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Los Angeles County comprises 4,058 square miles and is home to approximately 10 million residents (1), an estimated 59,000 (0.6%) of whom experience homelessness on a given night (2). In late 2018, Los Angeles County Department of Public Health (LAC DPH) was notified of a case of hepatitis A virus (HAV) infection in a person experiencing homelessness. LAC DPH conducted an investigation to determine the source of infection, identify additional cases, and identify contacts for postexposure prophylaxis (PEP). Over the next week, LAC DPH identified two additional hepatitis A cases in persons experiencing homelessness who knew one another socially and were known to congregate at a specific street intersection. To identify and respond rapidly to additional outbreak-associated cases, LAC DPH implemented enhanced surveillance procedures, including immediately obtaining specimens for molecular testing from all patients with suspected hepatitis A in the same geographic area. Enhanced surveillance identified four additional cases in persons linked to a senior living campus within two blocks of the intersection where the initial three patients reported congregating. These four cases were linked to the cluster in persons experiencing homelessness through HAV genotyping. Overall, DPH identified seven outbreak-associated hepatitis A cases during October 2018-January 2019. The DPH response to this community hepatitis A outbreak included conducting vaccination outreach to persons at risk, conducting environmental health outreach to restaurants in the outbreak area, and issuing health care provider alerts about the increased occurrence of hepatitis A. Implementation of near real-time molecular testing can improve hepatitis A outbreak responses by confirming HAV infections, linking additional cases to the outbreak, and informing the targeting of prevention efforts.

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.

Rocky Mountain spotted fever (RMSF) is a life-threatening tick-borne disease documented in North, Central, and South America. In California, RMSF is rare; nonetheless, recent fatal cases highlight ecological cycles of the two genera of ticks, Dermacentor and Rhipicephalus, known to transmit the disease. These ticks occur in completely different habitats (sylvatic and peridomestic, respectively) resulting in different exposure risks for humans. This study summarizes the demographic, exposure, and clinical aspects associated with the last 40 years of reported RMSF cases to the California Department of Public Health (CDPH). Seventy-eight RMSF cases with onsets from 1980 to 2019 were reviewed. The incidence of RMSF has risen in the last 20 years from 0.04 cases per million to 0.07 cases per million (a two-fold increase in reports), though the percentage of cases that were confirmed dropped significantly from 72% to 25% of all reported cases. Notably, Hispanic/Latino populations saw the greatest rise in incidence. Cases of RMSF in California result from autochthonous and out-of-state exposures. During the last 20 years, more cases reported exposure in Southern California or Mexico than in the previous 20 years. The driver of these epidemiologic changes is likely the establishment and expansion of Rhipicephalus sanguineus sensu lato ticks in Southern California and on-going outbreaks of RMSF in northern Mexico. Analysis of available electronically reported clinical data from 2011 to 2019 showed that 57% of reported cases presented with serious illness requiring hospitalization with a 7% mortality. The difficulty in recognizing RMSF is due to a non-specific clinical presentation; however, querying patients on the potential of tick exposure in both sylvatic and peridomestic environments may facilitate appropriate testing and treatment.

We identified an emerging SARS-CoV-2 variant by viral whole-genome sequencing of 2,172 nasal/nasopharyngeal swab samples from 44 counties in California, a state in the Western United States. Named B.1.427/B.1.429 to denote its 2 lineages, the variant emerged in May 2020 and increased from 0% to >50% of sequenced cases from September 2020 to January 2021, showing 18.6-24% increased transmissibility relative to wild-type circulating strains. The variant carries 3 mutations in the spike protein, including an L452R substitution. We found 2-fold increased B.1.427/B.1.429 viral shedding in vivo and increased L452R pseudovirus infection of cell cultures and lung organoids, albeit decreased relative to pseudoviruses carrying the N501Y mutation common to variants B.1.1.7, B.1.351, and P.1. Antibody neutralization assays revealed 4.0 to 6.7-fold and 2.0-fold decreases in neutralizing titers from convalescent patients and vaccine recipients, respectively. The increased prevalence of a more transmissible variant in California exhibiting decreased antibody neutralization warrants further investigation., A SARS-CoV-2 variant of concern bearing the L452R spike protein mutation is widely circulating in California, United States, and demonstrates increased transmissibility, infectivity, and avoidance of antibody neutralization.

We identified a novel SARS-CoV-2 variant by viral whole-genome sequencing of 2,172 nasal/nasopharyngeal swab samples from 44 counties in California. Named B.1.427/B.1.429 to denote its 2 lineages, the variant emerged around May 2020 and increased from 0% to >50% of sequenced cases from September 1, 2020 to January 29, 2021, exhibiting an 18.6-24% increase in transmissibility relative to wild-type circulating strains. The variant carries 3 mutations in the spike protein, including an L452R substitution. Our analyses revealed 2-fold increased B.1.427/B.1.429 viral shedding in vivo and increased L452R pseudovirus infection of cell cultures and lung organoids, albeit decreased relative to pseudoviruses carrying the N501Y mutation found in the B.1.1.7, B.1.351, and P.1 variants. Antibody neutralization assays showed 4.0 to 6.7-fold and 2.0-fold decreases in neutralizing titers from convalescent patients and vaccine recipients, respectively. The increased prevalence of a more transmissible variant in California associated with decreased antibody neutralization warrants further investigation.

In 2019, the United States (US) experienced the highest number of measles importations and cases in the postelimination era. More than a quarter of imported cases entered the US through California. Measles surveillance efforts in California resulted in the identification of 26 importations, 6 outbreaks, and 72 cases in 2019. Only genotype B3 and D8 measles strains were detected. Genotype-specific differences were noted in the incidence of vaccine failures, hospitalizations, and severe complications among cases. A targeted whole genome sequencing approach provided higher-resolution discrimination between epidemiologically linked and sporadically introduced strains than conventional N450 sequencing. Our report underscores the importance of ensuring appropriate measles vaccination status, especially prior to international travel to measles-endemic regions, and highlights the value of a strong measles surveillance system in minimizing outbreaks and preserving measles elimination status in the US.

The Pacific Coast tick (Dermacentor occidentalis Marx, 1892) is one of the most widely distributed and frequently encountered tick species in California. This tick is the primary vector of an unclassified spotted fever group rickettsial pathogen, designated currently as Rickettsia 364D, the etiologic agent of a recently recognized tick-borne rickettsiosis known as Pacific Coast tick fever. Despite intensified interest in this pathogen, important questions remain regarding its taxonomic status and possible variations in genotype among different strains that could influence its pathogenicity. Only the extensively passaged prototypical isolate (strain 364-D) is widely available to rickettsiologists and public health scientists worldwide. To achieve a larger, more geographically diverse, and contemporary collection of strains, 1,060 questing adult D. occidentalis ticks were collected from 18 sites across six counties in northern and southern California in 2016 and 2017. Fourteen ticks (1.3%) yielded DNA of Rickettsia 364D and from these, 10 unique isolates from Lake and Orange counties were obtained. Additionally, Rickettsia rhipicephali was detected in 108 (10.2%) ticks, from which eight isolates were obtained, and Rickettsia bellii in six (0.6%), from which three isolates were obtained. The panel of recently acquired, low-passage strains of Rickettsia 364D derived from this study could enhance opportunities for investigators to accurately determine the taxonomic standing of this agent and to develop specific diagnostic assays for detecting infections with Rickettsia 364D in ticks and humans.

We implemented subgenomic and whole-genome sequencing to support the investigation of a large hepatitis A virus outbreak among persons experiencing homelessness, users of illicit drugs, or both in California, USA, during 2017-2018. Genotyping data helped confirm case-patients, track chains of transmission, and monitor the effectiveness of public health control measures.

Background Substantial heterogeneity in measles outbreak sizes may be due to genotype-specific transmissibility. Using a branching process analysis, we characterize differences in measles transmission by estimating the association between genotype and the reproduction number R among postelimination California measles cases during 2000-2015 (400 cases, 165 outbreaks). Methods Assuming a negative binomial secondary case distribution, we fit a branching process model to the distribution of outbreak sizes using maximum likelihood and estimated the reproduction number R for a multigenotype model. Results Genotype B3 is found to be significantly more transmissible than other genotypes (P = .01) with an R of 0.64 (95% confidence interval [CI], .48-.71), while the R for all other genotypes combined is 0.43 (95% CI, .28-.54). This result is robust to excluding the 2014-2015 outbreak linked to Disneyland theme parks (referred to as "outbreak A" for conciseness and clarity) (P = .04) and modeling genotype as a random effect (P = .004 including outbreak A and P = .02 excluding outbreak A). This result was not accounted for by season of introduction, age of index case, or vaccination of the index case. The R for outbreaks with a school-aged index case is 0.69 (95% CI, .52-.78), while the R for outbreaks with a non-school-aged index case is 0.28 (95% CI, .19-.35), but this cannot account for differences between genotypes. Conclusions Variability in measles transmissibility may have important implications for measles control; the vaccination threshold required for elimination may not be the same for all genotypes or age groups.

Background Subacute sclerosing panencephalitis (SSPE) is a fatal complication of measles. We reviewed California cases from 1998-2015 to understand risk factors for SPPE and estimate incidence. Methods SSPE cases had clinically compatible symptoms and measles antibody detection in cerebrospinal fluid (CSF) or medical record documentation of SSPE. Cases were identified though a state death certificate search, Centers for Disease Control and Prevention reports, or investigations for undiagnosed neurologic disease. Measles detection in CSF was performed by serology at the California Department of Public Health or at clinical laboratories. Results Seventeen SSPE cases were identified. Males outnumbered females 2.4:1. Twelve (71%) cases had a history of measles-like illness; all 12 had illness prior to 15 months of age. Eight (67%) children were exposed to measles in California. SSPE was diagnosed at a median age of 12 years (3-35 years), with a latency period of 9.5 years (2.5-34 years). Among measles cases reported to CDPH during 1988-1991, the incidence of SSPE was 1:1367 for children

During measles outbreaks, it is important to be able to rapidly distinguish between measles cases and vaccine reactions to avoid unnecessary outbreak response measures such as case isolation and contact investigations. We have developed a real-time reverse transcription-PCR (RT-PCR) method specific for genotype A measles virus (MeV) (MeVA RT-quantitative PCR [RT-qPCR]) that can identify measles vaccine strains rapidly, with high throughput, and without the need for sequencing to determine the genotype. We have evaluated the method independently in three measles reference laboratories using two platforms, the Roche LightCycler 480 system and the Applied Biosystems (ABI) 7500 real-time PCR system. In comparison to the standard real-time RT-PCR method, the MeVA RT-qPCR showed 99.5% specificity for genotype A and 94% sensitivity for both platforms. The new assay was able to detect RNA from five currently used vaccine strains, AIK-C, CAM-70, Edmonston-Zagreb, Moraten, and Shanghai-191. The MeVA RT-qPCR assay has been used successfully for measles surveillance in reference laboratories, and it could be readily deployed to national and subnational laboratories on a wide scale.

Pacific Coast tick fever is a febrile illness associated with the bite of Dermacentor occidentalis and results from an infection due to the intracellular pathogen Rickettsia 364D (also known by the proposed name “Rickettsia philipii”). Current molecular methods for the detection of this pathogen rely on the amplification of a conserved spotted fever group rickettsial gene (ompA) followed by DNA sequencing of the amplicon to identify the species. This work describes the development of a Rickettsia 364D-specific TaqMan assay to simplify and accelerate the detection and identification processes. The assay demonstrated a sensitivity of 1 genomic copy per 4-μl sample and is highly specific for Rickettsia 364D. The utility of this assay for ecological and diagnostic samples was evaluated using banked specimens collected in a single-blind manner and yielded a clinical sensitivity and specificity of 100%. In conclusion, we describe the development and evaluation of a novel TaqMan real-time PCR assay for the detection and identification of Rickettsia 364D suitable for ecological and diagnostic applications.

Laboratory surveillance plays an important role in the detection and control of hepatitis A outbreaks and requires the application of rapid and accurate molecular diagnostic tools for hepatitis A virus (HAV) RNA detection, subgenotype identification, and sequence-based genotyping. We describe the development and validation of a triplex real-time, reverse transcription-PCR (triplex rRT-PCR) assay for the identification and discrimination of HAV subgenotypes IA, IB, and IIIA and a singleplex rRT-PCR assay designed to detect all HAV genotypes infecting humans. Overall, the accuracy, sensitivity, and specificity of the new assays were >97% for serum and plasma specimens collected during unrelated outbreaks of HAV in California and Michigan compared to a nested RT-PCR genotyping assay and the ISO 15216-1 rRT-PCR method for HAV detection. The new assays will permit the rapid detection of HAV RNA and discrimination among subgenotypes IA, IB, and IIIA in serum and plasma specimens, which will strengthen public health surveillance efforts for HAV outbreak detection and response.

Pacific Coast tick fever is a febrile illness associated with the bite of Dermacentor occidentalis and results from an infection due to the intracellular pathogen Rickettsia 364D (also known by the proposed name “Rickettsia philipii”). Current molecular methods for the detection of this pathogen rely on the amplification of a conserved spotted fever group rickettsial gene (ompA) followed by DNA sequencing of the amplicon to identify the species. This work describes the development of a Rickettsia 364D-specific TaqMan assay to simplify and accelerate the detection and identification processes. The assay demonstrated a sensitivity of 1 genomic copy per 4-μl sample and is highly specific for Rickettsia 364D. The utility of this assay for ecological and diagnostic samples was evaluated using banked specimens collected in a single-blind manner and yielded a clinical sensitivity and specificity of 100%. In conclusion, we describe the development and evaluation of a novel TaqMan real-time PCR assay for the detection and identification of Rickettsia 364D suitable for ecological and diagnostic applications.

Despite high coverage with measles, mumps, and rubella vaccine in the United States, outbreaks of mumps occur in close contact settings such as schools, colleges, and camps. Starting in late 2015, outbreaks were reported from several universities, and by the end of 2017, greater than 13,800 cases had been reported nation-wide. In 2013, the CDC and the Association of Public Health Laboratories contracted four Vaccine Preventable Diseases Reference Centers (VPD-RCs) to perform real-time reverse transcription PCR (RT-qPCR) to detect mumps RNA in clinical samples and to determine the genotype. Twelve genotypes of mumps virus are currently recognized by the World Health Organization, and the standard protocol for genotyping requires sequencing the entire gene coding for the small hydrophobic (SH) protein. Phylogenetic analysis of the 1862 mumps samples genotyped from 2015 through 2017 showed that the overall diversity of genotypes detected was low. Only 0.8 % of the sequences were identified as genotypes C, H, J, or K, and 0.5 % were identified as vaccine strains in genotypes A or N, while most sequences (98.7 %) were genotype G. The majority of the genotype G sequences could be included into one of two large groups with identical SH sequences. Within genotype G, a small number of phylogenetically significant outlier sequences were associated with epidemiologically distinct chains of transmission. These results demonstrate that molecular and epidemiologic data can be used to track transmission pathways of mumps virus; however, the limited diversity of the SH sequences may be insufficient for resolving transmission in all outbreaks.

In 2013, the national surveillance case definition for West Nile virus (WNV) disease was revised to remove fever as a criterion for neuroinvasive disease and require at most subjective fever for non-neuroinvasive disease. The aims of this project were to determine how often afebrile WNV disease occurs and assess differences among patients with and without fever. We included cases with laboratory evidence of WNV disease reported from four states in 2014. We compared demographics, clinical symptoms and laboratory evidence for patients with and without fever and stratified the analysis by neuroinvasive and non-neuroinvasive presentations. Among 956 included patients, 39 (4%) had no fever; this proportion was similar among patients with and without neuroinvasive disease symptoms. For neuroinvasive and non-neuroinvasive patients, there were no differences in age, sex, or laboratory evidence between febrile and afebrile patients, but hospitalisations were more common among patients with fever (P < 0.01). The only significant difference in symptoms was for ataxia, which was more common in neuroinvasive patients without fever (P = 0.04). Only 5% of non-neuroinvasive patients did not meet the WNV case definition due to lack of fever. The evidence presented here supports the changes made to the national case definition in 2013.

We report here the full coding sequence of nine paramyxovirus genomes, including two full-length mumps virus genomes (genotypes G and H) and seven measles virus genomes (genotypes B3 and D4, D8, and D9), from respiratory samples of patients from California, Virginia, and Alabama obtained between 2010 and 2014.

Doan et al. Genome Medicine (2016) 8:123 DOI 10.1186/s13073-016-0377-x ERRATUM Open Access Erratum to: Illuminating uveitis: metagenomic deep sequencing identifies common and rare pathogens Thuy Doan 1,2† , Michael R. Wilson 3,4† , Emily D. Crawford 3,5 , Eric D. Chow 3 , Lillian M. Khan 3 , Kristeene A. Knopp 3 , Brian D. O’Donovan 3 , Dongxiang Xia 6 , Jill K. Hacker 6 , Jay M. Stewart 2 , John A. Gonzales 1,2 , Nisha R. Acharya 1,2 and Joseph L. DeRisi 3* Erratum It has come to our attention that there is an error in Fig. 3a for this article [1]. The correct version of Fig. 3a can be found below. The red markers now reflect the se- quence differences. The text is correct. There was also a row omitted in Additional file 1: Table S1. The revised version can be found below. Additional file Additional file 1: Table S1. List of nucleotide substitutions identified in subject 6’s RV genome. The patient’s RV genome was aligned with the Stuttgart strain (GenBank DQ388280.1). A nucleotide change was considered a substitution only if the change was present in ≥ 4 reads or in 80% of the total reads at that nucleotide position. (PDF 120 kb) Author details Francis I. Proctor Foundation, University of California San Francisco, San Francisco, CA, USA. 2 Department of Ophthalmology, University of California San Francisco, San Francisco, CA, USA. 3 Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA, USA. Department of Neurology, University of California San Francisco, San Francisco, CA, USA. 5 Howard Hughes Medical Institute, Chevy Chase, MD, USA. 6 California Department of Public Health, Richmond, CA, USA. Received: 7 November 2016 Accepted: 7 November 2016 Reference 1. Doan T, Wilson MR, Crawford ED, Chow ED, Khan LM, Knopp KA, O’Donovan BD, Xia D, Hacker JK, Stewart JM, Gonzales JA, Archarya NR, DeRisi JL. Illuminating uveitis: metagenomic deep sequencing identifies common and rare pathogens. Genome Med. 2016;8:106. * Correspondence: joe@derisilab.ucsf.edu Equal contributors Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA, USA © The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Rickettsia philipii (type strain “Rickettsia 364D”), the etiologic agent of Pacific Coast tick fever (PCTF), is transmitted to people by the Pacific Coast tick, Dermacentor occidentalis. Following the first confirmed human case of PCTF in 2008, 13 additional human cases have been reported in California, more than half of which were pediatric cases. The most common features of PCTF are the presence of at least one necrotic lesion known as an eschar (100%), fever (85%), and headache (79%); four case-patients required hospitalization and four had multiple eschars. Findings presented here implicate the nymphal or larval stages of D. occidentalis as the primary vectors of R. philipii to people. Peak transmission risk from ticks to people occurs in late summer. Rickettsia philipii DNA was detected in D. occidentalis ticks from 15 of 37 California counties. Similarly, non-pathogenic Rickettsia rhipicephali DNA was detected in D. occidentalis in 29 of 38 counties with an average prevalence of 12.0% in adult ticks. In total, 5,601 ticks tested from 2009 through 2015 yielded an overall R. philipii infection prevalence of 2.1% in adults, 0.9% in nymphs and a minimum infection prevalence of 0.4% in larval pools. Although most human cases of PCTF have been reported from northern California, acarological surveillance suggests that R. philipii may occur throughout the distribution range of D. occidentalis., Author Summary Rickettsia philipii, a spotted fever group rickettsia, is the etiologic agent of Pacific Coast tick fever, an emerging tick-borne disease transmitted by the Pacific Coast tick (Dermacentor occidentalis). The Pacific Coast tick’s range, and thus potential for exposure to R. philipii, includes most of California, southern Oregon, and northern Baja California, Mexico. This study describes the clinical manifestations of the 14 human cases reported to date in California and the known acarological risk factors for encountering PCTF. While Rickettsia rickettsii, the agent of Rocky Mountain spotted fever (RMSF) is genetically similar to R. philipii, PCTF is a milder disease than RMSF; the two diseases share key clinical features such as headache and fever but differ in that PCTF presents with a localized eschar (necrotic wound) but without a petechial rash. Most case-patients have illness onset dates in the summer (peak: July and August) which is coincident with the activity period of immature D. occidentalis. Results from this study support the premise that larval and nymphal D. occidentalis are the vectors of PCTF to humans in California. While only three ticks have tested positive for R. rickettsii over decades of surveillance in California, the prevalence of R. philipii in D. occidentalis ticks averages 0.4% in larvae, 0.9% in nymphs, and 2.1% in adult ticks, indicating a higher risk of acquiring PCTF than RMSF statewide.

Background Ocular infections remain a major cause of blindness and morbidity worldwide. While prognosis is dependent on the timing and accuracy of diagnosis, the etiology remains elusive in ~50 % of presumed infectious uveitis cases. The objective of this study is to determine if unbiased metagenomic deep sequencing (MDS) can accurately detect pathogens in intraocular fluid samples of patients with uveitis. Methods This is a proof-of-concept study, in which intraocular fluid samples were obtained from five subjects with known diagnoses, and one subject with bilateral chronic uveitis without a known etiology. Samples were subjected to MDS, and results were compared with those from conventional diagnostic tests. Pathogens were identified using a rapid computational pipeline to analyze the non-host sequences obtained from MDS. Results Unbiased MDS of intraocular fluid produced results concordant with known diagnoses in subjects with (n = 4) and without (n = 1) uveitis. Samples positive for Cryptococcus neoformans, Toxoplasma gondii, and herpes simplex virus 1 as tested by a Clinical Laboratory Improvement Amendments-certified laboratory were correctly identified with MDS. Rubella virus was identified in one case of chronic bilateral idiopathic uveitis. The subject’s strain was most closely related to a German rubella virus strain isolated in 1992, one year before he developed a fever and rash while living in Germany. The pattern and the number of viral identified mutations present in the patient’s strain were consistent with long-term viral replication in the eye. Conclusions MDS can identify fungi, parasites, and DNA and RNA viruses in minute volumes of intraocular fluid samples. The identification of chronic intraocular rubella virus infection highlights the eye’s role as a long-term pathogen reservoir, which has implications for virus eradication and emerging global epidemics. Electronic supplementary material The online version of this article (doi:10.1186/s13073-016-0344-6) contains supplementary material, which is available to authorized users.

SUMMARYBackgroundOcular infections remain a major cause of blindness and morbidity worldwide. While prognosis is dependent on the timing and accuracy of diagnosis, the etiology remains elusive in ~ 50% of presumed infectious uveitis cases.1,2 We aimed to determine if unbiased metagenomic deep sequencing (MDS) can accurately detect pathogens in intraocular fluid samples of patients with uveitis.MethodsThis is a proof-of-concept study, in which intraocular fluid samples were obtained from 5 subjects with known diagnoses, and one subject with bilateral chronic uveitis without a known etiology. Samples were subjected to MDS, and results were compared with conventional diagnostic tests. Pathogens were identified using a rapid computational pipeline to analyze the non-host sequences obtained from MDS.FindingsUnbiased MDS of intraocular fluid produced results concordant with known diagnoses in subjects with (n=4) and without (n=1) uveitis. Rubella virus (RV) was identified in one case of chronic bilateral idiopathic uveitis. The subject’s strain was most closely related to a German RV strain isolated in 1992, one year before he developed a fever and rash while living in Germany.InterpretationMDS can identify fungi, parasites, and DNA and RNA viruses in minute volumes of intraocular fluid samples. The identification of chronic intraocular RV infection highlights the eye’s role as a long-term pathogen reservoir, which has implications for virus eradication and emerging global epidemics.

We describe 10 patients with 2009 H1N1 influenza and concurrent invasive group A streptococcal infection with marked associated morbidity and mortality. Seven patients required intensive care, 8 required mechanical ventilation, and 7 died. Five of the patients, including 4 of the fatalities, were previously healthy.

Endometritis and toxic shock syndrome associated with Clostridium sordellii have previously been reported after childbirth and, in one case, after medical abortion. We describe four deaths due to endometritis and toxic shock syndrome associated with C. sordellii that occurred within one week after medically induced abortions. Clinical findings included tachycardia, hypotension, edema, hemoconcentration, profound leukocytosis, and absence of fever. These cases indicate the need for physician awareness of this syndrome and for further study of its association with medical abortion.

Study objective The accurate diagnosis of influenza remains a diagnostic dilemma. We examine the performance of various strategies for diagnosing influenza infection in an unselected sample of adults during influenza season. Methods Consecutive adults presenting to a university emergency department or urgent care clinic between January and March 2002 with acute respiratory complaints were eligible for this prospective observational study. The performance of clinician judgment, a rapid influenza test, and a clinical prediction rule in predicting influenza infection was evaluated using referent standard of reverse transcriptase polymerase chain reaction. Statistical significance was assessed using McNemar's test of proportions. Results Fifty-three of 258 (21%) patients had a positive influenza reverse transcriptase polymerase chain reaction test. Overall, clinician judgment showed sensitivity of 29% (95% confidence interval [CI] 18% to 43%) and specificity of 92% (95% CI 87% to 95%). The rapid influenza test showed a sensitivity of 33% (95% CI 22% to 47%) and specificity of 98% (95% CI 96% to 99%). The clinical prediction rule showed a sensitivity of 40% (95% CI 27% to 54%) and specificity of 92% (95% CI 87% to 95%). Clinician judgment when patients presented within 48 hours showed a sensitivity of 67% (95% CI 39% to 86%) and specificity of 96% (95% CI 81% to 99%). Neither the rapid influenza test (P=.10) nor the clinical prediction rule (P=.42) was superior to clinician judgment alone in the diagnosis of influenza. Conclusion The suggestion that a clinical decision rule or a rapid influenza test is better than clinical judgment alone for the diagnosis of influenza in an unselected patient population is not supported by this study.

Background Adenoviruses are associated with sporadic infection and community and institutional outbreaks; they can cause especially severe disease in infants, young children, immunocompromised persons, and transplant recipients. Fifty-two adenovirus serotypes have been recognized and classified within 7 subgroups or species (A-G), with limited data available on associated clinical syndromes and disease severity in more than one-half of the known serotypes. Methods We describe the clinical presentation and virologic characterization of 1 adult and 2 pediatric patients admitted to 2 separate hospitals during April-May 2006 with severe acute respiratory tract infection. All patients had underlying chronic pulmonary disease; none were severely immunocompromised. All 3 experienced serious chronic sequelae or died. Results Adenovirus was isolated from all 3 case patients. Adenovirus serotype 14, a subspecies B2 serotype not previously associated with severe clinical illness, was confirmed by neutralization assay and sequencing of the hexon gene. Restriction enzyme analysis with BamHI, BglII, HindIII, and SmaI showed all 3 viruses to be identical and to belong to a new genome type that we have designated "Ad14a." Conclusions Our identification of severe respiratory illness due to a previously rarely reported adenovirus serotype may signify the emergence in the United States of a new genomic variant that has the potential to spread globally and cause epidemics. These case reports highlight the need for rapid diagnosis and improved surveillance, with serotyping and molecular characterization, to identify emerging variants of adenovirus, which may assist with targeted development of antiviral agents or type-specific vaccines.

Author(s): Louie, Janice K; Hacker, Jill K; Gonzales, Ralph; Mark, Jennifer; Maselli, Judy H; Yagi, Shigeo; Drew, W Lawrence | Abstract: BackgroundWith use of polymerase chain reaction (PCR) and a centrifugation-enhanced viral culture method, we characterized the viruses causing acute respiratory infection in adults during an influenza season.MethodsDuring January-March 2002, nasopharyngeal wash specimens from previously healthy adults presenting with respiratory symptoms were evaluated for viral pathogens with centrifugation-enhanced viral culture and PCR.ResultsThe diagnoses in 266 cases included unspecified upper respiratory infection (in 142 [54%] of the cases), acute bronchitis (42 [16%]), sinusitis (23 [9%]), pharyngitis (22 [8%]), and pneumonia (17 [6%]). The use of a shell vial assay and PCR identified a pathogen in 103 (39%) of the patients, including influenza A or B in 54, picornavirus in 28 (including rhinovirus in 24), respiratory syncytial virus (RSV) in 12, human metapneumovirus in 4, human coronavirus OC43 in 2, adenovirus in 2, parainfluenza virus type 1 in 1, and coinfection with influenza and parainfluenza virus type 1 in 2.ConclusionOur findings demonstrate that, even during the influenza season, rhinovirus and RSV are prevalent and must be considered in the differential diagnosis of adult acute respiratory infection before prescribing antiviral medication. Human coronavirus and human metapneumovirus did not play a substantial role. PCR was an especially useful tool in the identification of influenza and other viral pathogens not easily detected by traditional testing methods.

A 27-year-old woman presented to a hospital with symptoms resembling pyelonephritis; respiratory distress did not develop until nearly a day after admission and she subsequently died. The Unexplained Deaths and Critical Illnesses Project of the Centers for Disease Control and Prevention confirmed Sin Nombre virus infection by the results of serological testing and sequencing of the viral genome; staining of Sin Nombre virus antigen in the pulmonary capillaries was relatively weak.

Surveillance by the Unexplained Deaths and Critical Illnesses Project (UNEX) uncovered a novel presentation of adenovirus type 3 infection that satisfied the criteria for toxic shock-like syndrome in a 28-year-old immunocompetent man. Adenovirus may be a cause of toxic shock syndrome; surveillance systems such as UNEX may uncover additional causes of this and other clinically defined infectious syndromes.

We tested the role of the “spring-loaded” conformational change in the fusion mechanism of the influenza hemagglutinin (HA) by assessing the effects of 10 point mutants in the region of high coiled-coil propensity, HA2 54–81. The mutants included proline substitutions at HA2 55, 71, and 80, as well as a double proline substitution at residues 55 and 71. Mutants were expressed in COS or 293T cells and assayed for cell surface expression and structural features as well as for their ability to change conformation and induce fusion at low pH. We found the following: Specific mutations affected the precise carbohydrate structure and folding of the HA trimer. All of the mutants, however, formed trimers that could be expressed at the cell surface in a form that could be proteolytically cleaved from the precursor, HA0, to the fusion-permissive form, HA1-S-S-HA2. All mutants reacted with an antibody against the major antigenic site and bound red blood cells. Seven out of ten mutants displayed a wild-type (wt) or moderately elevated pH dependence for the conformational change. V55P displayed a substantial reduction (∼60– 80%) in the initial rate of lipid mixing. The other single mutants displayed efficient fusion with the same pH dependence as wt-HA. The double proline mutant V55P/ S71P displayed no fusion activity despite being well expressed at the cell surface as a proteolytically cleaved trimer that could bind red blood cells and change conformation at low pH. The impairment in fusion for both V55P and V55P/S71P was at the level of outer leaflet lipid mixing. We interpret our results in support of the hypothesis that the spring-loaded conformational change is required for fusion. An alternate model is discussed.

Objective In 2005, 4 Clostridium sordellii -associated toxic shock fatalities were reported in young Californian women after medical abortions. The true incidence of this rare disease is unknown, and a population-based study has never been performed. Additional clostridia-associated deaths were sought to describe associated clinical characteristics. Study Design Population-based death certificate review and a clinical case definition for clostridial-associated toxic shock identified women with likelihood of dying from a Clostridium infection. Formalin-fixed autopsy tissues underwent immunohistochemical and polymerase chain reaction assays. Results Thirty-eight women were suspected of having C sordellii -associated death. Five tested positive for Clostridium species: 3 for Clostridium perfringens , 1 for C sordellii , and 1 for both. Deaths occurred after the medical procedures for cervical dysplasia (n = 2), surgical abortion (n = 1), stillborn delivery (n = 1), and term live birth (n = 1). None had a medical abortion. Conclusion C sordellii and C perfringens are associated with undiagnosed catastrophic infectious gynecologic illnesses among women of childbearing age.

The G1 glycoprotein of California encephalitis (CE) virus plays a critical role in the infection of mosquito and mammalian cells. We found that CE virus enters baby hamster kidney (BHK-21) andAedes albopictus(C6/36) cells by the endocytic pathway. Ammonium chloride, a lysosomotropic amine that prevents release of virus from endosomes, inhibited infection of both cell types when added within 10 min after viral adsorption. In addition, infected cells formed polykaryons when the extracellular pH was lowered to 6.3; optimal fusion occurred at pH 5.8 and 6.0 (C6/36 and BHK-21 cells, respectively). Two neutralizing G1 MAbs, 6D5.5 and 7D4.5, inhibited low pH-induced syncytia formation without affecting viral attachment, suggesting a role for G1 in viral entry. Since viral fusion proteins have been demonstrated to undergo conformational changes at low pH, acid-induced changes in G1 and G2 were assessed. While both G1 and G2 demonstrated low pH-induced alterations in detergent binding, only G1 displayed an altered protease cleavage pattern at the fusion pH. These results indicate that the G1 protein of CE virus undergoes conformational changes necessary for low pH-mediated entry into both mosquito and mammalian cells.

A role for the large glycoprotein (G1) of California encephalitis (CE) virus was examined in the infection of baby hamster kidney (BHK-21) and Aedes albopictus (C6/36) cell lines and the mosquito Ae. dorsalis using G1 monoclonal antibodies (MAbs) and selective protein cleavage. Five MAbs neutralized CE viral infectivity in both cell lines. One MAb, 7D45, efficiently neutralized the peroral infection of Ae. dorsalis females fed CE virus in artificial bloodmeals. To determine if MAbs to G1 neutralized CE virus by sterically hindering the small glycoprotein (G2), portions of G1 were trypsinized, and viral infectivity was assayed in vivo and in vitro. Cleavage of G1 resulted in a complete loss of infectivity both in mosquitoes and in culture, even though a significant amount of G2 remained intact. The loss of infectivity by both neutralization with G1 MAbs and trypsinization indicates that the G1 protein of CE virus is required for infection of mosquito and mammalian cells in vitro and of mosquitoes by the peroral route.