Combination of mycological criteria: a better surrogate to identify COVID-19 associated pulmonary aspergillosis patients and evaluate prognosis?

Introduction: Diagnosis of COVID-19 associated pulmonary aspergillosis remains unclear especially in non-immunocompromised patients. The aim of this study was to evaluate seven mycological criteria and their combination in a large homogenous cohort of patients. Methods: All successive patients (n=176) hospitalized for COVID-19 requiring mechanical ventilation and who clinically worsened despite appropriate standard of care were included over a one-year period. Direct examination, culture, Aspergillus qPCR ( Af -qPCR) and galactomannan was performed on all respiratory samples (n=350). Serum galactomannan, ß-D-glucan and plasma Af -qPCR were also assessed. Criteria were analyzed alone or in combination in relation to mortality rate. Results: Mortality was significantly different in patients with 0, ≤2 and ≥3 positive criteria (logrank test, p=0.04) with death rate of 43.1, 58.1 and 76.4% respectively. Direct examination, plasma qPCR and serum galactomannan were associated with a 100% mortality rate. Bronchoalveolar lavage (BAL) galactomannan and positive respiratory sample culture were often found as isolated markers (28.1 and 34.1%) and poorly repeatable when a second sample was obtained. Aspergillus DNA was detected in 13.1% of samples (46/350) with significantly lower Cq when associated with at least one other criteria (30.2 vs 35.8) (p<0.001). Conclusion: Combination of markers and/or blood biomarkers and/or direct respiratory sample examination seems more likely to identify patients with CAPA. Af -qPCR may help identifying false positive results of BAL galactomannan testing and culture on respiratory samples while quantifying fungal burden accurately.

Duration of respiratory sample stability at -80ºC for SARS-CoV-2 PCR

Objective: To determine the stability of respiratory samples for SARS-CoV-2 PCR at standard laboratory ultra-freezer temperatures (-80°C). Methods: Five hundred and sixty-five archived, SARS-CoV-2 PCR positive patient specimens received at the Pathology Department of the Indus Hospital & Health Network between January 2021 and June 2021 were retested in June 2021. Samples had been stored at -70°C or below throughout this duration. Sample integrity following storage was assessed as the percentage of samples with reproducible results, and as consistency of cycle threshold (Ct) values between the original testing and the repeat testing. Results: Of the 565 samples evaluated in this study, 86% gave reproducible results upon retesting. However, there was no correlation between the duration of storage and result reproducibility, though the majority (69% for PCR Target-I and 78% for PCR Target-II respectively) of non-reproducible results had Ct values above 30. Similarly, there was a consistent increase of Ct values upon storage at ultra-freezer temperatures, though the effect again was more contingent upon freezing the sample in the ultra-freezer rather than the duration of storage. Conclusion: SARS-CoV-2 positive respiratory specimens for PCR can be stored for up to six months at -70°C or below without loss of sample integrity, though there is some loss of PCR-detected viral targets as evidenced by an immediate increased in the PCR-generated Ct values. In addition, samples with initial Ct values above 30 are more likely to give non-reproducible results. doi: https://doi.org/10.12669/pjms.38.ICON-2022.5777 How to cite this:Aijaz J, Naseer F, Dojki M, Jamal S. Duration of respiratory sample stability at -80ºC for SARS-CoV-2 PCR. Pak J Med Sci. 2022;38(2):393-398. doi: https://doi.org/10.12669/pjms.38.ICON-2022.5777 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

569. Characterization of COVID-19 Vaccine Breakthrough Infections in Metropolitan Detroit

Background Although COVID-19 vaccines are very effective, vaccine breakthrough infections have been reported, albeit rarely. When they do occur, people generally have milder COVID-19 illness compared to unvaccinated people. A total of 10,262 (0.01%) SARS-CoV-2 vaccine breakthrough infections had been reported as of April 30, 2021. The objective of this study was to evaluate the effectiveness of COVID-19 vaccines and characterize breakthrough infections in our patient population. Methods This was a retrospective review of all consecutive COVID-19 vaccine breakthrough infections at Henry Ford Health System (HFHS) in metropolitan Detroit, Michigan, from December 17, 2020 to June 7, 2021. Centers for Disease Control (CDC)'s breakthrough infection definition (detection of SARS-CoV-2 RNA or antigen in a respiratory sample ≥14 days after completion all recommended doses of COVID-19 vaccine) was used to identify cases. Vaccination status was extracted from the electronic medical records using Epic™ SlicerDicer. Results A total of 228,674 patients, including healthcare workers (HCW), were fully vaccinated in our healthcare system. We evaluate 299 patients for breakthrough infection but only 179 (0.08%) patients met the definition; 108 (60%) were female with median age of 59, 60 (33%) were HCW, and 11 (6%) were immunocompromised. The majority (92%) were asymptomatic (62 or 35%) or had mild/moderate illness (102 or 57%); 14 (8%) had severe or critical illness. The status of one patient was unknown. Of those who were symptomatic, 24 (13%) required hospitalization, and 3 (2%) required intensive unit care. One patient admitted for heart failure exacerbation died unexpectedly prior to being discharged. Nine had previous COVID-19 within 4 months but only one was symptomatic; this likely represented residual shedding in the asymptomatic patients. Conclusion COVID-19 vaccine was very effective among our patients and breakthrough infections were rare. Moreover, the vaccine reduced disease severity and mortality. Efforts should aim to increase vaccine uptake. Disclosures All Authors: No reported disclosures

Detection of Pneumocystis jirovecii in Hospitalized Children Less Than 3 Years of Age

Few data are available in the literature regarding Pneumocystis jirovecii infection in children under 3 years old. This retrospective cohort study aimed to describe medically relevant information among them. All children under 3 years old treated in the same medical units from April 2014 to August 2020 and in whom a P. jirovecii evaluation was undertaken were enrolled in the study. A positive case was defined as a child presenting at least one positive PCR for P. jirovecii in a respiratory sample. Medically relevant information such as demographical characteristics, clinical presentation, microbiological co-infections, and treatments were collected. The objectives were to describe the characteristics of these children with P. jirovecii colonization/infection to determine the key underlying diseases and risk factors, and to identify viral respiratory pathogens associated. The PCR was positive for P. jirovecii in 32 children. Cardiopulmonary pathologies (21.9%) were the most common underlying disease in them, followed by severe combined immunodeficiency (SCID) (18.8%), hyaline membrane disease (15.6%), asthma (9.4%) and acute leukaemia (6.3%). All SCID children were diagnosed with pneumocystis pneumonia. Co-infection with Pj/Rhinovirus (34.4%) was not significant. Overall mortality was 18.8%. Paediatric pneumocystis is not restricted to patients with HIV or SCID and should be considered in pneumonia in children under 3 years old.

A community-acquired Legionnaires’ disease outbreak caused by Legionella pneumophila serogroup 2: an uncommon event, Italy, August to October 2018

In September 2018 in Brescia province, northern Italy, an outbreak of Legionnaires' disease (LD) caused by Legionella pneumophila serogroup 2 (Lp2) occurred. The 33 cases (two fatal) resided in seven municipalities along the Chiese river. All cases were negative by urinary antigen test (UAT) and most were diagnosed by real-time PCR and serology. In only three cases, respiratory sample cultures were positive, and Lp2 was identified and typed as sequence type (ST)1455. In another three cases, nested sequence-based typing was directly applied to respiratory samples, which provided allelic profiles highly similar to ST1455. An environmental investigation was undertaken immediately and water samples were collected from private homes, municipal water systems, cooling towers and the river. Overall, 533 environmental water samples were analysed and 34 were positive for Lp. Of these, only three samples, all collected from the Chiese river, were Lp2 ST1455. If and how the river water could have been aerosolised causing the LD cases remains unexplained. This outbreak, the first to our knowledge caused by Lp2, highlights the limits of UAT for LD diagnosis, underlining the importance of adopting multiple tests to ensure that serogroups other than serogroup 1, as well as other Legionella species, are identified.

Virome of respiratory secretion from children with unknown etiological acute respiratory disease revealed recombinant human parechovirus and other significant viruses

Using viral metagenomics, viral nucleic acid in 30 respiratory secretion samples collected from children with unknown etiological acute respiratory disease were investigated. Sequences showing similarity to human parainfluenza virus 1, anellovirus, bocavirus, coxsackievirus A4, human parechovirus (HPeV), and alphaflexivirus were recovered from these samples. Complete genomes of one anellovirus, one coxsackievirus A4, three parechoviruses were determined from these libraries. The anellovirus (MW267851) phylogenetically clustered with an unpublished anellovirus (MK212032) from respiratory sample of a Vietnamese patient, forming a separate branch neighboring to strains within the genus Betatorquevirus. The genome of coxsackievirus A4 (MW267852) shares the highest sequence identity of 96.4% to a coxsackievirus A4 (MN964079) which was identified in clinical samples from children with Hand, Foot, and Mouth Disease (HFMD). Two (MW267853 and MW267854) of the three parechoviruses belong to HPeV-1 and the other one (MW267855) belongs to HPeV-6. Recombination analysis indicated that an HPeV-1 (MW267854) identified in this study is a putative recombinant occurred between HPeV-1 and HPeV-3. Whether these viruses have association with specific respiratory disease calls for further investigation.

A new SARS-CoV-2 variant poorly detected by RT-PCR on nasopharyngeal samples, with high lethality

Background: In early January 2021, an outbreak of nosocomial cases of COVID 19 emerged in Western France, with RT PCR tests repeatedly negative on nasopharyngeal samples but positive on lower respiratory tract samples. Whole genome sequencing (WGS) revealed a new variant, currently defining a novel SARS CoV 2 lineage: B.1.616. In March, WHO classified this variant as "under investigation" (VUI). We analyzed the characteristics and outcomes of COVID 19 cases related to this new variant. Methods: Clinical, virological, and radiological data were retrospectively collected from medical charts in the two hospitals involved. We enrolled patients with at least one of the following: i) positive SARS CoV 2 RT PCR on a respiratory sample; ii) seroconversion with anti SARS CoV 2 IgG/IgM; iii) suggestive symptoms and typical features of COVID 19 on chest CT scan. Cases were categorized as either: i) B.1.616; ii) variant of concern (VOC); iii) unknown. Findings: From January 1st to March 24th, 2021, 114 patients fulfilled the inclusion criteria: B.1.616 (n=34), VOC (n=32), and unknown (n=48). B.1.616 related cases were older than VOC related cases (81 years [73-88], vs 73 years [67-82], P<0.05) and their first RT PCR tests were less often positive (5/34, 15% vs 31/32, 97%, P<0.05). The B.1.616 variant was independently associated with severe disease (multivariable Cox model HR 4.2 [1.3 , 13.5], P=0.018), and increased lethality (logrank test P=0.01): 28day mortality 15/34 (44%) with B.1.616, vs. 5/32 (16%) for VOC, P=0.036. Interpretation: We report a nosocomial outbreak of COVID-19 cases related to a new variant, B.1.616, poorly detected by RT PCR on nasopharyngeal samples, with high lethality.

A qualitative assessment of the diagnosis and management of ventilator-associated pneumonia among critical care clinicians exploring opportunities for diagnostic stewardship

Background: Prompt diagnosis and intervention for ventilator-associated pneumonia (VAP) is critical but can lead to overdiagnosis and overtreatment. Objectives: We investigated healthcare provider (HCP) perceptions and challenges associated with VAP diagnosis, and we sought to identify opportunities for diagnostic stewardship. Methods: We conducted a qualitative study of 30 HCPs at a tertiary-care hospital. Participants included attending physicians, residents and fellows (trainees), advanced practice providers (APPs), and pharmacists. Interviews were composed of open-ended questions in 4 sections: (1) clinical suspicion and thresholds for respiratory culture ordering, (2) preferences for respiratory sample collection, (3) culture report interpretation, and (4) VAP diagnosis and treatment. Interviews transcripts were analyzed using Nvivo 12 software, and responses were organized into themes. Results: Overall, 10 attending physicians (75%) and 16 trainees (75%) trainees and APPs believed they were overdiagnosing VAP; this response was frequent among HCPs in practice 5–10 years (91%, n = 12). Increased identification of bacteria as a result of frequent respiratory culturing, misinterpretation of culture data, and fear of missing diagnosis were recognized as drivers of overdiagnosis and overtreatment. Although most HCPs rely on clinical and radiographic changes to initiate work-up, the fear of missing a diagnosis leads to sending cultures even in the absence of those changes. Conclusions: HCPs believe that VAP overdiagnosis and overtreatment are common due to fear of missing diagnosis, overculturing, and difficulty distinguishing colonization from infection. Although we identified opportunities for diagnostic stewardship, interventions influencing the ordering of cultures and starting antimicrobials will need to account for strongly held beliefs and ICU practices.

Evaluation of the timing of MRSA PCR nasal screening: How long can a negative assay be used to rule out MRSA-positive respiratory cultures?

Purpose Previous studies indicate that the polymerase chain reaction (PCR) nasal assay for methicillin-resistant Staphylococcus aureus (MRSA) has a consistently high (&gt;95%) negative predictive value (NPV) in ruling out MRSA pneumonia; however, optimal timing of PCR assay specimen and respiratory culture collection is unclear. Methods A study including 736 patients from a community hospital system was conducted. Patients were included if they had undergone MRSA nasal screening with a PCR assay and had documented positive respiratory culture results. Results In the full cohort, the MRSA PCR nasal screen assay was demonstrated to have an NPV of 94.9% (95% confidence interval [CI], 92.8%-96.5%) in ruling out MRSA-positive respiratory cultures. When evaluating the NPV by level of care (ie, where the MRSA PCR nasal assay sample was collected), no significant difference between values for samples collected in an intensive care unit vs medical/surgical units was identified (NPV [95%CI], 94.9% [92.7%-96.6%] vs 95.3% [88.4%-98.7%]).Additionally, NPV remained high with use of both invasive (NPV [95%CI], 96.8% [92.7%-99.0%]) and noninvasive (NPV [95%CI], 94.5% [91.7%-96.2%]) respiratory sampling methods. Finally, when evaluating the effect of time between MRSA PCR nasal screening and respiratory sample collection, we found high NPVs for all evaluated timeframes: within 24 hours, 93.8% (90.1%-96.4%); within 25 to 48 hours, 98.6% (92.7%-100.0%); within 49 hours to 7 days, 95.7% (91.4%-98.3%); within 8 to 14 days, 92.9% (85.1%-97.3%); and after more than 14 days, 95.5% (84.5%-99.4%). Conclusion We report high NPVs for up to 2 weeks between specimen collections, which allows clinicians to use a negative MRSA PCR nasal screen assay to rule out MRSA pneumonia, potentially leading to decreased exposure to MRSA-active antibiotics.