ABSTRACT
Flea-borne spotted fever and flea-borne (murine) typhus are rickettsioses caused by Rickettsia felis and Rickettsia typhi, respectively, and typically present as undifferentiated febrile illnesses. The relative contribution of these agents to flea-borne rickettsioses in California is unclear. We have developed a duplex reverse transcription real-time polymerase chain reaction (RT-rtPCR) assay targeting R. felis- and R. typhi-specific 23S ribosomal RNA single nucleotide polymorphisms to better understand the respective roles of these agents in causing flea-borne rickettsioses in California. This assay was compared with an established duplex R. felis- and R. typhi-ompB rt-PCR assay and was shown to have 1,000-fold and 10-fold greater analytical sensitivity for the detection of R. felis and R. typhi, respectively. Retrospective testing of clinical specimens with both assays established R. typhi as the major etiologic agent of flea-borne rickettsioses in California.
ABSTRACT
Fleaborne typhus (also known as murine typhus), a widely distributed vectorborne zoonosis caused by Rickettsia typhi, is a moderately severe, but infrequently fatal illness; among patients who receive doxycycline, the case-fatality rate is <1%. Fleaborne typhus is a mandated reportable condition in California. Reported fleaborne typhus cases in Los Angeles County have been increasing since 2010, with the highest number (171) reported during 2022. During June-October 2022, Los Angeles County Department of Public Health learned of three fleaborne typhus-associated deaths. This report describes the clinical presentation, illness course, and methods used to diagnose fleaborne typhus in these three cases. Severe fleaborne typhus manifestations among these cases included hemophagocytic lymphohistiocytosis, a rare immune hyperactivation syndrome that can occur in the infection setting; myocarditis; and septic shock with disseminated intravascular coagulation. Increased health care provider and public health awareness of the prevalence and severity of fleaborne typhus and of the importance of early doxycycline therapy is essential for prevention and treatment efforts.
Subject(s)
Typhus, Endemic Flea-Borne , Typhus, Epidemic Louse-Borne , Mice , Humans , Doxycycline/therapeutic use , Los Angeles/epidemiology , Typhus, Endemic Flea-Borne/epidemiology , Typhus, Endemic Flea-Borne/diagnosis , Typhus, Endemic Flea-Borne/microbiology , Rickettsia typhi , AnimalsABSTRACT
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.
Subject(s)
Disease Outbreaks , Measles Vaccine/administration & dosage , Measles virus , Measles/epidemiology , Adolescent , Adult , Aged , California/epidemiology , Child , DNA-Directed RNA Polymerases , Female , Genotype , Humans , Male , Measles virus/genetics , Measles virus/immunology , Measles virus/isolation & purification , Middle Aged , Molecular Epidemiology , Phylogeny , Sequence Analysis, DNA , United States/epidemiology , Young AdultABSTRACT
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.
Subject(s)
Disease Outbreaks , Mumps virus/genetics , Mumps/epidemiology , Viral Proteins/genetics , Genetic Variation , Genotype , Humans , RNA, Viral/genetics , United States/epidemiologyABSTRACT
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.
Subject(s)
Hepatitis A virus/classification , Hepatitis A virus/genetics , Hepatitis A/epidemiology , Hepatitis A/virology , Molecular Typing , California/epidemiology , Disease Outbreaks , Genotype , Hepatitis A/history , Hepatitis A/transmission , Hepatitis A virus/immunology , Hepatitis A virus/isolation & purification , History, 21st Century , Humans , Public Health Surveillance , Viral Proteins/genetics , Whole Genome SequencingABSTRACT
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.
Subject(s)
Genotyping Techniques/methods , Hepatitis A virus/classification , Hepatitis A virus/isolation & purification , Hepatitis A/diagnosis , Real-Time Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , California/epidemiology , Disease Outbreaks , Epidemiological Monitoring , Genotype , Hepatitis A/epidemiology , Hepatitis A/virology , Hepatitis A virus/genetics , Humans , Michigan/epidemiology , Molecular Epidemiology/methods , Sensitivity and SpecificityABSTRACT
In May 2016, an outbreak of Shiga toxin-producing Escherichia coli O157 infections occurred among children who had played in a stream flowing through a park. Analysis of E. coli isolates from the patients, stream water, and deer and coyote scat showed that feces from deer were the most likely source of contamination.
Subject(s)
Escherichia coli Infections/epidemiology , Escherichia coli Infections/microbiology , Escherichia coli O157/isolation & purification , Rivers/microbiology , Water Microbiology , Animals , California/epidemiology , Child, Preschool , Disease Outbreaks , Electrophoresis, Gel, Pulsed-Field , Escherichia coli Infections/transmission , Escherichia coli O157/classification , Escherichia coli O157/genetics , Humans , Infant , Multilocus Sequence Typing , Shiga-Toxigenic Escherichia coliABSTRACT
Occupationally acquired meningococcal disease is rare. Adherence to recommendations for safe handling of Neisseria meningitidis in the laboratory greatly reduces the risk for transmission to laboratory workers. A California microbiologist developed fatal serogroup B meningococcal disease after working with N. meningitidis patient isolates in a research laboratory (laboratory A). The California Department of Public Health (CDPH), the local health department, the California Division of Occupational Safety and Health (CalOSHA), and the federal Occupational Safety and Health Administration (OSHA) collaborated on an investigation of laboratory A, which revealed several breaches in recommended laboratory practice for safe handling of N. meningitidis, including manipulating cultures on the bench top. Additionally, laboratory workers had not been offered meningococcal vaccine in accordance with Advisory Committee on Immunization Practices (ACIP) recommendations and CalOSHA Aerosol Transmissible Diseases Standard requirements. In accordance with OSHA and CalOSHA regulations, laboratory staff members must receive laboratory biosafety training and use appropriate personal protective equipment, and those who routinely work with N. meningitidis isolates should receive meningococcal vaccine.
Subject(s)
Meningococcal Infections/diagnosis , Neisseria meningitidis/isolation & purification , Occupational Diseases/diagnosis , Adult , California , Fatal Outcome , Humans , Laboratories , MaleABSTRACT
We describe here the isolation and identification of a Shiga toxin 1 (Stx1)-producing Enterobacter cloacae strain, M12X01451, from a human clinical specimen. The bacterial isolate was identified as E. cloacae using a polyphasic approach that included phenotypic, genetic, and proteomic analyses. The M12X01451 stx1 was sequenced, and the holotoxin was found to share only 87% amino acid sequence identity with the nearest Stx1 subtype reference sequence. Sequence analysis of the regions immediately flanking stx1 displayed similarities with bacteriophage-related sequences, suggesting a prophage origin. The stx1 gene was a stable element within the M12X01451 genome, as demonstrated by real-time PCR detection following successive subculturing of the bacterial isolate. Culture supernatant from M12X01451 was cytotoxic to Vero cells but was not neutralized by an anti-Stx1 monoclonal antibody. In addition, Stx1 from M12X01451 demonstrated limited antigenicity with two commercially available lateral flow immunoassays. The M12X01451 Stx represents a new Stx1 subtype based on the degree of sequence dissimilarity with Stx1 subtype reference sequences and its limited reactivity with anti-Stx1 antibodies.
Subject(s)
Enterobacter cloacae/genetics , Enterobacter cloacae/isolation & purification , Shiga Toxin/genetics , Shiga Toxin/metabolism , Amino Acid Sequence , Animals , Bacterial Typing Techniques , Cell Survival/drug effects , Chlorocebus aethiops , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Enterobacteriaceae Infections/microbiology , Humans , Molecular Sequence Data , Prophages/genetics , Proteome/analysis , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Vero CellsABSTRACT
We report the development of a single-tube, multi-locus variable number tandem repeat analysis (MLVA) assay for simultaneous speciation and strain typing of Brucella, the etiologic agent of brucellosis. Our MLVA assay consists of eight loci, two of which are species-specific markers that allow for definitive identification of Brucella melitensis, B. abortus, and Brucella species while ruling out related pathogenic bacterial genera. The remaining six loci are moderately variable loci capable of discriminating between Brucella strains originating within our study area. We applied the assay to a collection of 110 B. melitensis isolates of primarily Mexican origin and to smaller sample sizes of four other Brucella species for a total of 161 isolates. Simpson's index of diversity was 0.985 for B. melitensis and 0.938 for B. abortus. The assay accurately distinguished seven epidemiologically-linked clusters of B. melitensis infections and ascertained the source of infection in several laboratory-acquired cases. This assay is accessible to limited-resource settings due to its technological and economical feasibility. The timely and accurate information provided by this assay will potentially aid brucellosis control efforts, improve patient outcomes, and reduce the occurrence of laboratory-acquired infections.
Subject(s)
Bacterial Typing Techniques/methods , Brucella melitensis/isolation & purification , Brucellosis/microbiology , Polymerase Chain Reaction/methods , Brucella melitensis/classification , Brucella melitensis/genetics , Humans , Minisatellite Repeats , PhylogenyABSTRACT
A comparative analysis of the Bordetella pertussis, B. bronchiseptica, and B. parapertussis genome assemblies permitted the identification of regions with significant sequence divergence and the design of two new real-time PCR assays, BP283 and BP485, for the specific detection of B. pertussis. The performance characteristics of these two assays were evaluated and compared to those of culture and an existing real-time PCR assay targeting the repetitive element IS481. The testing of 324 nasopharyngeal specimens indicated that, compared to culture, the BP283 assay had a sensitivity and specificity of 100 and 96.8% and the BP485 assay had a sensitivity and specificity of 92.3 and 97.1%. Notably, B. holmesii was isolated from two specimens that were positive by the IS481 assay but negative by the BP283 and BP485 assays. These two assays represent an improvement in specificity over those of PCR assays targeting only IS481 and may be duplexed or used in conjunction with existing PCR assays to improve the molecular detection of B. pertussis.
Subject(s)
Bacteriological Techniques/methods , Bordetella pertussis/genetics , Bordetella pertussis/isolation & purification , DNA Primers/genetics , DNA, Bacterial/genetics , Polymerase Chain Reaction/methods , Whooping Cough/diagnosis , DNA Transposable Elements , Humans , Nasopharynx/microbiology , Sensitivity and SpecificityABSTRACT
Members of the genus Brucella are known worldwide as pathogens of wildlife and livestock and are the most common organisms of zoonotic infection in humans. In general, brucellae exhibit a range of host specificity in animals that has led to the identification of at least seven Brucella species. The genomes of the various Brucella species are highly conserved, which makes the differentiation of species highly challenging. However, we found single-nucleotide polymorphisms (SNPs) in housekeeping and other genes that differentiated the seven main Brucella species or clades and thus enabled us to develop real-time PCR assays based around these SNPs. Screening of a diverse panel of 338 diverse isolates with these assays correctly identified each isolate with its previously determined Brucella clade. Six of the seven clade-specific assays detected DNA concentrations of less than 10 fg, indicating a high level of sensitivity. This SNP-based approach places samples into a phylogenetic framework, allowing reliable comparisons to be made among the lineages of clonal bacteria and providing a solid basis for genotyping. These PCR assays provide a rapid and highly sensitive method of differentiating the major Brucella groups that will be valuable for clinical and forensic applications.
Subject(s)
Bacterial Typing Techniques/methods , Brucella/classification , Brucella/genetics , DNA, Bacterial/genetics , Polymerase Chain Reaction/methods , Polymorphism, Single Nucleotide , Animals , DNA, Bacterial/chemistry , Genotype , Humans , Molecular Sequence Data , Sensitivity and Specificity , Sequence Analysis, DNAABSTRACT
BACKGROUND: Yersinia pestis, the etiologic agent of plague, was responsible for several devastating epidemics throughout history and is currently of global importance to current public heath and biodefense efforts. Y. pestis is widespread in the Western United States. Because Y. pestis was first introduced to this region just over 100 years ago, there has been little time for genetic diversity to accumulate. Recent studies based upon single nucleotide polymorphisms have begun to quantify the genetic diversity of Y. pestis in North America. METHODOLOGY/PRINCIPAL FINDINGS: To examine the evolution of Y. pestis in North America, a gapped genome sequence of CA88-4125 was generated. Sequence comparison with another North American Y. pestis strain, CO92, identified seven regions of difference (six inversions, one rearrangement), differing IS element copy numbers, and several SNPs. CONCLUSIONS/SIGNIFICANCE: The relatively large number of inverted/rearranged segments suggests that North American Y. pestis strains may be undergoing inversion fixation at high rates over a short time span, contributing to higher-than-expected diversity in this region. These findings will hopefully encourage the scientific community to sequence additional Y. pestis strains from North America and abroad, leading to a greater understanding of the evolutionary history of this pathogen.
Subject(s)
Biological Evolution , Genome, Bacterial , Yersinia pestis/genetics , Chromosome Inversion , Genetic Variation , Humans , Molecular Sequence Data , North America , Plague/epidemiology , Plague/genetics , Plague/microbiology , Polymorphism, Single NucleotideABSTRACT
Five Mycobacterium tuberculosis complex isolates in California were identified as M. africanum by spoligotyping, single nucleotide polymorphisms, a deletion mutation, and phenotypic traits, confirming it as a cause of tuberculosis in the United States. Three of the five patients from whom M. africanum was isolated had lived in Africa.
Subject(s)
Mycobacterium/classification , Mycobacterium/isolation & purification , Tuberculosis, Pulmonary/microbiology , Adult , Africa , Bacterial Typing Techniques , California , DNA, Bacterial/analysis , DNA, Bacterial/genetics , Female , Gene Deletion , Genotype , Humans , Male , Mycobacterium/genetics , Oligonucleotides/genetics , Phenotype , Polymorphism, Single NucleotideABSTRACT
The identification of Brucella can be a time-consuming and labor-intensive process that places personnel at risk for laboratory-acquired infection. Here, we describe a real-time PCR assay for confirmation of presumptive Brucella isolates. The assay was designed in a multiplex format that will allow the rapid identification of Brucella spp., B. abortus, and B. melitensis in a single test.
Subject(s)
Brucella abortus/isolation & purification , Brucella melitensis/isolation & purification , Brucella/isolation & purification , Polymerase Chain Reaction/methods , Base Sequence , Brucella/classification , Brucella/genetics , Brucella abortus/classification , Brucella abortus/genetics , Brucella melitensis/classification , Brucella melitensis/genetics , Computer Systems , DNA Primers , RNA, Bacterial/genetics , RNA, Ribosomal, 16S/geneticsABSTRACT
The incidence of serogroup Y meningococcal disease has increased recently in the United States. Here, we describe the development of a 5' exonuclease assay for the detection of serogroup Y Neisseria meningitidis and demonstrate the usefulness of this assay for resolving serogroup identification of strains that are resistant to conventional serogrouping and for the nonculture identification of serogroup Y meningococcal disease.