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BackgroundSaliva RT-PCR is an attractive alternative for the detection of SARS-CoV-2 in adults with much less known in children.MethodsChildren and adolescents with symptoms suggestive of COVID-19 were prospectively enrolled in a comparative clinical trial of saliva and nasopharyngeal (NP) RT-PCR between November and December 2020. Detection rates and sensitivities of saliva and NP RT-PCR were compared. Participants with discordant NP and saliva RT-PCR results including viral load (VL) were also analyzed.ResultOut of 405 patients enrolled, 397 patients had two tests performed. Mean age was 12.7 years (range 1.2-17.9). Detection rates were 22.9% (95%CI 18.8-27.1%) by saliva RT-PCR, 25.4% (21.2-29.7%) by NP RT-PCR, and 26.7% (22.4-31.1%) by any test. The sensitivity of saliva was 85.2% (78.2-92.1%) when using NP as the gold standard; in contrast, when saliva was considered the gold standard, the sensitivity of NP was 94.5% (89.8-99.2%).For a NP RT-PCR VL threshold of ≥103 and ≥104 copies/ml, sensitivity of saliva increases to 88.7% and 95.2% respectively. Sensitivity of saliva and NP swabs was respectively 89.5% and 95.3% in patient with symptoms less than 4 days (p=0.249) and 70.0% and 95.0% in those with symptoms ≥ 4 to 7 days (p=0.096). The 15 patients who had an isolated positive NP RT-PCR were significantly younger (p=0.034), had a lower NP VL (median 5.6×103 vs 3.9×107, p4; IQR 1.2×104-5.2×105; vs median 4.0×107cp/ml; IQR 8.6×105-1.x108; pConclusionSaliva PCR shows diagnostic performances close to NP RT-PCR for SARS-CoV2 detection in most symptomatic outpatient children and adolescents.

Multidrug resistance (MDR) has emerged in hospitals due to the use of several agents administered in combination or sequentially to the same individual. We reported earlier MDR inCandida lusitaniaeduring therapy with amphotericin B (AmB), azoles and candins. We used here comparative genomic approaches between the initial susceptible isolate and 4 other isolates with different MDR profiles. From a total of 18 non-synonymous SNPs (NSS) in genome comparisons with the initial isolate, six could be associated with MDR. One of the SNPs occurred in a putative transcriptional activator (MRR1) resulting in a V668G substitution in isolates resistant to azoles and 5-fluorocytosine (5-FC). We demonstrated by gene editing thatMMR1acted by upregulation ofMFS7(a multidrug transporter) in the presence of the V668G substitution.MFS7itself mediated not only azole resistance but also 5-FC resistance, which represents a novel resistance mechanism for this drug class. Three other distinct NSS occurred inFKS1(a glucan synthase that is targeted by candins) in three candin-resistant isolates. Lastly, two other NSS inERG3andERG4(ergosterol biosynthesis) resulting in non-sense mutations were revealed in AmB-resistant isolates, one of which accumulated the 2ERGNSS. AmB-resistant isolates lacked ergosterol and exhibited sterol profiles consistent withERG3andERG4defects. In conclusion, this genome analysis combined with genetics and metabolomics helped to decipher the resistance profiles identified in this clinical case. MDR isolates accumulated 6 different mutations conferring resistance to all antifungal agents used in medicine. This case study illustrates the capacity ofC. lusitaniaeto rapidly adapt under drug pressure within the host.ImportanceAntifungal resistance is an inevitable phenomenon when fungal pathogens get exposed to antifungal drugs. These drugs can be grouped in 4 distinct classes (azoles, candins, polyenes, pyrimidine analogs) and are used in different clinical settings. Failures in therapy implicates the sequential or combined use of these different drug classes, which can result in some cases in the development of multidrug resistance (MDR). MDR is particularly challenging in the clinic since it drastically reduces possible treatment alternatives. In this study, we report the rapid development of MDR inCandida lusitaniaein a patient, which became resistant to all known antifungal agents used up to now in medicine. To understand how MDR developed inC. lusitaniae, whole genome sequencing followed by comparative genome analysis was undertaken in sequential MDR isolates. This helped to detect all specific mutations linked to drug resistance and explained the different MDR patterns exhibited by the clinical isolates.