Clinical utility of SARS-CoV-2 subgenomic RT-PCR in a pediatric quaternary care setting.

BACKGROUND: During active transcription, SARS-CoV-2 generates subgenomic regions of viral RNA. While standard SARS-CoV-2 RT-PCR amplifies region(s) of genomic RNA, it cannot distinguish active infection from remnant viral genomic material. However, screening for subgenomic RNA (sgRNA) by RT-PCR may aid in the determination of actively transcribing virus. OBJECTIVES: To evaluate the clinical utility of SARS-CoV-2 sgRNA RT-PCR testing in a pediatric population. STUDY DESIGN: Retrospective analysis was performed on inpatients from February-September 2022 positive for SARS-CoV-2 by RT-PCR with a concomitant order for sgRNA RT-PCR. Chart abstractions were conducted to determine clinical outcomes, management, and infection prevention and control (IPC) practices. RESULTS: Of 95 SARS-CoV-2 positive samples from 75 unique patients, 27 (28.4%) were positive by sgRNA RT-PCR. A negative sgRNA RT-PCR test allowed for de-isolation in 68 (71.6%) patient episodes. Regardless of age or sex, a positive sgRNA RT-PCR result significantly correlated with disease severity (P = 0.007), generalized COVID-19 symptoms (P = 0.012), hospitalization for COVID-19 (P = 0.019), and immune status (P = 0.024). Moreover, sgRNA RT-PCR results prompted changes in management in 28 patients (37.3%); specifically, therapeutic escalation in 13/27 (48.1%) positives and de-escalation in 15/68 (22.1%) negatives. CONCLUSIONS: Taken together, these findings underscore the clinical utility of sgRNA RT-PCR testing in a pediatric population as we report significant associations between sgRNA RT-PCR results and clinical parameters related to COVID-19. These findings align with the proposed use of sgRNA RT-PCR testing to guide patient management and IPC practices in the hospital setting.

The Clinical Performance of the BioCode Respiratory Pathogen Panel for the Detection of Viruses and Bacteria from Nasopharyngeal Swabs.

Early detection of microbial pathogens causing respiratory tract infection plays a crucial role in clinical management. The BioCode Respiratory Pathogen Panel (BioCode RPP) utilizes reverse transcriptase PCR (RT-PCR) in combination with barcoded magnetic beads to amplify, detect, and identify respiratory pathogens. This panel qualitatively detects and identifies 14 viruses, including influenza virus A with H1 pdm09, H1, and H3 subtyping; influenza B; respiratory syncytial virus (RSV); human metapneumovirus; parainfluenza virus 1; parainfluenza virus 2; parainfluenza virus 3; parainfluenza virus 4; coronavirus (229E, NL63, OC43, and HKU1); adenovirus; and human rhinovirus/enterovirus, and 3 bacteria, including Chlamydia pneumoniae, Mycoplasma pneumoniae, and Bordetella pertussis. Reproducibility, which was assessed with contrived specimens containing 12 targets at 3 clinical sites, with 2 operators at each site for 5 days, was 99.4% for Flu A H3 and Flu B, 98.9% for RSV, and 100% for the remaining 9 targets assayed. A multicenter clinical trial evaluated the performance of the BioCode RPP with 2,647 nasopharyngeal swab specimens from 5 geographically distinct sites and revealed comparable performance between the BioCode RPP and FilmArray Respiratory Panel (FA-RP). Specifically, the positive percent agreements (PPAs) for various pathogens ranged between 80.8% and 100% compared with the FA-RP (1.7 and 2.0). Negative percent agreement ranged from 98.4% to 100% for BioCode RPP. The BioCode RPP also offers scalable automated testing capability of up to 96 specimens in a single run with total sample-to-result time under 5 h. The invalid rate of the BioCode RPP on initial testing was 1.0% (26/2,649). IMPORTANCE Early detection of microbial pathogens causing respiratory tract infection plays a crucial role in clinical management. The BioCode Respiratory Pathogen Panel (BioCode RPP) is a high-throughput test that utilizes RT-PCR in combination with barcoded magnetic beads to amplify, detect, and identify 17 respiratory pathogens, including 14 viruses and 3 bacteria. This study summarizes data generated from a multicenter clinical trial evaluating the performance of the BioCode RPP on 2,647 nasopharyngeal swab specimens from five geographically distinct sites.

Rapid Antigen Assays for SARS-CoV-2: Promise and Peril.

Though rapid antigen tests have historically problematic performance characteristics for the diagnosis of respiratory viral infections such as influenza, they have attained an unprecedented level of use in the context of the COVID-19 pandemic. Ease of use and scalability of rapid antigen tests has facilitated a democratization and scale of testing beyond anything reasonably achievable by traditional laboratory-based testing. In this chapter, we discuss the performance characteristics of rapid antigen testing for SARS-CoV-2 detection and their application to non-traditional uses beyond clinical diagnostic testing.

The Successes and Challenges of SARS-CoV-2 Molecular Testing in the United States.

SARS-CoV-2 was identified and diagnostic methods developed at an impressive speed due in great part to the wider use of molecular methods in 2019 compared with 2002 during the SARS pandemic. The development of rapid and novel molecular diagnostic assays, leveraging of the high adaptability of molecular tests, and the integration of SARS-CoV-2 genotyping into public health, clinical, and research laboratories have been some of the successes in SARS-CoV-2 molecular testing. The main challenges are related to regulatory hurdles, supply chain constraints, and laboratory preparation.

COVID-19 outcomes in children, adolescents and young adults with cancer.

Pediatric oncology patients are at risk for poor outcomes with respiratory viral infections. Outcome data for COVID-19 in children and young adults with cancer are needed; data are sparse for obese/overweight and adolescent and young adult subgroups. We conducted a single center cohort study of COVID-19 outcomes in patients younger than 25 years with cancer. Candidate hospitalization risk factors were analyzed via univariable and multivariable analyses. Eighty-seven patients with cancer and COVID-19 were identified. Most were Hispanic/Latinx (n = 63, 72%). Forty-two (48%) were overweight/obese. Anticancer therapy included chemotherapy only (n = 64, 74%), chimeric antigen receptor T-cells (CAR-T, n = 7), hematopoietic stem cell transplantation (HSCT, n = 12), or CAR-T and HSCT (n = 4). There was no COVID-19 related mortality. Twenty-six patients (30%) required COVID-19 related hospitalization; 4 required multiple hospitalizations. Nine (10%) had severe/critical infection; 6 needed intensive care. COVID-19 resulted in anticancer therapy delays in 22 (34%) of 64 patients on active therapy (median delay = 14 days). Factors associated with hospitalization included steroids within 2 weeks prior to infection, lymphopenia, previous significant non-COVID infection, and low COVID-19 PCR cycle threshold value. CAR-T recipients with B-cell aplasia tended to have severe/critical infection (3 of 7 patients). A COVID-19 antibody response was detected in 14 of 32 patients (44%). A substantial proportion of COVID-19 infected children and young adults with cancer require inpatient management; morbidity may be high in B-cell immunodeficiency. However, a majority of patients can be taken through chemotherapy without prolonged therapy delays. Viral load is a potential outcome predictor in COVID-19 in pediatric cancer.

The Hidden Crisis in the Times of COVID-19: Critical Shortages of Medical Laboratory Professionals in Clinical Microbiology.

The COVID pandemic has put a spotlight on laboratory medicine, showcasing how vital diagnostic testing is for society and the health care system. It has also brought to light and accelerated the critical shortage of trained and experienced laboratory personnel that has been felt for decades. The need for laboratory professionals is expected to grow by 11% between 2020 and 2030, a higher rate of growth than the overall average for all other health care occupations. Here, the background to this workforce shortage is reviewed. Some proposed actions to help address the issue are put forth, including increasing awareness of the medical laboratory science profession along with bolstering training opportunities and awareness of alternate routes to obtaining certification as a medical laboratory scientist. In addition, recent survey data specifically related to the employee shortages in microbiology are presented which demonstrate that 80% of microbiology laboratories have vacant positions and that filling these positions is challenging for a number of reasons, including a lack of qualified applicants.

Clinical and Infection Prevention Applications of Severe Acute Respiratory Syndrome Coronavirus 2 Genotyping: an Infectious Diseases Society of America/American Society for Microbiology Consensus Review Document.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged into a world of maturing pathogen genomics, with more than 2 million genomes sequenced at the time of writing. The rise of more transmissible variants of concern that impact vaccine and therapeutic effectiveness has led to widespread interest in SARS-CoV-2 evolution. Clinicians are also eager to take advantage of the information provided by SARS-CoV-2 genotyping beyond surveillance purposes. Here, we review the potential role of SARS-CoV-2 genotyping in clinical care. The review covers clinical use cases for SARS-CoV-2 genotyping, methods of SARS-CoV-2 genotyping, assay validation and regulatory requirements, and clinical reporting for laboratories, as well as emerging issues in clinical SARS-CoV-2 sequencing. While clinical uses of SARS-CoV-2 genotyping are currently limited, rapid technological change along with a growing ability to interpret variants in real time foretells a growing role for SARS-CoV-2 genotyping in clinical care as continuing data emerge on vaccine and therapeutic efficacy.

SARS-CoV-2 Transmission Dynamics in Households With Children, Los Angeles, California.

Objectives: Studies of household transmission of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) focused on households with children are limited. We investigated household secondary attack rate (SAR), transmission dynamics, and contributing factors in households with children. Materials and Methods: In this prospective case-ascertained study in Los Angeles County, California, all households members were enrolled if ≥1 member tested positive for SARS-CoV-2 by polymerase chain reaction (PCR). Nasopharyngeal PCRs, serology, and symptom data were obtained over multiple visits. Results: A total of 489 individuals in 105 households were enrolled from June to December 2020. The majority (77.3%) reported a household annual income of <$50,000, and most (92.9%) were of Hispanic/Latinx ethnicity. Children <18 years old accounted for 46.9% index cases, of whom 45.3% were asymptomatic. Household index cases were predominantly children during low community transmission and adults during the high community transmission period (χ2 = 7.647, p = 0.0036. The mean household SAR was 77.0% (95% CI: 69.4-84.6%). Child and adult index cases both efficiently transmitted SARS-CoV-2 within households [81.9%, (95% CI: 72.1-91.9%) vs. 72.4% (95% CI: 59.8-85.1%), p = 0.23]. Household income and pets were significantly associated with higher SAR in the multivariable analysis of household factors (p = 0.0013 and 0.004, respectively). Conclusions: The SAR in households with children in an urban setting with a large ethnic minority population is much higher than previously described. Children play important roles as index cases. SAR was disproportionately impacted by household income. Vaccination and public health efforts need special focus on children and vulnerable communities to help mitigate SARS-CoV-2 spread.

Clinical and Infection Prevention Applications of Severe Acute Respiratory Syndrome Coronavirus 2 Genotyping: An Infectious Diseases Society of America/American Society for Microbiology Consensus Review Document.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged into a world of maturing pathogen genomics, with >2 million genomes sequenced at this writing. The rise of more transmissible variants of concern that affect vaccine and therapeutic effectiveness has led to widespread interest in SARS-CoV-2 evolution. Clinicians are also eager to take advantage of the information provided by SARS-CoV-2 genotyping beyond surveillance purposes. Here, we review the potential role of SARS-CoV-2 genotyping in clinical care. The review covers clinical use cases for SARS-CoV-2 genotyping, methods of SARS-CoV-2 genotyping, assay validation and regulatory requirements, clinical reporting for laboratories, and emerging issues in clinical SARS-CoV-2 sequencing. While clinical uses of SARS-CoV-2 genotyping are currently limited, rapid technological change along with a growing ability to interpret variants in real time foretell a growing role for SARS-CoV-2 genotyping in clinical care as continuing data emerge on vaccine and therapeutic efficacy.

Preoperative SARS-CoV-2 Screening Fails to Detect Viral Particles Prior to Airway Surgery.

OBJECTIVES/HYPOTHESIS: Children have higher rates of asymptomatic SARS-CoV-2 infections or milder courses of infection, and their carrier status may potentially impact viral transmission to those providing them care. The aim of this study is to compare the existing COVID-19 preoperative screening protocols to the detection of SARS-CoV-2 viral particles in surgical samples. STUDY DESIGN: Cross-sectional study. METHODS: We conducted a prospective study with consecutive convenience sampling of children undergoing adenoidectomy between January and April 2021. Total nucleic acid was extracted from adenoid tissue and real-time reverse transcription-polymerase chain reaction was conducted to test for the presence of SARS-CoV-2 viral particles. Univariate logistic regression was used to summarize the effect size of variables of interest on the odds of having SARS-CoV-2 positive adenoid tissue. RESULTS: Forty adenoid samples were collected and 11 (27.5%) had a positive SARS-CoV-2 reverse transcriptase-polymerase chain reaction. Patients with positive adenoids were older (11.8 vs. 7.9 years, odds ratio: 1.3, P = .01) and more likely to have had a positive nasopharyngeal swab in the previous 90 days (4/11 or 36% vs. 0). CONCLUSION: These data are the first report on the presence of SARS-CoV-2 particles in pediatric adenoidectomy specimens, with a high percentage of patients showing evidence of viral particles within the adenoid. This finding calls in to question the utility of preoperative COVID screening protocols which have yet to be rigorously validated in asymptomatic patients and have the potential to delay patients' surgical care. LEVEL OF EVIDENCE: 3 Laryngoscope, 132:1665-1667, 2022.

Increased viral variants in children and young adults with impaired humoral immunity and persistent SARS-CoV-2 infection: A consecutive case series.

BACKGROUND: There is increasing concern that persistent infection of SARS-CoV-2 within immunocompromised hosts could serve as a reservoir for mutation accumulation and subsequent emergence of novel strains with the potential to evade immune responses. METHODS: We describe three patients with acute lymphoblastic leukemia who were persistently positive for SARS-CoV-2 by real-time polymerase chain reaction. Viral viability from longitudinally-collected specimens was assessed. Whole-genome sequencing and serological studies were performed to measure viral evolution and evidence of immune escape. FINDINGS: We found compelling evidence of ongoing replication and infectivity for up to 162 days from initial positive by subgenomic RNA, single-stranded RNA, and viral culture analysis. Our results reveal a broad spectrum of infectivity, host immune responses, and accumulation of mutations, some with the potential for immune escape. INTERPRETATION: Our results highlight the potential need to reassess infection control precautions in the management and care of immunocompromised patients. Routine surveillance of mutations and evaluation of their potential impact on viral transmission and immune escape should be considered.

High Prevalence of SARS-CoV-2 Genetic Variation and D614G Mutation in Pediatric Patients With COVID-19.

BACKGROUND: The full spectrum of the disease phenotype and viral genotype of coronavirus disease 2019 (COVID-19) have yet to be thoroughly explored in children. Here, we analyze the relationships between viral genetic variants and clinical characteristics in children. METHODS: Whole-genome sequencing was performed on respiratory specimens collected for all SARS-CoV-2-positive children (n = 141) between March 13 and June 16, 2020. Viral genetic variations across the SARS-CoV-2 genome were identified and investigated to evaluate genomic correlates of disease severity. RESULTS: Higher viral load was detected in symptomatic patients (P = .0007) and in children <5 years old (P = .0004). Genomic analysis revealed a mean pairwise difference of 10.8 single nucleotide variants (SNVs), and the majority (55.4%) of SNVs led to an amino acid change in the viral proteins. The D614G mutation in the spike protein was present in 99.3% of the isolates. The calculated viral mutational rate of 22.2 substitutions/year contrasts the 13.5 substitutions/year observed in California isolates without the D614G mutation. Phylogenetic clade 20C was associated with severe cases of COVID-19 (odds ratio, 6.95; P = .0467). Epidemiological investigation revealed major representation of 3 of 5 major Nextstrain clades (20A, 20B, and 20C) consistent with multiple introductions of SARS-CoV-2 in Southern California. CONCLUSIONS: Genomic evaluation demonstrated greater than expected genetic diversity, presence of the D614G mutation, increased mutation rate, and evidence of multiple introductions of SARS-CoV-2 into Southern California. Our findings suggest a possible association of phylogenetic clade 20C with severe disease, but small sample size precludes a definitive conclusion. Our study warrants larger and multi-institutional genomic evaluation and has implications for infection control practices.

Panels and Syndromic Testing in Clinical Microbiology.

Syndromic panels have allowed clinical microbiology laboratories to rapidly identify bacteria, viruses, fungi, and parasites and are now fully integrated into the standard testing practices of many clinical laboratories. To maximize the benefit of syndromic testing, laboratories must implement strict measures to ensure that syndromic panels are being used responsibly. This article discusses commercially available syndromic panels, the benefits and limitations of testing, and how diagnostic and laboratory stewardship can be used to optimize testing and improve patient care while keeping costs at a minimum.

Comparison of Upper Respiratory Viral Load Distributions in Asymptomatic and Symptomatic Children Diagnosed with SARS-CoV-2 Infection in Pediatric Hospital Testing Programs.

The distribution of upper respiratory viral loads (VL) in asymptomatic children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unknown. We assessed PCR cycle threshold (Ct) values and estimated VL in infected asymptomatic children diagnosed in nine pediatric hospital testing programs. Records for asymptomatic and symptomatic patients with positive clinical SARS-CoV-2 tests were reviewed. Ct values were (i) adjusted by centering each value around the institutional median Ct value from symptomatic children tested with that assay and (ii) converted to estimated VL (numbers of copies per milliliter) using internal or manufacturer data. Adjusted Ct values and estimated VL for asymptomatic versus symptomatic children (118 asymptomatic versus 197 symptomatic children aged 0 to 4 years, 79 asymptomatic versus 97 symptomatic children aged 5 to 9 years, 69 asymptomatic versus 75 symptomatic children aged 10 to 13 years, 73 asymptomatic versus 109 symptomatic children aged 14 to 17 years) were compared. The median adjusted Ct value for asymptomatic children was 10.3 cycles higher than for symptomatic children (P < 0.0001), and VL were 3 to 4 logs lower than for symptomatic children (P < 0.0001); differences were consistent (P < 0.0001) across all four age brackets. These differences were consistent across all institutions and by sex, ethnicity, and race. Asymptomatic children with diabetes (odds ratio [OR], 6.5; P = 0.01), a recent contact (OR, 2.3; P = 0.02), and testing for surveillance (OR, 2.7; P = 0.005) had higher estimated risks of having a Ct value in the lowest quartile than children without, while an immunocompromised status had no effect. Children with asymptomatic SARS-CoV-2 infection had lower levels of virus in their nasopharynx/oropharynx than symptomatic children, but the timing of infection relative to diagnosis likely impacted levels in asymptomatic children. Caution is recommended when choosing diagnostic tests for screening of asymptomatic children.

Saliva Is a Promising Alternative Specimen for the Detection of SARS-CoV-2 in Children and Adults.

Testing efforts for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been burdened by the scarcity of testing materials and personal protective equipment for health care workers. The simple and painless process of saliva collection allows for widespread testing, but enthusiasm is hampered by variable performance compared to that of nasopharyngeal swab (NPS) samples. We prospectively collected paired NPS and saliva samples from a total of 300 unique adult and pediatric patients. SARS-CoV-2 RNA was detected in 32.2% (97/300) of the individuals using the TaqPath COVID-19 Combo kit (Thermo Fisher). Performance of saliva and NPS was compared against the total number of positives regardless of specimen type. The overall concordances for saliva and NPS were 91.0% (273/300) and 94.7% (284/300), respectively. The values for positive percent agreement (PPA) for saliva and NPS were 81.4% (79/97) and 89.7% (87/97), respectively. Saliva yielded detection of 10 positive cases that were negative by NPS. For symptomatic and asymptomatic pediatric patients not previously diagnosed with COVID-19, the performances of saliva and NPS were comparable (PPA, 82.4% versus 85.3%). The overall values for PPA for adults were 83.3% and 90.7% for saliva and NPS, respectively, with saliva yielding detection of 4 fewer cases than NPS. However, saliva performance for symptomatic adults was identical to NPS performance (PPA of 93.8%). With lower cost and self-collection capabilities, saliva can be an appropriate sample choice alternative to NPS for detection of SARS-CoV-2 in children and adults.

Saliva is a promising alternative specimen for the detection of SARS-CoV-2 in children and adults.

Testing efforts for SARS-CoV-2 have been burdened by the scarcity of testing materials and personal protective equipment for healthcare workers. The simple and painless process of saliva collection allows for widespread testing, but enthusiasm is hampered by variable performance compared to nasopharyngeal swab (NPS) samples. We prospectively collected paired NPS and saliva samples from a total of 300 unique adult and pediatric patients. SARS-CoV-2 RNA was detected in 32.2% (97/300) of the individuals using the TaqPath COVID-19 Combo Kit (Thermo Fisher). Performance of saliva and NPS were compared against the total number of positives regardless of specimen type. The overall concordance for saliva and NPS was 91.0% (273/300) and 94.7% (284/300), respectively. The positive percent agreement (PPA) for saliva and NPS was 81.4% (79/97) and 89.7% (87/97), respectively. Saliva detected 10 positive cases that were negative by NPS. In symptomatic and asymptomatic pediatric patients not previously diagnosed with COVID-19, the performances of saliva and NPS were comparable (PPA: 82.4% vs 85.3%). The overall PPA for adults were 83.3% and 90.7% for saliva and NPS, respectively, with saliva detecting 4 cases less than NPS. However, saliva performance in symptomatic adults was identical to NPS (PPA of 93.8%). With lower cost and self-collection capabilities, saliva can be an appropriate alternative sample choice to NPS for detection of SARS-CoV-2 in children and adults. SUMMARY: Saliva is an acceptable alternative specimen compared to nasopharyngeal swabs for detection of SARS-CoV-2. Specifically, saliva demonstrated comparable performance to nasopharyngeal swabs in symptomatic and asymptomatic pediatric patients and in symptomatic adults.