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.

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Many detection methods have been used or reported for the diagnosis and/or surveillance of SARS-CoV-2. Among them, reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive, claiming detection of about 5 copies of viruses. However, it has been reported that only 47-59% of the positive cases were identified by RT-PCR, probably due to loss or degradation of virus RNA in the sampling process, or even mutation of the virus genome. Therefore, developing highly sensitive methods is imperative to ensure robust detection capabilities. With the goal of improving sensitivity and accommodate various application settings, we developed a multiplex-PCR-based method comprised of 172 pairs of specific primers, and demonstrated its efficiency to detect SARS-CoV-2 at low copy numbers. The assay produced clean characteristic target peaks of defined sizes, which allowed for direct identification of positives by electrophoresis. In addition, optional sequencing can provide further confirmation as well as phylogenetic information of the identified virus(es) for specific strain discrimination, which will be of paramount importance for surveillance purposes that represent a global health imperative. Finally, we also developed in parallel a multiplex-PCR-based metagenomic method that is amenable to detect SARS-CoV-2, with the additional benefit of its potential for uncovering mutational diversity and novel pathogens at low sequencing depth.

Background Full spectrum of disease phenotype and viral genotype of 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 on all SARS-CoV-2 positive children (n=141) between March 13 to 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=0.0007) and in children <5 years old (p=0.0004). Genomic analysis revealed a mean pairwise difference of 10.8 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 (p=0.0467, OR=6.95). 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 suggests 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.

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 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.

Effective response to the Coronavirus Disease 2019 (COVID-19) pandemic requires genomic resources and bioinformatics tools for genomic epidemiology and surveillance studies that involve characterizing full-length viral genomes, identifying origins of infections, determining the relatedness of viral infections, performing phylogenetic analyses, and monitoring the continuous evolution of the SARS-CoV-2 viral genomes. The Children's Hospital, Los Angeles (CHLA) COVID-19 Analysis Research Database (CARD) (https://covid19.cpmbiodev.net/) is a comprehensive genomic resource that provides access to full-length SARS-CoV-2 viral genomes and associated meta-data for over 30,000 (as of May 20, 2020) isolates collected from global sequencing repositories and the sequencing performed at the Center for Personalized Medicine (CPM) at CHLA. Reference phylogenetic trees of global and USA viral isolates were constructed and are periodically updated using selected high quality SARS-CoV-2 genome sequences. These provide the baseline and analytical context for identifying the origin of a viral infection, as well as the relatedness of SARS-CoV-2 genomes of interest. A web-based and interactive Phylogenetic Tree Browser supports flexible tree manipulation and advanced analysis based on keyword search while highlighting time series animation, as well as subtree export for graphical representation or offline exploration. A Virus Genome Tracker accepts complete or partial SARS-CoV-2 genome sequence, compares it against all available sequences in the database (>30,000 at time of writing), detects and annotates the variants, and places the new viral isolate within the global or USA phylogenetic contexts based upon variant profiles and haplotype comparisons, in a few seconds. The generated analysis can potentially aid in genomic surveillance to trace the transmission of any new infection. Using CHLA CARD, we demonstrate the identification of a candidate outbreak point where 13 of 31 CHLA internal isolates may have originated. We also discovered multiple indels of unknown clinical significance in the orf3a gene, and revealed a number of USA-specific variants and haplotypes.

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The OncoKids panel is an amplification-based next-generation sequencing assay designed to detect diagnostic, prognostic, and therapeutic markers across the spectrum of pediatric malignancies, including leukemias, sarcomas, brain tumors, and embryonal tumors. This panel uses low input amounts of DNA (20 ng) and RNA (20 ng) and is compatible with formalin-fixed, paraffin-embedded and frozen tissue, bone marrow, and peripheral blood. The DNA content of this panel covers the full coding regions of 44 cancer predisposition loci, tumor suppressor genes, and oncogenes; hotspots for mutations in 82 genes; and amplification events in 24 genes. The RNA content includes 1421 targeted gene fusions. We describe the validation of this panel by using a large cohort of 192 unique clinical samples that included a wide range of tumor types and alterations. Robust performance was observed for analytical sensitivity, reproducibility, and limit of detection studies. The results from this study support the use of OncoKids for routine clinical testing of a wide variety of pediatric malignancies.