Genotyping of a homogeneous group of Yersinia pestis strains isolated in the United States

Yersinia pestis, the causative agent of deadly plague, is considered a reemerging infectious disease and a significant biological terrorism threat. The present project focused on epidemiological investigation of the genetic variability of well-documented strains of Y. pestis from the United States by pulsed-field gel electrophoresis (PFGE) and restriction fragment length polymorphism (RFLP) analysis with insertion sequences IS100 and IS285 as probes. We examined 37 U.S. Y. pestis strains and isolates of a single ribotype, ribotype B, recovered between 1939 and 1998 from patients, animals, and fleas. Our results showed that all isolates had similar PFGE patterns, but minor differences such as missing, additional, and shifted bands were found among almost all strains if they came from different parent strains. The 37 strains and isolates were divided into 26 PFGE types. RFLP analysis with IS100 as a probe divided these strains and isolates into 16 types, with 43% belonging to IS100 type 1. Typing with IS285 as a probe was less specific and led to only four RFLP types, with 81% belonging to type 1. Similarity analysis with BioNumerics software showed that all strains shared >or=80, 86, and 91% similarities on dendrograms prepared from digitized PFGE, IS100 RFLP analysis, and IS285 RFLP analysis images, respectively. Our results demonstrate that PFGE offers an increased ability to discriminate between strains (Simpson's index of diversity, 0.98) and therefore can significantly improve epidemiological studies related to the origin of new plague isolates.     

Characterisation of Yersinia pestis isolates from natural foci of plague in the Republic of Georgia, and their relationship to Y. pestis isolates from other countries

Forty Yersinia pestis isolates from endemic foci of plague in the Republic of Georgia, and six Y. pestis isolates from neighbouring former Soviet Union countries, were analysed for their biochemical and phenotypic properties, and their genetic relatedness was compared with Y. pestis strains KIM and CO92 by pulsed-field gel electrophoresis (PFGE). In addition, 11 Y. pestis isolates from the USA, together with published nucleotide sequences from Y. pestis strains KIM, CO92 and 91001, were compared with the 46 isolates in the present collection using multilocus sequence typing (MLST), based on sequence data for the 16S rRNA, hsp60, glnA, gyrB, recA, manB, thrA and tmk loci. Four virulence gene loci (caf1, lcrV, psaA and pla) were also sequenced and analysed. Two sequence types (ST1 and ST2), which differed by a single nucleotide, were identified by MLST. With the exception of a single isolate (771G), all of the Georgian Y. pestis isolates belonged to ST2. PFGE also grouped the Georgian Y. pestis isolates separately from the non-Georgian isolates. Overall, PFGE discriminated the Y. pestis isolates more effectively than MLST. The sequences of three of the four virulence genes (lcrV, psaA and pla) were identical in all Georgian and non-Georgian isolates, but the caf1 locus was represented by two allele types, with caf1 NT1 being associated with the non-Georgian isolates and caf1 NT2 being associated with the Georgian isolates. These results suggest that Georgian Y. pestis isolates are of clonal origin.     

The mechanism of interaction between Yersinia pestis and erythrocytes, and its importance for the pathogenesis of plague

The ability of Yersinia pestis to get inside human and murine red blood cells (RBC) was found both in vivo and in vitro experiments. Due to oxidase and catalase activities, the microorganisms induced the denaturation of hemoglobin (Hb) through RBC oxidation to H2O2 in high concentration providing the formation of haemin and transformation of haem Fe2+ into the utilizable form, Fe3+. This phenomenon was found to be common in vitro for all Y. pestis strains used in the study independently of Pgm phenotype and plasmid content, including vaccine Pgm(-) Y. pestis EV NIIEG and plasmidless Pgm(+) Y. pestis PKR-133 stains. This, probably, allows the bacteria to use Hb as an essential source of iron and porphyrins for de novo synthesis of DNA followed by effective multiplication in the mammalian organism. A correlation between the loss of the ability of RBC to transport O2 to organs and tissues and the development of progressive tissue hypoxia with specific clinical features of metHb accumulation and haemorrhagic syndrome was shown. The participation of Y. pestis phospholipases (A and D) in the destruction of RBC membranes and translocation of plague bacilli into RBC, as well as the phenomenon of polysaccharide chain lengthening depending on cultivation conditions of Y. pestis bacteria, are discussed.     

Role of the Yersinia pestis Ail protein in preventing a protective polymorphonuclear leukocyte response during bubonic plague

The ability of Yersinia pestis to forestall the mammalian innate immune response is a fundamental aspect of plague pathogenesis. In this study, we examined the effect of Ail, a 17-kDa outer membrane protein that protects Y. pestis against complement-mediated lysis, on bubonic plague pathogenesis in mice and rats. The Y. pestis ail mutant was attenuated for virulence in both rodent models. The attenuation was greater in rats than in mice, which correlates with the ability of normal rat serum, but not mouse serum, to kill ail-negative Y. pestis in vitro. Intradermal infection with the ail mutant resulted in an atypical, subacute form of bubonic plague associated with extensive recruitment of polymorphonuclear leukocytes (PMN or neutrophils) to the site of infection in the draining lymph node and the formation of large purulent abscesses that contained the bacteria. Systemic spread and mortality were greatly attenuated, however, and a productive adaptive immune response was generated after high-dose challenge, as evidenced by high serum antibody levels against Y. pestis F1 antigen. The Y. pestis Ail protein is an important bubonic plague virulence factor that inhibits the innate immune response, in particular the recruitment of a protective PMN response to the infected lymph node.     

Infections prevented by increasing HIV serostatus awareness in the United States, 2001 to 2004

BACKGROUND: Persons living with HIV (PLWH) who are aware of their HIV status are more likely than serostatus-unaware PLWH to take precautions to prevent HIV transmission to their partners. The estimates of the Centers for Disease Control and Prevention (CDC) indicate that the proportion of PLWH who were aware of their serostatus increased between 2001 and 2004. The epidemiologic consequences of this increase in serostatus awareness are unknown. METHODS: We developed a basic model of the US HIV epidemic from 2001 to 2004. Using this model, we calculated the number of incident infections that would have occurred in 2002 to 2004 had the proportion of PLWH who were aware of their serostatus remained at its 2001 level rather than increasing between 2001 and 2004. We then compared this incidence estimate with the CDC's estimated total of 120,000 incident infections in 2002 to 2004 to determine the number of infections prevented by the increase in serostatus awareness. RESULTS: The increase from 2001 to 2004 in the proportion of PLWH who were aware of their serostatus can be credited with preventing nearly 6000 incident HIV infections in the 3-year period from 2002 to 2004. Sensitivity analyses indicated a plausible range of 4000 to 8700 prevented infections. CONCLUSION: This analysis demonstrates the important epidemiologic benefits of increasing the proportion of PLWH who are aware of their HIV status.     

Quantities of Yersinia pestis in fleas (Siphonaptera: Pulicidae, Ceratophyllidae, and Hystrichopsyllidae) collected from areas of known or suspected plague activity

We used a quantitative competitive polymerase chain reaction (PCR) (QC-PCR) to determine bacterial loads in 669 fleas collected in areas of confirmed and suspected plague epizootics. Fleas were collected out of rodent burrows (67.9%) and off of captured animals (24.1%) and rodent carcasses (8.1%). An initial PCR screening assay indicated that 12.1% (81/669) of all fleas were positive for Yersinia pestis. Fleas collected from burrows had significantly lower (chi2 = 264.9, P < 0.0001) infection rates (6.8%) but significantly higher (Student t-test, P < 0.0001) bacterial loads (mean = 10(5.6) Y. pestis per flea) than fleas collected off of rodent carcasses (infection rate = 92.6%; mean bacterial load = 10(4.8) Y. pestis per flea). None of the fleas collected off of captured animals were positive for Y. pestis by PCR, although seven of the 176 captured animals were serologically positive for Y. pestis.     

Molecular Characterization of Methicillin-Susceptible Staphylococcus aureus Clinical Isolates in the United States, 2004 to 2010

While much is known about the geographic distribution of different clonal types of methicillin-resistant Staphylococcus aureus (MRSA), few studies have assessed the molecular epidemiology of methicillin-susceptible S. aureus (MSSA), despite its continued clinical importance. In each U.S. Census region, reference laboratories collected successive MSSA isolates from patients with invasive or superficial staphylococcal infections for use in the Tigecycline Evaluation and Surveillance Trial. All isolates from the periods of 2004 to 2005 and 2009 to 2010 underwent antimicrobial susceptibility testing and characterization of their staphylococcal protein A (spa) type. Of the 708 isolates analyzed, 274 spa types were identified and divided into 15 genetic clusters. The most common clones were spa t002 (n = 63, 8.9%) and t008 (n = 56, 7.9%). While the distribution of the predominant spa types did not differ by U.S. Census region or time period, spa t008 was nearly twice as common in community skin and soft tissue infections than in nosocomial bloodstream infections (11.1% versus 5.6%, respectively; P = 0.008). Despite such differences, both community and nosocomial settings had diverse staphylococcal clonal types representing all major spa clusters. In contrast to those of MRSA, MSSA infectious isolates show wide genetic diversity without clear geographical or temporal clustering. Notably, the prevalent MSSA strains (spa t002 and spa t008) are analogous to the predominant MRSA clones, further demonstrating the importance of both lineages.     

Regional models of genetic services in the United States

PurposeTo outline structures for regional genetic services support centers that improve access to clinical genetic services.MethodsA workgroup (WG) and advisory committee (AC) (1) conducted a comprehensive review of existing models for delivering health care through a regional infrastructure, especially for genetic conditions; (2) analyzed data from a needs assessment conducted by the National Coordinating Center (NCC) to determine important components of a regional genetic services support center; and (3) prioritized components of a regional genetic services support system.ResultsAnalysis of identified priorities and existing regional systems led to development of eight models for regional genetic services support centers. A hybrid model was recommended that included an active role for patients and families, national data development and collection, promotion of efficient and quality genetic clinical practices, healthcare professional support for nongeneticists, and technical assistance to healthcare professionals.ConclusionGiven the challenges in improving access to genetic services, especially for underserved populations, regional models for genetic services support centers offer an opportunity to improve access to genetic services to local populations. Although a regional model can facilitate access, some systemic issues exist—e.g., distribution of a workforce trained in genetics—that regional genetic services support centers cannot resolve.     

Use of DNA hybridizations probes for detection of the plague bacillus (Yersinia pestis) in fleas (Siphonaptera: Pulicidae and Ceratophyllidae)

The detection of active plaque in nature relies primarily on demonstration of the etiologic agent of the disease. Yersinia pestis, in the flea vectors and susceptible mammalian hosts. A live animal assay is currently used for identification of a Y. pestis virulence antigen that is not expressed in the flea. We have found that DNA hybridization probes specific for Y. pestis, used in very simple sample preparation schemes, allow detection of Y. pestis in three species of fleas as well as tissues of experimentally infected mice at minimum concentrations of 1 x 10(6) bacilli/ml. We detected Y. pestis in 22 of 90 (24%) experimentally infected Xenopsylla cheopis (Rothschild), 13 of 25 (52%) Thrassis bacchi (Rothschild), and 9 of 25 (36%) Diamanus montanus (Baker), but no hybridization signals were observed from fleas that had fed on normal mice. The probe technique indicated infection in 9 of 10 potentially infected liver and spleen samples and none of the 5 control samples. Our techniques permit definitive diagnosis in 48 h.     

HIV incidence in the United States, 1978-1999

CONTEXT: HIV incidence measurements, which reflect recent or current transmission, are valuable for monitoring the epidemic and evaluating prevention programs. OBJECTIVES: To summarize HIV incidence patterns and trends in U.S. population groups. DATA SOURCES: Publications in English from 1980 through mid-2000. STUDY SELECTION AND STATISTICAL METHODS: We searched the literature for reports of HIV incidence in the United States. Locally weighted scatterplot smoothing was used to generate smooth curves to estimate trends in incidence. Spearman rank correlation was used to estimate the correlation coefficient between prevalence and incidence. DATA SYNTHESIS: In 74 eligible reports, HIV incidence varied widely (0.002-19.8 per 100 person-years [py]) depending on risk group. Among men who have sex with men (MSM), HIV incidence peaked in the early 1980s (5-20/100 py) and then declined but remained high during the 1990s (2-4/100 py). Among injection drug users (IDUs), incidence decreased since the mid-1980s but differed by geographic area; in the 1990s, incidence remained high in the East (1-3/100 py) but was lower in the West (<0.5/100 py). Throughout the late 1980s and 1990s, incidence was low and stable in broader populations (blood donors: <0.01/100 py; military personnel: 0.01-0.07/100 py). The correlation between HIV incidence and prevalence was strong in populations with a prevalence less than 1% (r = 0.94, p<.0001), moderate in populations with a prevalence from 1% to less than 10% (r = 0.57, p<.0001), and weak in populations with a prevalence at least 10% (r = 0.23, p=.09). CONCLUSIONS: HIV prevention in the United States should continue to focus on MSM and IDUs. HIV incidence measurements should be considered for monitoring HIV transmission in MSM, IDUs, and other populations in which seroprevalence is high.     

Board-certified physicians in the United States, 1971-1986

BACKGROUND. This is our third report covering the census of U.S. physicians over a 15-year period. The present report updates the information for 1980 to 1986. METHODS. Most of our data are based on published information from the Association of American Medical Colleges, the Educational Council for Foreign Medical Graduates, the American Board of Medical Specialties, and the National Resident Matching Program. Data on board-certified physicians were obtained from the Division of Survey and Data Resources of the American Medical Association and are not published elsewhere. RESULTS. After a steep rise in the 1970s, the annual number of physicians receiving licenses increased at a slower rate. The numbers of new board diplomas in medicine and primary care continued to grow. In other non-surgical clinical specialties there was less growth, and in certain fields of surgery the numbers declined. The board-certified percentage of all practitioners increased slightly (74 to 79 percent). About 14 to 16 percent of all active physicians are still in their residency and fellowship years. The percentage of all practitioners under the age of 35 who are women has increased from 8.4 percent in 1967 to 25.2 percent in 1986. The enrollment of some residency programs is currently more than 50 percent women. CONCLUSIONS. The work force of physicians did not grow as rapidly in the 1980s as in the 1970s. This nonlinearity of growth and massive changes in the epidemiology and treatment of disease render predictions about the need for or the numbers of physicians a decade hence unreliable.     

A non-invasive in vivo imaging system to study dissemination of bioluminescent Yersinia pestis CO92 in a mouse model of pneumonic plague

The gold standard in microbiology for monitoring bacterial dissemination in infected animals has always been viable plate counts. This method, despite being quantitative, requires sacrificing the infected animals. Recently, however, an alternative method of in vivo imaging of bioluminescent bacteria (IVIBB) for monitoring microbial dissemination within the host has been employed. Yersinia pestis is a Gram-negative bacterium capable of causing bubonic, septicemic, and pneumonic plague. In this study, we compared the conventional counting of bacterial colony forming units (cfu) in the various infected tissues to IVIBB in monitoring Y. pestis dissemination in a mouse model of pneumonic plague. By using a transposon mutagenesis system harboring the luciferase (luc) gene, we screened approximately 4000 clones and obtained a fully virulent, luc-positive Y. pestis CO92 (Y. pestis-luc2) reporter strain in which transposition occurred within the largest pMT1 plasmid which possesses murine toxin and capsular antigen encoding genes. The aforementioned reporter strain and the wild-type CO92 exhibited similar growth curves, formed capsule based on immunofluorescence microscopy and flow cytometry, and had a similar LD₅₀. Intranasal infection of mice with 15 LD₅₀ of CO92-luc2 resulted in animal mortality by 72 h, and an increasing number of bioluminescent bacteria were observed in various mouse organs over a 24–72 h period when whole animals were imaged. However, following levofloxacin treatment (10 mg/kg/day) for 6 days 24 h post infection, no luminescence was observed after 72 h of infection, indicating that the tested antimicrobial killed bacteria preventing their detection in host peripheral tissues. Overall, we demonstrated that IVIBB is an effective and non-invasive way of monitoring bacterial dissemination in animals following pneumonic plague having strong correlation with cfu, and our reporter CO92-luc2 strain can be employed as a useful tool to monitor the efficacy of antimicrobial countermeasures in real time.