A mutation responsible for impaired detection by the Xpert SARS-CoV-2 assay independently emerged in different lineages during the SARS-CoV-2 pandemic.

BACKGROUND: COVID-19 diagnosis lies on the detection of SARS-CoV-2 on nasopharyngeal specimens by RT-PCR. The Xpert-Xpress SARS-CoV-2 assay provides results in less than one hour from specimen reception, which makes it suitable for clinical/epidemiological circumstances that require faster responses. The analysis of a COVID-19 outbreak suspected in the neonatology ward from our institution showed that the Ct values obtained for the targeted genes in the Xpert assay were markedly different within each specimen (N Ct value > 20 cycles above the E Ct value). RESULTS: We identified the mutation C29200T in the N gene as responsible for an impairment in the N gene amplification by performing whole genome sequencing of the specimens involved in the outbreak (Omicron variant). Subsequently, a retrospective analysis of all specimens sequenced in our institution allowed us to identify the same SNP as responsible for similar impairments in another 12 cases (42% of the total cases reported in the literature). Finally, we found that the same SNP emerged in five different lineages independently, throughout almost all the COVID-19 pandemic. CONCLUSIONS: We demonstrated for the first time the impact of this SNP on the Xpert assay, when harbored by new Omicron variants. We extend our observation period throughout almost all the COVID-19 pandemic, offering the most updated observations of this phenomenon, including sequences from the seventh pandemic wave, until now absent in the reports related to this issue. Continuous monitoring of emerging SNPs that could affect the performance of the most commonly used diagnostic tests, is required to redesign the tests to restore their correct performance.

Systematic genomic analysis of SARS-CoV-2 co-infections throughout the pandemic and segregation of the strains involved.

BACKGROUND: SARS-CoV-2 recombinants involving the divergent Delta and Omicron lineages have been described, and one of them, "Kraken" (XBB.1.5), has recently been a matter of concern. Recombination requires the coexistence of two SARS-CoV-2 strains in the same individual. Only a limited number of studies have focused on the identification of co-infections and are restricted to co-infections involving the Delta/Omicron lineages. METHODS: We performed a systematic identification of SARS-CoV-2 co-infections throughout the pandemic (7609 different patients sequenced), not biassed towards the involvement of highly divergent lineages. Through a comprehensive set of validations based on the distribution of allelic frequencies, phylogenetic consistency, re-sequencing, host genetic analysis and contextual epidemiological analysis, these co-infections were robustly assigned. RESULTS: Fourteen (0.18%) co-infections with ≥ 8 heterozygous calls (8-85 HZs) were identified. Co-infections were identified throughout the pandemic and involved an equal proportion of strains from different lineages/sublineages (including pre-Alpha variants, Delta and Omicron) or strains from the same lineage. Co-infected cases were mainly unvaccinated, with mild or asymptomatic clinical presentation, and most were at risk of overexposure associated with the healthcare environment. Strain segregation enabled integration of sequences to clarify nosocomial outbreaks where analysis had been impaired due to co-infection. CONCLUSIONS: Co-infection cases were identified throughout the pandemic, not just in the time periods when highly divergent lineages were co-circulating. Co-infections involving different lineages or strains from the same lineage were occurring in the same proportion. Most cases were mild, did not require medical assistance and were not vaccinated, and a large proportion were associated with the hospital environment.

Frequent Emergence of Resistance Mutations Following Complex Intra-Host Genomic Dynamics in SARS-CoV-2 Patients Receiving Sotrovimab.

The emergence of the Omicron variant of SARS-CoV-2 represented a challenge to the treatment of COVID-19 using monoclonal antibodies. Only Sotrovimab maintained partial activity, allowing it to be used in high-risk patients infected with the Omicron variant. However, reports of resistance mutations to Sotrovimab demand efforts to better understand the intra-patient emergence of Sotrovimab resistance. A retrospective genomic analysis was conducted on respiratory samples from immunocompromised patients infected with SARS-CoV-2 who received Sotrovimab at our hospital between December 2021 and August 2022. The study involved 95 sequential specimens from 22 patients (1 to 12 samples/patient; 3 to 107 days post-infusion; threshold cycle [CT] ≤ 32). Resistance mutations (in P337, E340, K356, and R346) were detected in 68% of cases; the shortest time to detection of a resistance mutation was 5 days after Sotrovimab infusion. The dynamics of resistance acquisition were highly complex, with up to 11 distinct amino acid changes in specimens from the same patient. In two patients, the mutation distribution was compartmentalized in respiratory samples from different sources. This is the first study to examine the acquisition of Sotrovimab resistance in the BA.5 lineage, enabling us to determine the lack of genomic or clinical differences between Sotrovimab resistance in BA.5 relative to that in BA.1/2. Across all Omicron lineages, the acquisition of resistance delayed SARS-CoV-2 clearance (40.67 versus 19.5 days). Close, real-time genomic surveillance of patients receiving Sotrovimab should be mandatory to facilitate early therapeutic interventions.

Early SARS-CoV-2 Reinfections Involving the Same or Different Genomic Lineages, Spain.

Centers for Disease Control and Prevention guidelines consider SARS-CoV-2 reinfection when sequential COVID-19 episodes occur >90 days apart. However, genomic diversity acquired over recent COVID-19 waves could mean previous infection provides insufficient cross-protection. We used genomic analysis to assess the percentage of early reinfections in a sample of 26 patients with 2 COVID-19 episodes separated by 20-45 days. Among sampled patients, 11 (42%) had reinfections involving different SARS-CoV-2 variants or subvariants. Another 4 cases were probable reinfections; 3 involved different strains from the same lineage or sublineage. Host genomic analysis confirmed the 2 sequential specimens belonged to the same patient. Among all reinfections, 36.4% involved non-Omicron, then Omicron lineages. Early reinfections showed no specific clinical patterns; 45% were among unvaccinated or incompletely vaccinated persons, 27% were among persons <18 years of age, and 64% of patients had no risk factors. Time between sequential positive SARS-CoV-2 PCRs to consider reinfection should be re-evaluated.

Pneumocystis jirovecii Pneumonia Diagnostic Approach: Real-Life Experience in a Tertiary Centre.

Pneumocystis jirovecii pneumonia (PJP) in immunocompromised patients entails high mortality and requires adequate laboratory diagnosis. We compared the performance of a real time-PCR assay against the immunofluorescence assay (IFA) in the routine of a large microbiology laboratory. Different respiratory samples from HIV and non-HIV-infected patients were included. The retrospective analysis used data from September 2015 to April 2018, which included all samples for which a P. jirovecii test was requested. A total of 299 respiratory samples were tested (bronchoalveolar lavage fluid (n = 181), tracheal aspirate (n = 53) and sputum (n = 65)). Forty-eight (16.1%) patients fulfilled the criteria for PJP. Five positive samples (10%) had only colonization. The PCR test was found to have a sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 96%, 98%, 90% and 99%, compared to 27%, 100%, 100% and 87%, for the IFA, respectively. PJ-PCR sensitivity and specificity were >80% and >90% for all tested respiratory samples. Median cycle threshold values in definite PJP cases were 30 versus 37 in colonized cases (p < 0.05). Thus, the PCR assay is a robust and reliable test for the diagnosis PJP in all respiratory sample types. Ct values of ≥36 could help to exclude PJP diagnosis.

Demographic, clinical and microbiological characteristics of the first 30 human monkeypox confirmed cases attended in a tertiary hospital in Madrid (Spain), during the May-June 2022 international outbreak / Características demográficas, clínicas y microbiológicas de los primeros 30 casos humanos confirmados de viruela del mono atendidos en un hospital terciario de Madrid (España), durante el brote internacional de mayo-junio de 2022

The present outbreak of Human Monkeypox (HMPX) that has begun in May 2022 and has spread across all continents in less than two months has qualitative and quantitative characteristics that make it different from the pattern of human disease previously caused by this virus. It has spread with enormous ease, affects almost exclusively adults, behaves as a sexually transmitted disease and focuses on very specific groups and transmission conditions. The high incidence in the city of Madrid in males that have sex with males (MSM) has allowed us to observe and report the experience with the first 30 cases diagnosed in our institution. Patients presented with febrile symptoms, genital and paragenital skin lesions reminiscent of smallpox, but less extensive and severe. The disease may also cause proctitis, pharyngitis and perioral lesions. The PCR test for diagnostic confirmation has been shown to be very sensitive and effective, not only in skin lesions but also in blood and other fluids such as pharyngeal, rectal exudates and blood. A very high proportion of patients with HMPX also have other sexually transmitted diseases that must be actively detected in this context. The spontaneous evolution of our patients has been good and hospitalization has been practically unnecessary. Transmission to non-sexual cohabitants and health personnel has been nonexistent and the lesions have disappeared in less than 30 days without leaving sequelae and no need for specific antiviral treatment. (AU)

El actual brote de la enfermedad por el virus de la viruela del mono humana (HMPX), que ha comenzado en mayo de 2022 y se ha extendido por todos los continentes en menos de dos meses, tiene unas características cualitativas y cuantitativas que lo diferencian del patrón de enfermedad humana causado anteriormente por este virus. Se ha extendido con enorme facilidad, afecta casi exclusivamente a adultos, se comporta como una enfermedad de transmisión sexual y se centra en grupos y condiciones de transmisión muy específicas. La alta incidencia en la ciudad de Madrid en varones que tienen sexo con varones (HSH) nos ha permitido observar y comunicar la experiencia con los primeros 30 casos diagnosticados en nuestra institución. Los pacientes se presentaron con síntomas febriles, lesiones cutáneas genitales y paragenitales que recuerdan a la viruela, pero menos extensas y graves. La enfermedad también puede causar proctitis, faringitis y lesiones periorales. La prueba PCR para la confirmación del diagnóstico ha demostrado ser muy sensible y eficaz, no sólo en las lesiones cutáneas sino también en la sangre y otros fluidos como los exudados faríngeos y rectales y la sangre. Una proporción muy elevada de pacientes con HMPX presentan también otras enfermedades de transmisión sexual que deben ser detectadas activamente en este contexto. La evolución espontánea de nuestros pacientes ha sido buena y la hospitalización ha sido prácticamente innecesaria. La transmisión a convivientes no sexuales y al personal sanitario ha sido inexistente y las lesiones han desaparecido en menos de 30 días sin dejar secuelas y sin necesidad de tratamiento antiviral específico. (AU)

Cost analysis of disease including treatment with dalbavancin in a Spanish hospital: ECODAL ANALYSIS.

INTRODUCTION AND AIM: Dalbavancin is an antibiotic with activity against gram-positive bacteria that allows early discharge of patients requiring intravenous therapy. Outpatient treatment helps offset hospitalisation costs associated with standard intravenous treatment. Our objective was to assess the cost of disease management, including treatment with dalbavancin, in a Spanish hospital for 1 year, and the hypothetical costs associated with treatment with other therapeutic alternatives to dalbavancin. METHODS: A single-centre, observational, retrospective post-hoc analysis was conducted based on electronic medical records analysing all patients who received dalbavancin treatment throughout 1 year; cost analysis was performed for the whole process. In addition, three scenarios designed on the basis of real clinical practice by clinical experts were hypothesised: (i) individual therapeutic alternative to dalbavancin, (ii) all patients treated with daptomycin, and (iii) all days of dalbavancin as outpatient treatment transformed into hospital stay. Costs were obtained from the hospital. RESULTS: Thirty-four patients were treated with dalbavancin; their mean age was 57.9 years, and 70.6% were men. The main reasons for dalbavancin use were outpatient management (61.7%, n = 21) and ensuring treatment adherence (26.5%, n = 9). The main indications were: osteoarticular infection (32.4%) and infective endocarditis (29.4%). One-half (50%) of the infections were due to Staphylococcus aureus (23.5% were methicillin resistant). All patients achieved clinical resolution, and no costs associated with dalbavancin-associated adverse events or re-admissions were reported. The mean total cost of treatment was 22,738€ per patient, with the greatest expenditures in interventions (8,413€) and hospital stay (6,885€). The mean cost of dalbavancin treatment was 3,936€; without dalbavancin, this cost could have been increased to 3,324-11,038€ depending on the scenario, mainly due to hospital stays. MAIN LIMITATION: Limited sample size obtained from a single centre. CONCLUSION: The economic impact of the management of these infections is high. The cost of dalbavancin is offset by the decreased length of stay.

Rapid Identification of Relevant Microbial Strains by Identifying Multiple Marker Single Nucleotide Polymorphisms via Amplicon Sequencing: Epidemic Monkeypox Virus as a Proof of Concept.

Despite the proven value of applying genomic data for epidemiological purposes, commonly used high-throughput sequencing formats are not adapted to the response times required to intervene and finally control outbreaks. In this study, we propose a fast alternative to whole-genome sequencing (WGS) to track relevant microbiological strains: nanopore sequencing of multiple amplicons including strain marker single nucleotide polymorphisms (SNPs). As a proof a concept, we evaluated the performance of our approach to offer a rapid response to the most recent public health global alarm, the monkeypox virus (MPXV) global outbreak. Through a multisequence alignment, a list of 42 SNPs were extracted as signature makers for this outbreak. Twenty primer pairs were designed to amplify in a multiplex PCR the regions including 22 of these SNPs. Amplicon pools were sequenced in a MinION device, and SNPs were called in real time by an in-house bioinformatic pipeline. A total of 120 specimens (95 MPXV-PCR positive, Ct values from 14 to 39) were selected. In 67.37% of the positive subset, all 22 SNPs were called. After excluding low viral load specimens, in 92% of samples ≥11 outbreak SNPs were called. No false positives were observed in any of the 25 negative specimens. The total turnaround time required for this strategy was 5 hours, and the cost per sample was 14 euros. Nanopore sequencing of multiple amplicons harboring signature SNPs escapes the targeting limitations of strain-specific PCRs and offers a powerful alternative to systematic WGS, paving the way to real-time genomic epidemiology and making immediate intervention possible to finally optimize transmission control. IMPORTANCE Nanopore sequencing of multiple amplicons harboring signature single nucleotide polymorphisms (SNPs) escapes the targeting limitations of strain-specific PCRs and offers a powerful alternative to systematic whole-genome analysis, paving the way to real-time genomic epidemiology and making immediate intervention possible to finally optimize transmission control.

The relevance of multiple clinical specimens in the diagnosis of monkeypox virus, Spain, June 2022.

A monkeypox virus (MPXV) outbreak has been ongoing worldwide since May 2022. The role of specimens other than skin lesions for MPXV diagnosis is unknown. We evaluated 140 different clinical specimens by real-time PCR. The highest positivity rates (97%) were from skin lesions of any part of the body, followed by plasma, pharyngeal and anal swabs. Testing specimens from multiple sites may improve the sensitivity and reduce false-negative test results.

Invasive pulmonary aspergillosis in the COVID-19 era: An expected new entity.

OBJECTIVES: Information on the recently COVID-19-associated pulmonary aspergillosis (CAPA) entity is scarce. We describe eight CAPA patients, compare them to colonised ICU patients with coronavirus disease 2019 (COVID-19), and review the published literature from Western countries. METHODS: Prospective study (March to May, 2020) that included all COVID-19 patients admitted to a tertiary hospital. Modified AspICU and European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria were used. RESULTS: COVID-19-associated pulmonary aspergillosis was diagnosed in eight patients (3.3% of 239 ICU patients), mostly affected non-immunocompromised patients (75%) with severe acute respiratory distress syndrome (ARDS) receiving corticosteroids. Diagnosis was established after a median of 15 days under mechanical ventilation. Bronchoalveolar lavage was performed in two patients with positive Aspergillus fumigatus cultures and galactomannan (GM) index. Serum GM was positive in 4/8 (50%). Thoracic CT scan findings fulfilled EORTC/MSG criteria in one case. Isavuconazole was used in 4/8 cases. CAPA-related mortality was 100% (8/8). Compared with colonised patients, CAPA subjects were administered tocilizumab more often (100% vs. 40%, p = .04), underwent longer courses of antibacterial therapy (13 vs. 5 days, p = .008), and had a higher all-cause mortality (100% vs. 40%, p = .04). We reviewed 96 similar cases from recent publications: 59 probable CAPA (also putative according modified AspICU), 56 putative cases and 13 colonisations according AspICU algorithm; according EORTC/MSG six proven and two probable. Overall, mortality in the reviewed series was 56.3%. CONCLUSIONS: COVID-19-associated pulmonary aspergillosis must be considered a serious and potentially life-threatening complication in patients with severe COVID-19 receiving immunosuppressive treatment.