A cloud-compatible bioinformatics pipeline for ultrarapid pathogen identification from next-generation sequencing of clinical samples.

Unbiased next-generation sequencing (NGS) approaches enable comprehensive pathogen detection in the clinical microbiology laboratory and have numerous applications for public health surveillance, outbreak investigation, and the diagnosis of infectious diseases. However, practical deployment of the technology is hindered by the bioinformatics challenge of analyzing results accurately and in a clinically relevant timeframe. Here we describe SURPI ("sequence-based ultrarapid pathogen identification"), a computational pipeline for pathogen identification from complex metagenomic NGS data generated from clinical samples, and demonstrate use of the pipeline in the analysis of 237 clinical samples comprising more than 1.1 billion sequences. Deployable on both cloud-based and standalone servers, SURPI leverages two state-of-the-art aligners for accelerated analyses, SNAP and RAPSearch, which are as accurate as existing bioinformatics tools but orders of magnitude faster in performance. In fast mode, SURPI detects viruses and bacteria by scanning data sets of 7-500 million reads in 11 min to 5 h, while in comprehensive mode, all known microorganisms are identified, followed by de novo assembly and protein homology searches for divergent viruses in 50 min to 16 h. SURPI has also directly contributed to real-time microbial diagnosis in acutely ill patients, underscoring its potential key role in the development of unbiased NGS-based clinical assays in infectious diseases that demand rapid turnaround times.

Implementation of California COVIDNet - a multi-sector collaboration for statewide SARS-CoV-2 genomic surveillance.

Introduction: The SARS-CoV-2 pandemic represented a formidable scientific and technological challenge to public health due to its rapid spread and evolution. To meet these challenges and to characterize the virus over time, the State of California established the California SARS-CoV-2 Whole Genome Sequencing (WGS) Initiative, or "California COVIDNet". This initiative constituted an unprecedented multi-sector collaborative effort to achieve large-scale genomic surveillance of SARS-CoV-2 across California to monitor the spread of variants within the state, to detect new and emerging variants, and to characterize outbreaks in congregate, workplace, and other settings. Methods: California COVIDNet consists of 50 laboratory partners that include public health laboratories, private clinical diagnostic laboratories, and academic sequencing facilities as well as expert advisors, scientists, consultants, and contractors. Data management, sample sourcing and processing, and computational infrastructure were major challenges that had to be resolved in the midst of the pandemic chaos in order to conduct SARS-CoV-2 genomic surveillance. Data management, storage, and analytics needs were addressed with both conventional database applications and newer cloud-based data solutions, which also fulfilled computational requirements. Results: Representative and randomly selected samples were sourced from state-sponsored community testing sites. Since March of 2021, California COVIDNet partners have contributed more than 450,000 SARS-CoV-2 genomes sequenced from remnant samples from both molecular and antigen tests. Combined with genomes from CDC-contracted WGS labs, there are currently nearly 800,000 genomes from all 61 local health jurisdictions (LHJs) in California in the COVIDNet sequence database. More than 5% of all reported positive tests in the state have been sequenced, with similar rates of sequencing across 5 major geographic regions in the state. Discussion: Implementation of California COVIDNet revealed challenges and limitations in the public health system. These were overcome by engaging in novel partnerships that established a successful genomic surveillance program which provided valuable data to inform the COVID-19 public health response in California. Significantly, California COVIDNet has provided a foundational data framework and computational infrastructure needed to respond to future public health crises.

Expanded Molecular Testing on Patients with Suspected West Nile Virus Disease.

Most diagnostic testing for West Nile virus (WNV) disease is accomplished using serologic testing, which is subject to cross-reactivity, may require cumbersome confirmatory testing, and may fail to detect infection in specimens collected early in the course of illness. The objective of this project was to determine whether a combination of molecular and serologic testing would increase detection of WNV disease cases in acute serum samples. A total of 380 serum specimens collected ≤7 days after onset of symptoms and submitted to four state public health laboratories for WNV diagnostic testing in 2014 and 2015 were tested. WNV immunoglobulin M (IgM) antibody and RT-PCR tests were performed on specimens collected ≤3 days after symptom onset. WNV IgM antibody testing was performed on specimens collected 4-7 days after onset and RT-PCR was performed on IgM-positive specimens. A patient was considered to have laboratory evidence of WNV infection if they had detectable WNV IgM antibodies or WNV RNA in the submitted serum specimen. Of specimens collected ≤3 days after symptom onset, 19/158 (12%) had laboratory evidence of WNV infection, including 16 positive for only WNV IgM antibodies, 1 positive for only WNV RNA, and 2 positive for both. Of specimens collected 4-7 days after onset, 21/222 (9%) were positive for WNV IgM antibodies; none had detectable WNV RNA. These findings suggest that routinely performing WNV RT-PCR on acute serum specimens submitted for WNV diagnostic testing is unlikely to identify a substantial number of additional cases beyond IgM antibody testing alone.

New rabies virus variant in Mexican immigrant.

A novel rabies virus was identified after death in a man who had immigrated from Oaxaca, Mexico, to California, USA. Despite the patient's history of exposure to domestic and wild carnivores, molecular and phylogenetic characterizations suggested that the virus originated from insectivorous bats. Enhanced surveillance is needed to elucidate likely reservoirs.

Emerging epidemiology of bat-associated cryptic cases of rabies in humans in the United States.

In the United States, during the past half-century, the number of humans to die of rabies dramatically decreased to an average of 1-2 per year. Although the number of deaths is low, most deaths occur because individuals are unaware that they had been exposed to and infected with rabies virus, and, therefore, they do not seek effective postexposure treatment. Molecular epidemiological studies have linked most of these cryptic rabies exposures to rabies virus variants associated with insectivorous bats. In particular, virus variants associated with 2 relatively reclusive species, the silver-haired bat (Lasionycteris noctivagans) and the eastern pipistrelle (Pipistrellus subflavus), are the unexpected culprits of most cryptic cases of rabies in humans.

Rickettsia spp. 364D causing a cluster of eschar-associated illness, California.

We describe the clinical course of the first 3 pediatric cases infected with Rickettsia spp. 364D. Although the pathogen was identified in California ticks decades ago, only recently have human cases been documented. Clinical features are generally mild, characterized by eschar, fever, headache, malaise and lymphadenopathy. Antigenic similarity among rickettsiae leads to cross-reactive antibody responses; definitive diagnosis requires molecular methods.

Fatal transplant-associated west nile virus encephalitis and public health investigation-california, 2010.

BACKGROUND: In December 2010, a case of West Nile virus (WNV) encephalitis occurring in a kidney recipient shortly after organ transplantation was identified. METHODS: A public health investigation was initiated to determine the likely route of transmission, detect potential WNV infections among recipients from the same organ donor, and remove any potentially infected blood products or tissues. Available serum, cerebrospinal fluid, and urine samples from the organ donor and recipients were tested for WNV infection by nucleic acid testing and serology. RESULTS: Two additional recipients from the same organ donor were identified, their clinical and exposure histories were reviewed, and samples were obtained. WNV RNA was retrospectively detected in the organ donor's serum. After transplantation, the left kidney recipient had serologic and molecular evidence of WNV infection and the right kidney recipient had prolonged but clinically inapparent WNV viremia. The liver recipient showed no clinical signs of infection but had flavivirus IgG antibodies; however, insufficient samples were available to determine the timing of infection. No remaining infectious products or tissues were identified. CONCLUSIONS: Clinicians should suspect WNV as a cause of encephalitis in organ transplant recipients and report cases to public health departments for prompt investigation of the source of infection. Increased use of molecular testing and retaining pretransplantation sera may improve the ability to detect and diagnose transplant-associated WNV infection in organ transplant recipients.

Emerging pattern of rabies deaths and increased viral infectivity.

Most human rabies deaths in the United States can be attributed to unrecognized exposures to rabies viruses associated with bats, particularly those associated with two infrequently encountered bat species (Lasionycteris noctivagans and Pipistrellus subflavus). These human rabies cases tend to cluster in the southeastern and northwestern United States. In these regions, most rabies deaths associated with bats in nonhuman terrestrial mammals are also associated with virus variants specific to these two bat species rather than more common bat species; outside of these regions, more common bat rabies viruses contribute to most transmissions. The preponderance of rabies deaths connected with the two uncommon L. noctivagans and P. subflavus bat rabies viruses is best explained by their evolution of increased viral infectivity.