Single nucleotide variation catalog from clinical isolates mapped on tertiary and quaternary structures of ESX-1-related proteins reveals critical regions as putative Mtb therapeutic targets.

Proteins encoded by the ESX-1 genes of interest are essential for full virulence in all Mycobacterium tuberculosis complex (Mtbc) lineages, the pathogens causing the highest mortality worldwide. Identifying critical regions in these ESX-1-related proteins could provide preventive or therapeutic targets for Mtb infection, the game changer needed for tuberculosis control. We analyzed a compendium of whole genome sequences of clinical Mtb isolates from all lineages from >32,000 patients and identified single nucleotide polymorphisms. When mutations corresponding to all non-synonymous single nucleotide polymorphisms were mapped on structural models of the ESX-1 proteins, fully conserved regions emerged. Some could be assigned to known quaternary structures, whereas others could be predicted to be involved in yet-to-be-discovered interactions. Some mutants had clonally expanded (found in >1% of the isolates); these mutants were mostly located at the surface of globular domains, remote from known intra- and inter-molecular protein-protein interactions. Fully conserved intrinsically disordered regions of proteins were found, suggesting that these regions are crucial for the pathogenicity of the Mtbc. Altogether, our findings highlight fully conserved regions of proteins as attractive vaccine antigens and drug targets to control Mtb virulence. Extending this approach to the whole Mtb genome as well as other microorganisms will enhance vaccine development for various pathogens. IMPORTANCE: We mapped all non-synonymous single nucleotide polymorphisms onto each of the experimental and predicted ESX-1 proteins' structural models and inspected their placement. Varying sizes of conserved regions were found. Next, we analyzed predicted intrinsically disordered regions within our set of proteins, finding two putative long stretches that are fully conserved, and discussed their potential essential role in immunological recognition. Combined, our findings highlight new targets for interfering with Mycobacterium tuberculosis complex virulence.

Nationwide Harmonization Effort for Semi-Quantitative Reporting of SARS-CoV-2 PCR Test Results in Belgium.

From early 2020, a high demand for SARS-CoV-2 tests was driven by several testing indications, including asymptomatic cases, resulting in the massive roll-out of PCR assays to combat the pandemic. Considering the dynamic of viral shedding during the course of infection, the demand to report cycle threshold (Ct) values rapidly emerged. As Ct values can be affected by a number of factors, we considered that harmonization of semi-quantitative PCR results across laboratories would avoid potential divergent interpretations, particularly in the absence of clinical or serological information. A proposal to harmonize reporting of test results was drafted by the National Reference Centre (NRC) UZ/KU Leuven, distinguishing four categories of positivity based on RNA copies/mL. Pre-quantified control material was shipped to 124 laboratories with instructions to setup a standard curve to define thresholds per assay. For each assay, the mean Ct value and corresponding standard deviation was calculated per target gene, for the three concentrations (107, 105 and 103 copies/mL) that determine the classification. The results of 17 assays are summarized. This harmonization effort allowed to ensure that all Belgian laboratories would report positive PCR results in the same semi-quantitative manner to clinicians and to the national database which feeds contact tracing interventions.

Comparison of the Quantitative DiaSorin Liaison Antigen Test to Reverse Transcription-PCR for the Diagnosis of COVID-19 in Symptomatic and Asymptomatic Outpatients.

We evaluated the quantitative DiaSorin Liaison severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen test in symptomatic and asymptomatic individuals consulting their general practitioners (GPs) during a period of stable intense virus circulation (213/100,000 habitants per day). Leftover reverse transcription-PCR (RT-PCR) positive (n = 204) and negative (n = 210) nasopharyngeal samples were randomly selected among fresh routine samples collected from patients consulting their GPs. Samples were tested on Liaison XL according to the manufacturer's instructions. Equivocal results were considered negative. The overall sensitivity and specificity of the Liaison antigen test compared to RT-PCR were 65.7% (95% confidence interval [CI], 58.9% to 71.9%) and 100% (CI, 97.8% to 100%). Sensitivity in samples with viral loads of ≥105, ≥104, and ≥103 copies/ml were 100% (CI, 96.3% to 100.0%), 96.5% (CI, 91.8% to 98.7%), and 87.4% (CI, 81.3% to 91.5%), respectively. All samples with ≤103 copies/ml were antigen negative. The ratio of antigen concentration to viral load in samples with ≥103 copies/ml was comparable in symptomatic and asymptomatic individuals (P = 0.58). The proportion of RT-PCR-positive participants with a high viral load (≥105 copies/ml) was not significantly higher in symptomatic than in asymptomatic participants (63.9% [CI, 54.9% to 72.0%] versus 51.9% [CI, 41.1% to 62.6%]; P = 0.11), but the proportion of participants with a low viral load (<103 copies/ml) was significantly higher in asymptomatic than in symptomatic RT-PCR-positive participants (35.4% [CI, 25.8% to 46.4%] versus 14.3% [CI, 9.0% to 21.8%]; P < 0.01). Sensitivity and specificity in samples with a viral load of ≥104 copies/ml were 96.5% and 100%. The correlation of antigen concentration with viral load was comparable in symptomatic and asymptomatic individuals.

Triazole-Resistance in Environmental Aspergillus fumigatus in Latin American and African Countries.

Triazole-resistance has been reported increasingly in Aspergillus fumigatus. An international expert team proposed to avoid triazole monotherapy for the initial treatment of invasive aspergillosis in regions with >10% environmental-resistance, but this prevalence is largely unknown for most American and African countries. Here, we screened 584 environmental samples (soil) from urban and rural locations in Mexico, Paraguay, and Peru in Latin America and Benin and Nigeria in Africa for triazole-resistant A. fumigatus. Samples were screened using triazole-containing agars and confirmed as triazole-resistant by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth dilution reference method. Isolates were further characterized by cyp51A sequencing and short-tandem repeat typing. Fungicide presence in samples was likewise determined. Among A. fumigatus positive samples, triazole-resistance was detected in 6.9% (7/102) of samples in Mexico, 8.3% (3/36) in Paraguay, 9.8% (6/61) in Peru, 2.2% (1/46) in Nigeria, and none in Benin. Cyp51A gene mutations were present in most of the triazole-resistant isolates (88%; 15/17). The environmentally-associated mutations TR34/L98H and TR46/Y121F/T289A were prevalent in Mexico and Peru, and isolates harboring these mutations were closely related. For the first time, triazole-resistant A. fumigatus was found in environmental samples in Mexico, Paraguay, Peru, and Nigeria with a prevalence of 7-10% in the Latin American countries. Our findings emphasize the need to establish triazole-resistance surveillance programs in these countries.

First case of Candida auris infection in Belgium in a surgical patient from Kuwait.

Objective and importance: Candida auris is a relatively new yeast species and an emerging opportunistic pathogen. It was first reported in 2009 in East Asia, as a difficult-to-identify Candida species of uncertain clinical relevance. In recent years, it has appeared globally as a cause of invasive infections, not infrequently eliciting nosocomial outbreaks. Species identification in clinical laboratories has been challenging, as traditional phenotypic and biochemical methods have been generally unreliable. Clinical management is often complicated by multidrug resistance in many isolates. Additionally, C. auris has demonstrated an unusual ability for persistence in the hospital environment and in asymptomatic patients. We present the first Belgian case of C. auris infection along with a brief review of the literature.Clinical presentation: A patient was referred from Kuwait for surgical treatment after a complicated bariatric procedure. Few days after transferral, she developed a catheter-related blood stream infection with C. auris. We obtained a low-confidence identification of C. auris with the Bruker Biotyper MALDI-TOF MS system (Bruker Corporation, Billerica, MA, U.S.A.), and of Candida haemulonii with the Vitek YST identification system, version 7.01 (bioMérieux, Marcy-L'Etoile, France). Definite identification was obtained using Internal Transcribed Spacer (ITS) sequencing. As most C. auris isolates, our strain was resistant to fluconazole, and the patient was eventually treated with catheter removal and anidulafungin therapy. We documented persistence of C. auris clones with acquired echinocandin resistance in our patient up to 18 months after the infection.Conclusion: Clinicians and microbiologists should be aware of this globally emerging yeast, that poses important challenges in identification, treatment and hospital infection control.

Hospital Laboratory Survey for Identification of Candida auris in Belgium.

Candida auris is a difficult-to-identify, emerging yeast and a cause of sustained nosocomial outbreaks. Presently, not much data exist on laboratory preparedness in Europe. To assess the ability of laboratories in Belgium and Luxembourg to detect this species, a blinded C. auris strain was included in the regular proficiency testing rounds organized by the Belgian public health institute, Sciensano. Laboratories were asked to identify and report the isolate as they would in routine clinical practice, as if grown from a blood culture. Of 142 respondents, 82 (57.7%) obtained a correct identification of C. auris. Of 142 respondents, 27 (19.0%) identified the strain as Candida haemulonii. The remaining labs that did not obtain a correct identification (33/142, 23.2%), reported other yeast species (4/33) or failed to obtain a species identification (29/33). To assess awareness about the infection-control implications of the identification, participants were requested to indicate whether referral of this isolate to a reference laboratory was desirable in a clinical context. Over one-third of all respondents (54/142, 38.0%) stated that they would not send the isolate to a reference laboratory, neither for epidemiological reasons nor for identification confirmation or antifungal susceptibility testing. This comprised 41.5% of the laboratories that submitted an identification of C. auris (34/82). Awareness among Belgian microbiologists, therefore, remains inadequate more than two years after C. auris' emergence in European clinics. Our data confirm high rates of misidentifications with commonly used identification methods. Programs for raising awareness in European hospitals may be warranted.