 lineage (equivalent to RFLP genotype ToxoDB#20), that is a widespread lineage in Africa.DiscussionThis is the first genetic characterization of T. gondii isolated from a wild carnivore on the African continent and the first report of T. gondii in Black-backed Jackal. The Africa 4 lineage was also confirmed in the region of Southern Africa for the first time.

Background Toxoplasmosis is a widespread zoonosis caused by the intracellular protozoan parasite Toxoplasma gondii. Limited epidemiological information is available about the prevalence of T. gondii in sheep in Romania, and a high incidence would have implications for both the economy and public health. To our knowledge, no studies are available about the T. gondii strains circulating in lambs. The objective of this study was to assess the prevalence of T. gondii in sheep (serology), lambs (serology, bioassay, PCR) and sheep abortions (PCR) in Romania. Moreover, the study aimed to perform the genetic characterization of T. gondii isolates from lambs. Methods Serum samples collected from 2650 sheep (2067 adults and 583 lambs) were tested for anti- T. gondii antibodies (IgG) using a commercial ELISA kit. Likewise, 328 pairs of diaphragmatic muscle-serum samples were collected from lambs aged between 2 and 4 months. Lamb serum samples were analyzed using MAT for anti- T. gondii antibody detection. The diaphragm tissue samples from MAT-positive lambs (at a dilution ≥ 1:25) were bioassayed in mice. The T. gondii strains were genotyped using 15 microsatellites markers. Additionally, brain and heart samples from 76 sheep abortions were analyzed for T. gondii DNA by polymerase chain reaction (PCR) targeting the 529-bp repeat region (REP529). Results The results showed that more than half of the tested sheep were T. gondii seropositive (53.5%). The seroprevalence was significantly higher in adults (61.1%) than in lambs (26.4%). The seroprevalence of T. gondii infection in slaughtered lambs, by MAT, was 37.5% (123/328). There were bioassayed in mice 56 diaphragmatic tissues from 123 seropositive lambs. Toxoplasma gondii strains were isolated from 18 (32.1%) lambs intended for human consumption. All T. gondii strains were confirmed by PCR. Six strains were genotyped using 15 microsatellite markers and belonged to genotype II. Toxoplasma gondii DNA was detected in 11.8% (9/76) of sheep abortions. Conclusions The present study showed the presence of T. gondii in sheep in all the regions considered in the study. The high prevalence of T. gondii infection in sheep and lambs, demonstrated by serology, molecular analysis and bioassay, highlighted that there is an important risk of human infection in consuming raw or undercooked sheep/lamb meat. Graphical Abstract

De novo mutations in the UL56 terminase subunit and its associated phenotypes were studied in the context of cytomegalovirus (CMV) transplant recipients clinically resistant to DNA-polymerase inhibitors, naive to letermovir. R246C was the only UL56 variant detected by standard and deep sequencing, located within the letermovir-resistance-associated region (residues 230-370). R246C emerged in 2/80 transplant recipients (1 hematopoietic and 1 heart) since first cytomegalovirus replication and responded transiently to various alternative antiviral treatments in vivo. Recombinant phenotyping showed R246C conferred an advanced viral fitness and was sensitive to ganciclovir, cidofovir, foscarnet, maribavir, and letermovir. These results demonstrate a low rate (2.5%) of natural occurring polymorphisms within the letermovir-resistant-associated region before its administration. Identification of high replicative capacity variants in patients not responding to treatment or experiencing relapses could be helpful to guide further therapy and dosing of antiviral molecules. IMPORTANCE We provide comprehensive data on the clinical correlates of both CMV genotypic follow-up by standard and deep sequencing and the clinical outcomes, as well as recombinant phenotypic results of this novel mutation. Our study emphasizes that the clinical follow-up in combination with genotypic and phenotypic studies is essential for the assessment and optimization of patients experiencing HCMV relapses or not responding to antiviral therapy. This information may be important for other researchers and clinicians working in the field to improve the care of transplant patients since drug-resistant CMV infections are an important emerging problem even with the new antiviral development.

Toxoplasmosis, one of the most prevalent parasitic infections in humans and animals, is caused by the intracellular protozoan parasite Toxoplasma gondii. Small mammals play a key role as intermediate reservoir hosts in the maintenance of the T. gondii life cycle. In this study, we estimated the molecular prevalence and provide genetic diversity data for T. gondii in 632 small mammals sampled in four areas of Cotonou city, Benin. Both the brain and heart of each individual were screened through T. gondii-targeting qPCR, and positive samples were then genotyped using a set of 15 T. gondii-specific microsatellites. Prevalence data were statistically analyzed in order to assess the relative impact of individual host characteristics, spatial distribution, composition of small mammal community, and urban landscape features. An overall T. gondii molecular prevalence of 15.2% was found and seven genotypes, all belonging to the Africa 1 lineage, could be retrieved from the invasive black rat Rattus rattus and the native African giant shrew Crocidura olivieri. Statistical analyses did not suggest any significant influence of the environmental parameters used in this study. Rather, depending on the local context, T. gondii prevalence appeared to be associated either with black rat, shrew, or mouse abundance or with the trapping period. Overall, our results highlight the intricate relationships between biotic and abiotic factors involved in T. gondii epidemiology and suggest that R. rattus and C. olivieri are two competent reservoirs for the Africa 1 lineage, a widespread lineage in tropical Africa and the predominant lineage in Benin.

Background This study describes the genotypic and phenotypic characterization of novel human cytomegalovirus (HCMV) genetic variants of a cohort of 94 clinically resistant HCMV patients. Methods and results Antiviral-resistant mutations were detected in the UL97, UL54, and UL56 target genes of 25 of 94 (26.6%) patients. The genotype-phenotype correlation study resolved the status of 5 uncharacterized UL54 deoxyribonucleic acid polymerase (G441S, A543V, F460S, R512C, A928T) and 2 UL56 terminase (F345L, P800L) mutations found in clinical isolates. A928T conferred high, triple resistance to ganciclovir, foscarnet, and cidofovir, and A543V had 10-fold reduced susceptibility to cidofovir. Viral growth assays showed G441S, A543V, F345L, and P800L impaired viral growth capacities compared with wild-type AD169 HCMV. Three-dimensional modeling predicted A543V and A928T phenotypes but not R512C, reinforcing the need for individual characterization of mutations by recombinant phenotyping. Conclusions Extending mutation databases is crucial to optimize treatments and to improve the assessment of patients with resistant/refractory HCMV infection.

The isolation and molecular typing of Toxoplasma gondii strains provide an essential basis for a better understanding of the parasite’s genetic diversity, determinants of its geographical distribution and associated risks to human health. In this study, we isolated and genetically characterized T. gondii strains from domestic animals in Southern and coastal area of Tunisia. Blood, hearts and/or brains were collected from 766 domestic animals (630 sheep and 136 free-range chickens). Strain isolation from these samples was performed using mouse bioassay and genotyping was carried out with a multiplex PCR technique using 15 microsatellite markers. Thirty viable strains of T. gondii were successfully isolated from tissues of sheep (19/142) and chickens (11/33). In addition, 3 strains could be successfully genotyped from animal tissues for which mouse bioassay was unsuccessful. A large predominance of type II strains (n = 29) was found in the sampled regions, followed by type III (n = 3) and, for the first time in Tunisia, a single isolate of Africa 4 lineage from a sheep. Analyses of population genetics showed the presence of a divergent population of type II lineage in Tunisia, supporting limited recent migrations of strains between Tunisia and other countries of the world.

Two transplant recipients (1 kidney and 1 hematopoietic stem cell) received maribavir (MBV) after cytomegalovirus (CMV) infection clinically resistant to standard therapy. Both patients achieved CMV DNA clearance within 30 and 18 days; however, the UL97 C480F variant emerged, causing recurrent CMV infection after a cumulative 2 months of MBV and 15 or 4 weeks of ganciclovir treatment, respectively. C480F was not detected under ganciclovir before MBV treatment. Recombinant phenotyping showed that C480F conferred the highest level of MBV resistance and ganciclovir cross-resistance, with impaired viral growth. Clinical follow-up and genotypic and phenotypic studies are essential for the assessment and optimization of patients with suspected MBV resistance.

Toxoplasma gondii is a zoonotic protozoan with a worldwide occurrence, but the determinants of the current pattern in the geographical distribution of T. gondii lineages and strains remain poorly understood. To test the influence of human trade on T. gondii populations, we conducted a population genetic study of 72 T. gondii animal isolates from Senegal, a West African country in which the ongoing inland progress of invasive murine hosts (introduced in port cities of Senegal since the 16th century by European sailors) is well described. Isolates were mainly collected on free-range poultry, which are considered as relevant bioindicators of T. gondii strain diversity in the domestic environment. Sampling was conducted in two port cities of Senegal (Dakar and Saint-Louis) and in one inland region (Kedougou). Population genetic analyses using 15 microsatellite markers revealed different patterns between port cities where lineages non-virulent for mice (type II, type III, and Africa 4) were predominant, and Kedougou where the mouse-virulent Africa 1 lineage was the most common. By considering the current spatial pattern in the inland progress of invasive rodents in Senegal, our results suggest that the invasive house mouse Mus musculus domesticus counter-selects the Africa 1 lineage in the invaded areas. The comparison of the microsatellite alleles of type II strains from Senegal to type II strains from other areas in Africa and Western Europe, using discriminant analysis of principal components and Network analysis, point to a mainly Western European origin of the type II lineage in Senegal. Collectively, these findings suggest that human-mediated intercontinental migrations of murine hosts are important vectors of T. gondii strains. Differential susceptibility of endemic and introduced murine hosts to various T. gondii strains probably determines the persistence of these strains in the environment, and therefore their availability for human and animal infection.