Implementation of Salmonella serotype determination using pulsed-field gel electrophoresis in a state public health laboratory.

We examined the use of pulsed-field gel electrophoresis (PFGE) to predict serotype for Salmonella isolates. Between 2012 and 2014 we assessed 4481 isolates, resulting in >90% assigned serotypes. PFGE is efficient for determining serotype in the majority of cases and results in expedited serotype determination, as well as cost savings.

Enhanced identification and characterization of non-O157 Shiga toxin-producing Escherichia coli: a six-year study.

Non-O157 Shiga toxin-producing Escherichia coli (STEC) are emerging pathogens with the potential to cause serious illness and impact public health due to diagnostic challenges. Between 2005 and 2010, the Wadsworth Center (WC), the public health laboratory of the New York State (NYS) Department of Health, requested that Shiga toxin enzyme immunoassay (EIA)-positive stool enrichment broths and/or stool specimens be submitted by clinical and commercial reference laboratories testing NYS patient specimens. A total of 798 EIA-positive specimens were received for confirmation and serotyping, and additionally a subset of STEC was assessed for the presence of six virulence genes (stx1, stx2, eaeA, hlyA, nleA, and nleB) by real-time polymerase chain reaction. We confirmed 591 specimens as STEC, 164 (28%) as O157 STEC, and 427 (72%) as non-O157 STEC. Of the non-O157 STEC serogroups identified, over 70% were O103, O26, O111, O45, O121, or O145. During this time period, WC identified and characterized a total of 1282 STEC received as E. coli isolates, stool specimens, or EIA broths. Overall, the STEC testing identified 59% as O157 STEC and 41% as non-O157 STEC; however, out of 600 isolates submitted to the WC as E. coli cultures, 543 (90%) were identified as O157 STEC. This report summarizes a 6-year study utilizing enhanced STEC testing that resulted in increased identification and characterization of non-O157 STEC in NYS. Continued utilization of enhanced STEC testing may lead to effective and timely outbreak response and improve monitoring of trends in STEC disease epidemiology.

Morphological and molecular characterizations of psychrophilic fungus Geomyces destructans from New York bats with White Nose Syndrome (WNS).

BACKGROUND: Massive die-offs of little brown bats (Myotis lucifugus) have been occurring since 2006 in hibernation sites around Albany, New York, and this problem has spread to other States in the Northeastern United States. White cottony fungal growth is seen on the snouts of affected animals, a prominent sign of White Nose Syndrome (WNS). A previous report described the involvement of the fungus Geomyces destructans in WNS, but an identical fungus was recently isolated in France from a bat that was evidently healthy. The fungus has been recovered sparsely despite plentiful availability of afflicted animals. METHODOLOGY/PRINCIPAL FINDINGS: We have investigated 100 bat and environmental samples from eight affected sites in 2008. Our findings provide strong evidence for an etiologic role of G. destructans in bat WNS. (i) Direct smears from bat snouts, Periodic Acid Schiff-stained tissue sections from infected tissues, and scanning electron micrographs of bat tissues all showed fungal structures similar to those of G. destructans. (ii) G. destructans DNA was directly amplified from infected bat tissues, (iii) Isolations of G. destructans in cultures from infected bat tissues showed 100% DNA match with the fungus present in positive tissue samples. (iv) RAPD patterns for all G. destructans cultures isolated from two sites were indistinguishable. (v) The fungal isolates showed psychrophilic growth. (vi) We identified in vitro proteolytic activities suggestive of known fungal pathogenic traits in G. destructans. CONCLUSIONS/SIGNIFICANCE: Further studies are needed to understand whether G. destructans WNS is a symptom or a trigger for bat mass mortality. The availability of well-characterized G. destructans strains should promote an understanding of bat-fungus relationships, and should aid in the screening of biological and chemical control agents.

Genotypic characterization of environmental isolates of Cryptococcus gattii from Puerto Rico.

Cryptococcus neoformans and Cryptococcus gattii are found in distinct environments with some overlap around different parts of the world. However, no systematic surveys of these two pathogens have been reported from Puerto Rico, a tropical island uniquely situated between mainland USA and countries in South America. We carried out an exhaustive environmental survey in southwestern Puerto Rico for pathogenic Cryptococcus species. Twenty-two presumptive isolates of C. gattii from cacti and tree detritus were characterized in detail by physiological and molecular methods and seventeen strains were confirmed as C. gattii. Cryptococcus gattii isolates were haploid and majority of them were MATa [corrected] strains. Sixteen out of seventeen C. gattii isolates belonged to VGII/AFLP6 genotype while one isolate was a VGIV/AFLP7 genotype. The results are significant as Puerto Rico strains are distinct from VGIII/AFLP5 strains reported from Southern California, but similar to C. gattii VGII/AFLP6 molecular type implicated in recent outbreaks of cryptococcosis in Pacific Northwest and British Columbia, Canada, but different in its M13 fingerprinting, and a common genotype in South America.

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.

Design and implementation of a protocol for the detection of Legionella in clinical and environmental samples.

Our laboratory has developed a novel real-time polymerase chain reaction (PCR) assay for the detection of Legionella pneumophila and differentiation from other Legionella spp. in clinical and environmental samples. The 23S rRNA gene was used as a target to detect all Legionella spp., and the mip gene was targeted for the specific detection of L. pneumophila in this multiplex Taqman real-time PCR assay. The 23S rRNA gene is a novel target for Legionella testing; it detects all species and serogroups of Legionella without the contamination issues that accompany the use of the 16S rRNA gene as a target. This assay provides an analytical sensitivity of <1 colony-forming unit and a specificity of 100%. Because culture is important and provides a means for molecular typing via pulsed-field gel electrophoresis (PFGE), we developed a testing algorithm that includes both the new real-time PCR assay and culture for clinical and environmental samples and applied this algorithm during a period of 3 years. Of the 64 clinical samples received by our laboratory for Legionella testing during this period, PCR was found to be an essential diagnostic tool because only 13.3% (2/15) clinical samples that were determined to be L. pneumophila were detected by culture during this period. Of the 276 environmental samples received for Legionella testing during this period, 140 were found to be positive for L. pneumophila. Of these 140 samples, 69.3% were detected by both PCR and culture methods, 29.3% were positive by PCR alone, and 1.4% were positive by culture methods alone. We feel these results indicate that our algorithm, including both PCR and culture, should be used for environmental samples. Among both the clinical and environmental Legionella samples identified by PCR, a subset was not suitable for culture because of issues of lengthy transport, antimicrobial treatment, or bacterial overgrowth. Samples like these are commonly submitted to our laboratory, so the use of our testing algorithm combining these methods is critical. We conclude that molecular and culture methods must be used in combination to provide the best and most comprehensive approach to laboratory detection and investigation of legionellosis.

A multistate outbreak of Serratia marcescens bloodstream infection associated with contaminated intravenous magnesium sulfate from a compounding pharmacy.

BACKGROUND: In contrast to pharmaceutical manufacturers, compounding pharmacies adhere to different quality-control standards, which may increase the likelihood of undetected outbreaks. In 2005, the Centers for Disease Control and Prevention received reports of cases of Serratia marcescens bloodstream infection occurring in patients who underwent cardiac surgical procedures in Los Angeles, California, and in New Jersey. An investigation was initiated to determine whether there was a common underlying cause. METHODS: A matched case-control study was conducted in Los Angeles. Case record review and environmental testing were conducted in New Jersey. The Centers for Disease Control and Prevention performed a multistate case-finding investigation; isolates were compared using pulsed-field gel electrophoresis analysis. RESULTS: Nationally distributed magnesium sulfate solution (MgSO(4)) from compounding pharmacy X was the only significant risk factor for S. marcescens bloodstream infection (odds ratio, 6.4; 95% confidence interval, 1.1-38.3) among 6 Los Angeles case patients and 18 control subjects. Five New Jersey case patients received MgSO(4) from a single lot produced by compounding pharmacy X; culture of samples from open and unopened 50-mL bags in this lot yielded S. marcescens. Seven additional case patients from 3 different states were identified. Isolates from all 18 case patients and from samples of MgSO(4) demonstrated indistinguishable pulsed-field gel electrophoresis patterns. Compounding pharmacy X voluntarily recalled the product. Neither the pharmacy nor the US Food and Drug Administration could identify a source of contamination in their investigations of compounding pharmacy X. CONCLUSIONS: A multistate outbreak of S. marcescens bloodstream infection was linked to contaminated MgSO(4) distributed nationally by a compounding pharmacy. Health care personnel should take into account the different quality standards and regulation of compounded parenteral medications distributed in large quantities during investigations of outbreaks of bloodstream infection.

A large, multiple-restaurant outbreak of infection with Shigella flexneri serotype 2a traced to tomatoes.

BACKGROUND: Foodborne outbreaks of Shigella infection are uncommon and tomatoes are an unusual vehicle. We describe a large, multiple-restaurant outbreak of Shigella flexneri serotype 2a infection that was associated with tomatoes. METHODS: We conducted nationwide surveillance and a case-control study, collected fecal specimens for culture, and measured the survival of the outbreak strain of S. flexneri in tomatoes. RESULTS: We interviewed 306 of 886 ill restaurant patrons and 167 control subjects. Matched univariate analysis showed that several food items were associated with illness, but only tomatoes remained significant in multivariate models. Illness peaked at each restaurant within 24 h after the arrival of hand-sorted bruised and overripe tomatoes from a new distributor; all patient isolates that were tested were indistinguishable by PFGE. Sliced tomatoes from the distributor were inoculated with the outbreak strain, and viable S. flexneri were recovered for 72 h. CONCLUSION: To prevent such outbreaks, persons with shigellosis should be excluded from handling food at all points along the distribution chain.

Multistate outbreak of Listeria monocytogenes infection linked to delicatessen turkey meat.

BACKGROUND: Despite a decreasing incidence of listeriosis in the United States, molecular subtyping has increased the number of recognized outbreaks. In September 2000, the New York City Department of Health identified a cluster of infections caused by Listeria monocytogenes isolates with identical molecular subtypes by pulsed-field gel electrophoresis (PFGE) and ribotyping. METHODS: To determine the magnitude of the outbreak and identify risk factors for infection, we notified state health departments and conducted a case-control study. A case was defined as a patient or mother-infant pair infected with Listeria monocytogenes whose isolate yielded the outbreak PFGE pattern. Controls were patients infected with Listeria monocytogenes whose isolate yielded a different PFGE pattern. Patients were asked about food and drink consumed during the 30 days before the onset of illness. RESULTS: Between May and December 2000, there were 30 clinical isolates of Listeria monocytogenes with identical PFGE patterns identified in 11 US states. Cases of infection caused by these isolates were associated with 4 deaths and 3 miscarriages. A case-control study implicated sliced processed turkey from a delicatessen (Mantel-Haenszel odds ratio, 8.0; 95% confidence interval, 1.2-43.3). A traceback investigation identified a single processing plant as the likely source of the outbreak, and the company voluntarily recalled 16 million pounds of processed meat. The same plant had been identified in a Listeria contamination event that had occurred more than a decade previously. CONCLUSIONS: Prevention of persistent L. monocytogenes contamination in food processing plants presents a critical challenge to food safety professionals.

Detection, isolation, and molecular subtyping of Escherichia coli O157:H7 and Campylobacter jejuni associated with a large waterborne outbreak.

The largest reported outbreak of waterborne Escherichia coli O157:H7 in the United States occurred in upstate New York following a county fair in August 1999. Culture methods were used to isolate E. coli O157:H7 from specimens from 128 of 775 patients with suspected infections. Campylobacter jejuni was also isolated from stools of 44 persons who developed diarrheal illness after attending this fair. There was one case of a confirmed coinfection with E. coli O157:H7 and C. jejuni. Molecular detection of stx(1) and stx(2) Shiga toxin genes, immunomagnetic separation (IMS), and selective culture enrichment were utilized to detect and isolate E. coli O157:H7 from an unchlorinated well and its distribution points, a dry well, and a nearby septic tank. PCR for stx(1) and stx(2) was shown to provide a useful screen for toxin-producing E. coli O157:H7, and IMS subculture improved recovery. Pulsed-field gel electrophoresis (PFGE) was used to compare patient and environmental E. coli O157:H7 isolates. Among patient isolates, 117 of 128 (91.5%) were type 1 or 1a (three or fewer bands different). Among the water distribution system isolates, 13 of 19 (68%) were type 1 or 1a. Additionally, PFGE of C. jejuni isolates revealed that 29 of 35 (83%) had indistinguishable PFGE patterns. The PFGE results implicated the water distribution system as the main source of the E. coli O157:H7 outbreak. This investigation demonstrates the potential for outbreaks involving more than one pathogen and the importance of analyzing isolates from multiple patients and environmental samples to develop a better understanding of bacterial transmission during an outbreak.

Issues associated with and recommendations for using PCR to detect outbreaks of pertussis.

Two outbreaks of respiratory tract illness associated with prolonged cough occurring in 1998 and 1999 in New York State were investigated. A PCR test for Bordetella pertussis was primarily used by a private laboratory to confirm 680 pertussis cases. Several clinical specimens had positive culture results for B. pertussis during both outbreaks, which confirmed that B. pertussis was circulating during the outbreaks. However, testing by the New York State Department of Health reference laboratory suggested that some of the PCR results may have been falsely positive. In addition, features of the outbreak that suggested that B. pertussis may not have been the primary agent of infection included a low attack rate among incompletely vaccinated children and a significant amount of illness among patients testing PCR negative for B. pertussis. These investigations highlight the importance of appropriate clinical laboratory quality assurance programs, of the limitations of the PCR test, and of interpreting laboratory results in context of clinical disease.