Development of real-time PCR assays for specific detection of hmsH, hmsF, hmsR, and irp2 located within the 102-kb pgm locus of Yersinia pestis.

Virulent isolates of three pathogenic Yersinia species (Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica) harbor a 102-kb chromosomal region which encodes elements critical for virulence. A 35-kb high pathogenicity island is contained in this region, is a known virulence determinant, contains irp1 and irp2 iron-regulating genes. An additional segment, the 68-kb high pathogenicity island, contains genetic elements responsible for conferring the Y. pestis pigmentation phenotype on Congo red agar at 28 °C. Collectively, these contiguous segments are referred to as the pigmentation (pgm) locus, the absence of which results in strain attenuation and exemption from CDC Select Agent status. In this study, we developed a set of four real-time PCR assays to detect the presence or absence of multiple virulence genes located within this region. Specifically, we designed TaqMan(®) PCR assays to individually detect three hemin storage genes (hmsH, hmsF, and hmsR) which are genetic elements that confer the pigmentation phenotype, as well as the iron-regulating status of 25 Y. pestis isolates (representing 23 different strains), thus establishing a molecular based assay capable of determining the pgm status of candidate Y. pestis isolates. Included in the validation process, was a comparison of these real-time PCR assays and newly developed conventional PCR assays targeting much larger areas of the 102-kb region (including one assay spanning hmsR and hmsF, one spanning hmsH and hsmF, one targeting hmsF, and one targeting irp2). There was high concordance between the conventional and real-time PCR assays for all Y. pestis strains tested. The results from the comparative analysis document the specificity and sensitivity of the real-time PCR assays and further solidify the ostensible benefits of real-time PCR over conventional PCR.

Regulation of the subcellular distribution of key cellular RNA-processing factors during permissive human cytomegalovirus infection.

Alternative splicing and polyadenylation of human cytomegalovirus (HCMV) immediate-early (IE) pre-mRNAs are temporally regulated and rely on cellular RNA-processing factors. This study examined the location and abundance of essential RNA-processing factors, which affect alternative processing of UL37 IE pre-mRNAs, during HCMV infection. Serine/threonine protein kinase 1 (SRPK1) phosphorylates serine/arginine-rich proteins, necessary for pre-spliceosome commitment. It was found that HCMV infection progressively increased the abundance of cytoplasmic SRPK1, which is regulated by subcellular partitioning. The essential polyadenylation factor CstF-64 was similarly increased in abundance, albeit in the nucleus, proximal to and within viral replication compartments (VRCs). In contrast, the location of polypyrimidine tract-binding protein (PTB), known to adversely affect splicing of HCMV major IE RNAs, was temporally regulated during infection. PTB co-localized with CstF-64 in the nucleus at IE times. By early times, PTB was detected in punctate cytoplasmic sites of some infected cells. At late times, PTB relocalized to the nucleus, where it was notably excluded from HCMV VRCs. Moreover, HCMV infection induced the formation of nucleolar stress structures, fibrillarin-containing caps, in close proximity to its VRCs. PTB exclusion from HCMV VRCs required HCMV DNA synthesis and/or late gene expression, whereas the regulation of SRPK1 subcellular distribution did not. Taken together, these results indicated that HCMV increasingly regulates the subcellular distribution and abundance of essential RNA-processing factors, thereby altering their ability to affect the processing of viral pre-mRNAs. These results further suggest that HCMV infection selectively induces sorting of nucleolar and nucleoplasmic components.

Impact of Plasmodium falciparum infection on haematological parameters in children living in Western Kenya.

BACKGROUND: Malaria is the commonest cause of childhood morbidity in Western Kenya with varied haematological consequences. The t study sought to elucidate the haematological changes in children infected with malaria and their impact on improved diagnosis and therapy of childhood malaria. METHODS: Haematological parameters in 961 children, including 523 malaria-infected and 438 non-malaria infected, living in Kisumu West District, an area of malaria holoendemic transmission in Western Kenya were evaluated. RESULTS: The following parameters were significantly lower in malaria-infected children; platelets, lymphocytes, eosinophils, red blood cell count and haemoglobin (Hb), while absolute monocyte and neutrophil counts, and mean platelet volume (MPV) were higher in comparison to non-malaria infected children. Children with platelet counts of <150,000/uL were 13.8 times (odds ratio) more likely to have malaria. Thrombocytopaenia was present in 49% of malaria-infected children and was associated with high parasitaemia levels, lower age, low Hb levels, increased MPV and platelet aggregate flag. Platelet aggregates were more frequent in malaria-infected children (25% vs. 4%, p<0.0001) and associated with thrombocytopaenia rather than malaria status. CONCLUSION: Children infected with Plasmodium falciparum malaria exhibited important changes in some haematological parameters with low platelet count and haemoglobin concentration being the two most important predictors of malaria infection in children in our study area. When used in combination with other clinical and microscopy, these parameters could improve malaria diagnosis in sub-patent cases.

An outbreak of multidrug-resistant Acinetobacter baumannii-calcoaceticus complex infection in the US military health care system associated with military operations in Iraq.

BACKGROUND: We investigated an outbreak of multidrug-resistant Acinetobacter baumannii-calcoaceticus complex infection among US service members injured in Iraq. METHODS: The investigation was conducted in Iraq and Kuwait, in the 2 military hospitals where the majority of injured service members were initially treated. After initially characterizing the outbreak, we evaluated 3 potential sources of infection for the period March 2003 to December 2004. The evaluation included screening samples that were obtained from the skin of patients for the presence of colonization and assessing the soil and health care environments for the presence of A. baumanii-calcoaceticus complex organisms. Isolates obtained from samples from patients in US Military treatment facilities, as well as environmental isolates, were genotypically characterized and compared using pulsed-field gel electrophoresis. RESULTS: A. baumanii-calcoaceticus complex organisms were present on the skin in only 1 (0.6%) of 160 patients who were screened and in 1 (2%) of 49 soil samples. A. baumanii-calcoaceticus complex isolates were recovered from treatment areas in 7 of the 7 field hospitals sampled. Using pulsed-field gel electrophoresis, we identified 5 cluster groups in which isolates from patients were related to environmental isolates. One cluster included hospitalized patients who had not been deployed to Iraq. Among the clinical isolates, only imipenem, polymyxin B, and colistin demonstrated reliable in vitro antimicrobial activity. Generally, the environmental isolates were more drug susceptible than were the clinical isolates. CONCLUSIONS: Our findings suggest that environmental contamination of field hospitals and infection transmission within health care facilities played a major role in this outbreak. On the basis of these findings, maintaining infection control throughout the military health care system is essential. Novel strategies may be required to prevent the transmission of pathogens in combat field hospitals.

Identification of Acinetobacter species and genotyping of Acinetobacter baumannii by multilocus PCR and mass spectrometry.

Members of the genus Acinetobacter are ubiquitous in soil and water and are an important cause of nosocomial infections. A rapid method is needed to genotype Acinetobacter isolates to determine epidemiology and clonality during infectious outbreaks. Multilocus PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) is a method that uses the amplicon base compositions to genotype bacterial species. In order to identify regions of the Acinetobacter genome useful for this method, we sequenced regions of six housekeeping genes (trpE, adk, efp, mutY, fumC, and ppa) from 267 isolates of Acinetobacter. Isolates were collected from infected and colonized soldiers and civilians involved in an outbreak in the military health care system associated with the conflict in Iraq, from previously characterized outbreaks in European hospitals, and from culture collections. Most of the isolates from the Iraqi conflict were Acinetobacter baumannii (189 of 216 isolates). Among these, 111 isolates had genotypes identical or very similar to those associated with well-characterized A. baumannii isolates from European hospitals. Twenty-seven isolates from the conflict were found to have genotypes representing different Acinetobacter species, including 8 representatives of Acinetobacter genomospecies 13TU and 13 representatives of Acinetobacter genomospecies 3. Analysis by the PCR/ESI-MS method using nine primer pairs targeting the most information-rich regions of the trpE, adk, mutY, fumC, and ppa genes distinguished 47 of the 48 A. baumannii genotypes identified by sequencing and identified at the species level at least 18 Acinetobacter species. Results obtained with our genotyping method were essentially in agreement with those obtained by pulse-field gel electrophoresis analysis. The PCR/ESI-MS genotyping method required 4 h of analysis time to first answer with additional samples subsequently analyzed every 10 min. This rapid analysis allows tracking of transmission for the implementation of appropriate infection control measures on a time scale previously not achievable.

Comparison of 10 different hemostatic dressings in an aortic injury.

BACKGROUND: Uncontrolled hemorrhage is the leading preventable cause of death on the battlefield. Similarly, hemorrhage accounts for 80% of all deaths within the first 48 hours of injury in civilian trauma patients. New methods of hemostasis are required to reduce hemorrhagic mortality. The purpose of this study was to compare nine hemostatic dressings for their efficacy in controlling bleeding from an otherwise fatal aortic injury in a pig model. Each hemostatic dressing was compared with the current standard U.S. Army field gauze dressing for a 1-hour period. METHODS: Fifty-nine anesthetized pigs were instrumented with catheters and splenectomized. Nine test dressings (n = 5 per group) and two control groups (gauze, n = 9; suture, n = 5) were applied to a 4.4-mm aortotomy through the spraying jet of blood, and direct pressure was held for 4 minutes and then released. Survival, blood loss, and other variables were measured over a 1-hour period. RESULTS: All animals with fibrin dressing and those receiving suture repair (five of five in both groups) survived the 1-hour observation period with minimal bleeding in the postocclusion period (< 37 mL). Those in the other dressing groups exsanguinated within 10 minutes, except for two animals in the gauze group surviving 1 hour. CONCLUSION: With one 4-minute application, a single fibrin dressing stopped bleeding from an aortotomy, which was equivalent to sutured repair. No other test group exhibited any evidence of significant hemostatic efficacy.