A novel hybridization capture method for direct whole genome sequencing of clinical specimens to inform Legionnaires' disease investigations.

The unprecedented precision and resolution of whole genome sequencing (WGS) can provide definitive identification of infectious agents for epidemiological outbreak tracking. WGS approaches, however, are frequently impeded by low pathogen DNA recovery from available primary specimens or unculturable samples. A cost-effective hybrid capture assay for Legionella pneumophila WGS analysis directly on primary specimens was developed. DNA from a diverse range of sputum and autopsy specimens PCR-positive for L. pneumophila serogroup 1 (LPSG1) was enriched with this method, and WGS was performed. All tested specimens were determined to be enriched for Legionella reads (up to 209,000-fold), significantly improving the discriminatory power to compare relatedness when no clinical isolate was available. We found the WGS data from some enriched specimens to differ by less than five single-nucleotide polymorphisms (SNPs) when compared to the WGS data of a matched culture isolate. This testing and analysis retrospectively provided previously unconfirmed links to environmental sources for clinical specimens of sputum and autopsy lung tissue. The latter provided the additional information needed to identify the source of these culture-negative cases associated with the South Bronx 2015 Legionnaires' disease (LD) investigation in New York City. This new method provides a proof of concept for future direct clinical specimen hybrid capture enrichment combined with WGS and bioinformatic analysis during outbreak investigations.IMPORTANCELegionnaires' disease (LD) is a severe and potentially fatal type of pneumonia primarily caused by inhalation of Legionella-contaminated aerosols from man-made water or cooling systems. LD remains extremely underdiagnosed as it is an uncommon form of pneumonia and relies on clinicians including it in the differential and requesting specialized testing. Additionally, it is challenging to obtain clinical lower respiratory specimens from cases with LD, and when available, culture requires specialized media and growth conditions, which are not available in all microbiology laboratories. In the current study, a method for Legionella pneumophila using hybrid capture by RNA baiting was developed, which allowed us to generate sufficient genome resolution from L. pneumophila serogroup 1 PCR-positive clinical specimens. This new approach offers an additional tool for surveillance of future LD outbreaks where isolation of Legionella is not possible and may help solve previously unanswered questions from past LD investigations.

Shiga Toxin-Producing Escherichia coli Testing in New York 2011-2022 Reveals Increase in Non-O157 Identifications.

Shiga toxin-producing Escherichia coli (STEC) are an important cause of bacterial enteric infection. STEC strains cause serious human gastrointestinal disease, which may result in life-threatening complications such as hemolytic uremic syndrome. They have the potential to impact public health due to diagnostic challenges of identifying non-O157 strains in the clinical laboratory. The Wadsworth Center (WC), the public health laboratory of the New York State Department of Health, has isolated and identified non-O157 STEC for decades. A shift from initially available enzyme immunoassay testing to culture-independent diagnostic tests (CIDTs) has increased the uptake of testing at clinical microbiology laboratories. This testing change has resulted in an increased number of specimen submissions to WC. During a 12-year period between 2011 and 2022, WC received 5037 broths and/or stool specimens for STEC confirmation from clinical microbiology laboratories. Of these, 3992 were positive for Shiga toxin genes (stx1 and/or stx2) by real-time PCR. Furthermore, culture methods were utilized to isolate, identify, and characterize 2925 STEC from these primary specimens. Notably, WC observed a >200% increase in the number of STEC specimens received in 2021-2022 compared with 2011-2012 and an 18% increase in the number of non-O157 STEC identified using the same methodologies. During the past decade, the WC testing algorithm has been updated to manage the increase in specimens received, while also navigating the novel COVID-19 pandemic, which took priority over other testing for a period of time. This report summarizes updated methods for confirmation, surveillance, and outbreak detection of STEC and describes findings that may be related to our algorithm updates and the increased use of CIDTs, which is starting to elucidate the true incidence of non-O157 STEC.

Spatiotemporal distribution of Borrelia miyamotoi (Spirochaetales: Spirochaetaceae) and coinfection with other tick-borne pathogens in host-seeking Ixodes scapularis (Acari: Ixodidae) from New York State, USA.

Blacklegged ticks (Ixodes scapularis Say, Acari: Ixodidae) were collected from 432 locations across New York State (NYS) during the summer and autumn of 2015-2020 to determine the prevalence and geographic distribution of Borrelia miyamotoi (Spirochaetales: Spirochaetaceae) and coinfections with other tick-borne pathogens. A total of 48,386 I. scapularis were individually analyzed using a multiplex real-time polymerase chain reaction assay to simultaneously detect the presence of Bo. miyamotoi, Borrelia burgdorferi (Spirochaetales: Spirochaetaceae), Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae), and Babesia microti (Piroplasmida: Babesiidae). Overall prevalence of Bo. miyamotoi in host-seeking nymphs and adults varied geographically and temporally at the regional level. The rate of polymicrobial infection in Bo. miyamotoi-infected ticks varied by developmental stage, with certain co-infections occurring more frequently than expected by chance. Entomological risk of exposure to Bo. miyamotoi-infected nymphal and adult ticks (entomological risk index [ERI]) across NYS regions in relation to human cases of Bo. miyamotoi disease identified during the study period demonstrated spatial and temporal variation. The relationship between select environmental factors and Bo. miyamotoi ERI was explored using generalized linear mixed effects models, resulting in different factors significantly impacting ERI for nymphs and adult ticks. These results can inform estimates of Bo. miyamotoi disease risk and further our understanding of Bo. miyamotoi ecological dynamics in regions where this pathogen is known to occur.

Laboratory diagnosis of bacterial meningitis by direct detection, serotyping and Next Generation Sequencing: How 10 years of testing in New York State has evolved to improve laboratory diagnosis and public health.

Since 2005, the Wadsworth Center (WC) has provided molecular testing on cerebrospinal fluid (CSF) and whole blood specimens in close collaboration with epidemiologists in New York State and New York City. In this study, we analyzed 10 years of data to demonstrate the significant value of utilizing molecular methods to assess patient specimens for etiologic agents of bacterial meningitis. A comprehensive molecular testing algorithm to detect and serotype/serogroup bacterial agents known to cause bacterial meningitis (Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae and Streptococcus agalactiae) has evolved, and retrospective specimen testing has been essential for each improvement. Over a ten-year span from 2010 to 2019 the WC received 831 specimens from 634 patients with suspected bacterial meningitis. Real-time PCR was positive for at least one of the agents in 223 (27%) specimens from 183 patients (29%). Of the 223 positives, 146 (66%) were further characterized by real-time PCR into serogroup/serotype. Additionally, examination of 131 paired specimens of CSF and whole blood from the same patients found better detection in CSF, but whole blood is a useful alternative for diagnosis when CSF is not available. For specimens initially PCR-negative, 16S rDNA Sanger sequencing was requested by the submitter for 146 cases resulting in the identification of bacterial agents in an additional 24 (16%) specimens. In a retrospective study, Next Generation Sequencing (NGS) was evaluated for the detection of pathogens in 53 previously tested PCR-negative CSF specimens and identified bacteria in 14 (26%) specimens. This molecular testing algorithm has provided clinicians a diagnosis when culture is negative with the potential to guide therapy. It has also aided public health in determining when antibiotic prophylaxis was needed, augmented surveillance data to yield a fuller picture of community prevalence, and highlighted gaps in the spectrum of agents that cause bacterial meningitis.

A novel chemical lysis method for maximum release of DNA from difficult-to-lyse bacteria.

Molecular detection of microorganisms requires releasing DNA from cells. However, since certain microbial organisms are refractory to lysis by chemical or enzymatic methods, mechanical lysis by bead-beating is typically employed to disrupt difficult-to-lyse microbes. A newly developed chemical lysis method called sporeLYSE enables release of DNA from difficult-to-lyse microbes without bead-beating. The sporeLYSE method was compared to bead-beating and an alkaline/detergent lysis solution for releasing DNA from microbes grown in vitro, including surrogates of Category A bioterrorism agents. sporeLYSE released 83% to 100% of DNA from Mycobacterium smegmatis, Francisella philomiragia, Yersinia enterocolitica, Bacillus thuringiensis, Pseudomonas aeruginosa, Moraxella catarrhalis and Klebsiella pneumoniae. qPCR results indicated that sporeLYSE extracted an equal or greater amount of DNA than either bead-beating or alkaline/detergent lysis from Gram-positive and Gram-negative bacteria. When sporeLYSE was used to extract DNA from saliva and sputum spiked with M. smegmatis and M. tuberculosis, respectively, the qPCR Ct values were 4-8 cycles lower than those for extractions via alkaline/detergent lysis and heat. Mean Ct values for sporesLYSE extractions from spores of Clostridium difficile and C. botulinum were approximately two cycles lower than those of MagNA Pure DNA extractions. Our results suggest that sporeLYSE is an easy-to-use liquid reagent that can efficiently release large amounts of DNA from a variety of bacteria, including spores.

Insights into the long-term persistence of Legionella in facilities from whole-genome sequencing.

We investigated the value of whole-genome sequencing (WGS) and single nucleotide polymorphism (SNP) analyses in determining the relationships among and evolutionary rates of Legionella species with long-term persistence in three healthcare facilities. We examined retrospective clinical and environmental isolates of Legionella micdadei and Legionella pneumophila serogroup 1 isolates with identical PFGE DNA fingerprints sampled over the course of up to 18 years. WGS analyses demonstrated that heterogeneous populations of Legionella were present within each facility despite displaying the same PFGE profiles. Additionally, clustering of some clinical isolates with those from a separate but related institution exposed a source of infection not previously detected, underscoring the importance of considering phylogenetic relationships when assessing epidemiological links. The data supported an average substitution rate of 0.80 SNPs per genome per year for L. micdadei but a reliable estimate for L. pneumophila serogroup 1 could not be obtained due to complicating factors such as non-chronological links among isolates and inadequate sampling depths. While the substitution rate for L. micdadei is consistent with previous estimates for L. pneumophila, the lack of a temporal signal in our sequence data for L. pneuomphila serogroup 1 isolates suggests either insufficient change to provide an estimate or variable evolutionary rates, which could reflect the presence of both actively dividing and viable but non-culturable Legionella spp. in the built environment. This study highlights the increased discriminatory power of WGS SNP analysis as compared to PFGE, emphasizes the need for extended sampling, and provides insight into the evolution of Legionella from longitudinal investigations.

Legionnaires' Disease Outbreak Caused by Endemic Strain of Legionella pneumophila, New York, New York, USA, 2015.

During the summer of 2015, New York, New York, USA, had one of the largest and deadliest outbreaks of Legionnaires' disease in the history of the United States. A total of 138 cases and 16 deaths were linked to a single cooling tower in the South Bronx. Analysis of environmental samples and clinical isolates showed that sporadic cases of legionellosis before, during, and after the outbreak could be traced to a slowly evolving, single-ancestor strain. Detection of an ostensibly virulent Legionella strain endemic to the Bronx community suggests potential risk for future cases of legionellosis in the area. The genetic homogeneity of the Legionella population in this area might complicate investigations and interpretations of future outbreaks of Legionnaires' disease.

Neisseria dumasiana sp. nov. from human sputum and a dog's mouth.

Three independent isolates of Gram-reaction-negative cocci collected from two New York State patients and a dog's mouth in California were subjected to a polyphasic analysis. The 16S rRNA gene sequence similarity among these isolates is 99.66 to 99.86 %. The closest species with a validly published name is Neisseria zoodegmatis (98.7 % 16S rRNA gene sequence similarity) with six additional species of the genus Neisseria with greater than 97 % similarity. Average nucleotide identity (ANI) and genome-to-genome distance calculator (GGDC 2.0) analysis on whole genome sequence data support the three novel isolates as being from a single species that is distinct from all other closely related species of the genus Neisseria. Phylogenetic analysis of 16S rRNA gene sequences and ribosomal multilocus sequence typing (rMLST) indicate the novel species belongs in the genus Neisseria. This assignment is further supported by the predominant cellular fatty acids composition of C16 : 0, summed feature 3 (C16 : 1ω7c/C15 : 0iso 2-OH), and C18 : 1ω7c, and phenotypic characters. The name Neisseria dumasiana sp. nov. is proposed, and the type strain is 93087T (=DSM 104677T=LMG 30012 T).

Detection of Borrelia miyamotoi and other tick-borne pathogens in human clinical specimens and Ixodes scapularis ticks in New York State, 2012-2015.

Borrelia miyamotoi (Bm) is a recently emerging bacterial agent transmitted by several species of ixodid ticks. Diagnosis of Bm infection can be challenging, as the organism is not easily cultivable. We have developed and validated a multiplex real-time PCR to simultaneously identify Bm infection and the agents causing human granulocytic anaplasmosis and human monocytic ehrlichiosis, Anaplasma phagocytophilum and Ehrlichia chaffeensis, respectively. The assay is 100% specific; highly sensitive, detecting 11 gene copies of Bm DNA in both whole blood and cerebral spinal fluid; and provides rapid results in less than two hours. A retrospective study of 796 clinical specimens collected between the years 2012 and 2014 and a prospective study of 366 clinical specimens were performed utilizing this novel assay to evaluate the frequency of Bm infection in New York State (NYS). Eight clinical specimens (1%) were found to be positive for Bm, 216 were positive for A. phagocytophilum, and 10 were positive for E. chaffeensis. Additionally, we tested 411 I. scapularis ticks collected in NYS during 2013 and 2014 in a separate multiplex real-time PCR to determine the prevalence of Bm, A. phagocytophilum, Borrelia burgdorferi s.s., and Borrelia species. Our results indicated rates of 1.5%, 27%, 19.7%, and 8.8% respectively. The ability to monitor both the frequency and geographic distribution of Bm cases and the prevalence and geographic distribution of Bm in ticks will help create a better understanding of this emerging tick-borne pathogen.

Cutaneous polyarteritis nodosa localized to a region of lymphedema secondary to Streptococcus viridans cellulitis and multiple surgeries.

Cutaneous polyarteritis nodosa (CPAN) is a chronic, indolent, single organ arteritis that generally presents with lower extremity nodules and/or livedo racemosa, accompanied by malaise and arthralgias. CPAN is often triggered by infection, commonly Group A streptococcal species, and is considered an autoimmune reaction. Scarring from surgery and obliterative lymphangiitis from bacterial cellulitis are the causes of lymphedema. Lymphedematous skin is predisposed to autoimmune disorders. Herein we report a 53-year-old woman who developed CPAN restricted to a localized area of the right upper arm-shoulder that had undergone multiple surgeries, complicated by episodes of Streptococcus viridans cellulitis. Clinically, a 15 cm diameter plaque exhibited violaceous, reticulate margins, subtle papules and nodules and central livedo racemosa. Biopsy showed numerous foci of arteritis in active, subacute and reparative stages. In addition, a broad zone of fibrosis replaced the deep dermis-subcutis zone and harbored numerous dilated lymphatic vessels scar lymphedema. Treatment consisted of high potency topical corticosteroids under occlusion; remission after 3 months therapy and follow-up. CPAN primarily affects the lower legs, a region of frequently affected by phlebolymphedema. This report of CPAN localized to an area of scar lymphedema underscores the importance of lymphatic function in the pathogenesis of CPAN.

Direct molecular testing to assess the incidence of meningococcal and other bacterial causes of meningitis among persons reported with unspecified bacterial meningitis.

Confirmed and probable cases of invasive Neisseria meningitidis (Nm) infection are reportable in New York City. We conducted a study to identify Nm among culture-negative reports of bacterial and viral meningitis. During the study period, 262 reports of suspected meningitis were eligible. Cerebrospinal fluid (CSF) specimens from 138 patients were obtained for testing. No Nm cases were detected. Results from real-time polymerase chain reaction and 16S on CSF specimens were concordant with hospital microbiology findings in 80%; however, other pathogenic organisms were detected in 14 culture-negative specimens. New York City's surveillance system appears to be effective at capturing cases of Nm meningitis. Nucleic acid testing is useful for detecting the presence of bacterial DNA when antibiotic therapy precedes lumbar puncture or bacterial cultures are negative. It remains unanswered whether culture-negative cases of Nm bacteremia are being missed by reportable disease surveillance.

Postmortem diagnosis of invasive meningococcal disease.

We diagnosed invasive meningococcal disease by using immunohistochemical staining of embalmed tissue and PCR of vitreous humor from 2 men in New York City. Because vitreous humor is less subject than other body fluids to putrefaction, it is a good material for postmortem analysis.

Primary cutaneous marginal zone lymphoma associated with juxta-articular fibrotic nodules in a teenager.

Primary cutaneous marginal zone lymphoma (PCMZL) has rarely been reported in teenagers and is occasionally associated with Borrelia burgdorferi infection. Juxta-articular fibrotic nodules represent a unique, localized fibrosing response to spirochete infections, namely Borreliosis. Herein, we report a 15-year-old healthy boy who presented with a 4-year history of progressive acquisition of asymptomatic, erythematous nodules, ≤ 3 cm, beginning with his right forearm (3), then right arm (1) and lastly his right inner thigh (1). Biopsy showed PCMZL in three of five samples, and inflamed, fibrotic nodules, near the elbow in two. The bottom heavy lymphomatous nodules consisted of mostly small CD20+ CD43+ lymphocytes, some with plasmacytoid features. Mature plasma cells were lambda light chain restricted by in situ hybridization. The juxta-articular fibrotic nodules were located in the deep dermis and subcutis, had peripheral plasma cell-rich infiltrates, and showed nodular sclerosis (morphea profunda-like) in one, and lamellar and angiocentric sclerosis in the other reminiscent of quiescent lesions of chronic localized fibrosing leukocytoclastic vasculitis. Immunohistochemistry for B. burgdorferi revealed rare positive organisms; however, polymerase chain reaction (PCR) and serology were negative for B. burgdorferi as were serologic and/or PCR assays for Bartonella henselae, Ba. quintana, Ehrlichia chaffeensis, Treponema pallidum, Helicobacter pylori and Babesia microti. No evidence of extracutaneous disease was found by the review of systems and imaging studies. A 4-week trial of doxycycline therapy failed, whereas intralesional (IL) corticosteroid therapy induced rapid regression of his nodules. After two local recurrences, also treated with IL corticosteroids, he is well, without cutaneous disease, 20 months later. A literature review of 19 pediatric cases PCMZL reveals a similar natural history as adult PCMZL. Despite negative serology and PCR for B. burgdorferi, the occurrence of ipsilateral juxta-articular fibrotic nodules, positive B. burgdorferi immunohistochemistry and rapid response to IL corticosteroids implicate the presence of a replicative or non-replicative infectious (spirochetal) antigen in the initiation and promotion of this teenager's PCMZL.

Utilization of a real-time PCR approach for Haemophilus influenzae serotype determination as an alternative to the slide agglutination test.

Our laboratory has developed a simple two-step multiplex real-time PCR for use on isolates of Haemophilus influenzae for molecular serotype identification and the detection of capsular gene targets. The assay consists of a 2-plex real-time PCR targeting the capsule transport gene (bexA), and serotype b specific gene (bcsB), and a 5-plex real-time PCR detecting serotypes a, c, d, e, and f targeting Region II serotype-specific genes. Both real-time PCR assays are highly sensitive (<8 CFU) for all serotypes and 100% specific when tested by a panel of more than 40 bacterial organisms. A retrospective study of 214 isolates received between 1998 and 2011 were tested and compared against the traditional slide agglutination test (SAT) resulting in 100% concordance. We demonstrate that this two-step real-time PCR approach is more sensitive than previously published PCR assays and provides a simple alternative to the SAT. Reliable, rapid and sensitive H. influenzae serotyping is critical for identifying new emerging strains for epidemiological surveillance.

Clinical and laboratory characteristics of Clostridium difficile infection in patients with discordant diagnostic test results.

The aim of this study was to compare the clinical and laboratory characteristics of Clostridium difficile infection (CDI) in patients with discordant test results for the cytotoxin assay (CYT) and PCR assays. A retrospective study from May to August 2008 and March to May 2010 was performed. CDI was diagnosed in 128 patients. PCR increased the yield of C. difficile cases by 2-fold compared to that of the CYT assay. Fifty-six cases (44%) were detected by PCR only (CYT negative). Forty-nine percent of patients with non-NAP1 strains were detected by PCR only, compared to 28% of those infected with NAP1 strains (P < 0.05). No significant differences were found in the clinical severity of illness and outcome among patients that tested positive for CDI by both tests (CYT and PCR) compared to those that tested positive by PCR only.

Biodiversity of Clostridium botulinum type E associated with a large outbreak of botulism in wildlife from Lake Erie and Lake Ontario.

The genetic relatedness of Clostridium botulinum type E isolates associated with an outbreak of wildlife botulism was studied using random amplification of polymorphic DNA (RAPD). Specimens were collected from November 2000 to December 2008 during a large outbreak of botulism affecting birds and fish living in and around Lake Erie and Lake Ontario. In our present study, a total of 355 wildlife samples were tested for the presence of botulinum toxin and/or organisms. Type E botulinum toxin was detected in 110 samples from birds, 12 samples from fish, and 2 samples from mammals. Sediment samples from Lake Erie were also examined for the presence of C. botulinum. Fifteen of 17 sediment samples were positive for the presence of C. botulinum type E. Eighty-one C. botulinum isolates were obtained from plants, animals, and sediments; of these isolates, 44 C. botulinum isolates produced type E toxin, as determined by mouse bioassay, while the remaining 37 isolates were not toxic for mice. All toxin-producing isolates were typed by RAPD; that analysis showed 12 different RAPD types and multiple subtypes. Our study thus demonstrates that multiple genetically distinct strains of C. botulinum were involved in the present outbreak of wildlife botulism. We found that C. botulinum type E is present in the sediments of Lake Erie and that a large range of bird and fish species is affected.

Rapid molecular characterization of Clostridium difficile and assessment of populations of C. difficile in stool specimens.

Our laboratory has developed testing methods that use real-time PCR and pyrosequencing analysis to enable the rapid identification of potential hypervirulent Clostridium difficile strains. We describe a real-time PCR assay that detects four C. difficile genes encoding toxins A (tcdA) and B (tcdB) and the binary toxin genes (cdtA and cdtB), as well as a pyrosequencing assay that detects common deletions in the tcdC gene in less than 4 h. A subset of historical and recent C. difficile isolates (n = 31) was also analyzed by pulsed-field gel electrophoresis to determine the circulating North American pulsed-field (NAP) types that have been isolated in New York State. Thirteen different NAP types were found among the 31 isolates tested, 13 of which were NAP type 1 strains. To further assess the best approach to utilizing our conventional and molecular methods, we studied the populations of C. difficile in patient stool specimens (n = 23). Our results indicated that 13% of individual stool specimens had heterogeneous populations of C. difficile when we compared the molecular characterization results for multiple bacterial isolates (n = 10). Direct molecular analysis of stool specimens gave results that correlated well with the results obtained with cultured stool specimens; the direct molecular analysis was rapid, informative, and less costly than the testing of multiple patient stool isolates.