Multiplex real-time PCR for detection of Campylobacter, Salmonella, and Shigella.

Infectious diarrhea can be classified based on its clinical presentation as noninflammatory or inflammatory disease. In developing countries, among inflammatory diarrhea cases, Shigella is the most common cause, followed by Campylobacter and Salmonella. Because the time frame in which treatment choices must be made is short and conventional stool cultures lack good sensitivity, there is a need for a rapid, sensitive, and inexpensive detection technique. The purpose of our study was to develop a multiplex real-time PCR procedure to simultaneously identify Campylobacter spp., Salmonella spp., and Shigella spp. Primers were designed to amplify the invA, ipaH, and 16S rRNA genes simultaneously in a single reaction to detect Salmonella, Shigella, and Campylobacter, respectively. Using this approach, we correctly identified 102 of 103 strains of the targeted enteropathogens and 34 of 34 other pathogens. The melting temperatures were 82.96 ± 0.05 °C for invA, 85.56 ± 0.28 °C for ipaH, and 89.21 ± 0.24 °C for 16S rRNA. The limit of accurate quantification for the assay in stool samples was 10(4) CFU g(-1); however, the limit of detection was 10(3) CFU g(-1). This assay is a simple, rapid, inexpensive, and reliable system for the practical detection of these three enteropathogens in clinical specimens.

Genetic diversity of locus of enterocyte effacement genes of enteropathogenic Escherichia coli isolated from Peruvian children.

The aim of this study was to determine the frequency and allele associations of locus of enterocyte effacement encoded esp and tir genes among 181 enteropathogenic Escherichia coli (EPEC) strains (90 diarrhoea-associated and 91 controls) isolated from Peruvian children under 18 months of age. We analysed espA, espB, espD and tir alleles by PCR-RFLP. EPEC strains were isolated with higher frequency from healthy controls (91/424, 21.7%) than from diarrhoeal samples (90/936, 9.6%) (P<0.001); 28.9% of diarrhoeal and 17.6% of control samples were typical EPEC (tEPEC). The distribution of espA alleles (alpha, beta, beta2 and gamma) and espD alleles (alpha, beta, gamma and a new variant, espD-N1) between tEPEC and atypical EPEC (aEPEC) was significantly different (P<0.05). espD-alpha was more common among acute episodes (P<0.05). espB typing resulted in five alleles (alpha, beta, gamma and two new sub-alleles, espB-alpha2 and espB-alpha3), while tir-beta and tir-gamma2 were the most common intimin receptor subtypes. Seventy-two combinations of espA, espB, espD and tir alleles were found; the most prevalent combination was espA-beta, espB-beta, espD-beta, tir-beta (34/181 strains), which was more frequent among tEPEC strains (P<0.05). Our findings indicate that there is a high degree of heterogeneity among EPEC strains isolated from Peruvian children and that aEPEC and tEPEC variants cluster.

Clinical and radiological features of respiratory syncytial virus in solid organ transplant recipients: a single-center experience.

BACKGROUND: Respiratory syncytial virus (RSV) infections range from upper respiratory illness to severe lower respiratory disease. There is no universally accepted treatment for RSV in solid organ transplant (SOT) recipients. METHODS: Retrospective review of adult SOT patients with RSV infections, between January 2007 and December 2009, in a single transplant center was performed. RESULTS: During the 3-year period, a total of 24 adults developed RSV infection, including 12 (50%) SOT recipients (5 kidneys, 4 livers, and 3 lungs). Most cases were seen in 2009 during the influenza H1N1 pandemic, likely as a result of increased testing. In 83% of the cases, the diagnosis was based on RSV antigen detection, which was also used to follow subsequent shedding (mean duration: 20.6 days). Most of the cases presented with lower respiratory disease and required hospitalization. All the patients were on at least two classes of immunosuppressive drugs. We observed a lower lymphocyte count in patients with lower respiratory tract infection. Computed tomography was superior to chest x-ray in demonstrating pulmonary disease, with the most common findings being pulmonary nodules and ground-glass opacities. Novel radiographic findings were small cavities and pleural effusions. No co-infections were documented, and no mortality could be attributed to RSV. Inhaled or oral ribavirin was administered in 67% of the cases, with variations in the treatment regimens. CONCLUSION: SOT recipients accounted for half of all adult cases of RSV at our institution. Type and length of treatment varied widely, and we cannot conclude that outcomes differed between treatments with oral or inhaled ribavirin. Current therapeutic management of RSV in SOT is empiric, and can be rather expensive and difficult, without clear evidence of effectiveness.

Phylogenetic relationships of Shiga toxin-producing Escherichia coli isolated from Peruvian children.

The aim of this study was to determine the prevalence, virulence factors (stx, eae, ehxA and astA) and phylogenetic relationships [PFGE and multilocus sequence typing (MLST)] of Shiga toxin-producing Escherichia coli (STEC) strains isolated from four previous cohort studies in 2212 Peruvian children aged <36 months. STEC prevalence was 0.4 % (14/3219) in diarrhoeal and 0.6 % (15/2695) in control samples. None of the infected children developed haemolytic uraemic syndrome (HUS) or other complications of STEC. stx1 was present in 83 % of strains, stx2 in 17 %, eae in 72 %, ehxA in 59 % and astA in 14 %. The most common serotype was O26 : H11 (14 %) and the most common seropathotype was B (45 %). The strains belonged mainly to phylogenetic group B1 (52 %). The distinct combinations of alleles across the seven MLST loci were used to define 13 sequence types among 19 STEC strains. PFGE typing of 20 STEC strains resulted in 19 pulsed-field patterns. Comparison of the patterns revealed 11 clusters (I-XI), each usually including strains belonging to different serotypes; one exception was cluster VI, which gathered exclusively seven strains of seropathotype B, clonal group enterohaemorrhagic E. coli (EHEC) 2 and phylogenetic group B1. In summary, STEC prevalence was low in Peruvian children with diarrhoea in the community setting. The strains were phylogenetically diverse and associated with mild infections. However, additional studies are needed in children with bloody diarrhoea and HUS.

Allelic variability of critical virulence genes (eae, bfpA and perA) in typical and atypical enteropathogenic Escherichia coli in Peruvian children.

Enteropathogenic Escherichia coli (EPEC) is a leading cause of infantile diarrhoea in developing countries. The aim of this study was to describe the allelic diversity of critical EPEC virulence genes and their association with clinical characteristics. One hundred and twenty EPEC strains isolated from a cohort diarrhoea study in Peruvian children were characterized for the allele type of eae (intimin), bfpA (bundlin pilin protein of bundle-forming pilus) and perA (plasmid encoded regulator) genes by PCR-RFLP. Atypical EPEC strains (eae+, bfp-) were the most common pathotype in diarrhoea (54/74, 73 %) and control samples from children without diarrhoea (40/46, 87 %). Overall, there were 13 eae alleles; the most common were beta (34/120, 28 %), theta (24/120, 20 %), kappa (14/120, 12 %) and mu (8/120, 7 %). There were five bfpA alleles; the most common were beta1/7 (10/26), alpha3 (7/26) and beta5 (3/26). There were three perA alleles: beta (8/16), alpha (7/16) and gamma (1/16). The strains belonged to 36 distinct serogroups; O55 was the most frequent. The gamma-intimin allele was more frequently found in diarrhoea episodes of longer duration (>7 days) than those of shorter duration (3/26, 12 % vs 0/48, 0 %, P<0.05). The kappa-intimin allele had the highest clinical severity score in comparison with other alleles (P<0.05). In Peruvian children, the virulence genes of EPEC strains are highly variable. Further studies are needed to evaluate additional virulence markers to determine whether relationships exist between specific variants and clinical features of disease.

Mycobacterium abscessus infection in solid organ transplant recipients: report of three cases and review of the literature.

Mycobacterium abscessus is an ubiquitous organism found in the environment. This rapidly growing mycobacterium infrequently causes disease in humans; however, in immunocompromised hosts, disease can range from localized cutaneous lesions to disseminated infection. The organism is resistant to most antimycobacterial drugs and therapy can be limited by drug interactions. The exact incidence of M. abscessus infection among solid organ transplant (SOT) recipients is unknown; data are only available from previously reported cases in the literature. We describe 3 cases of M. abscessus infection in SOT recipients diagnosed within a 5-month period. One of the cases followed multi-visceral transplantation, the first such case to be reported in the literature. An epidemiological investigation did not reveal significant commonalities among the cases, and pulsed-field gel electrophoresis of genomic DNA of the case isolates confirmed their non-identity. All cases improved with antibiotic therapy, most notably with the new glycylcycline, tigecycline, along with surgical intervention in 2 of the cases. In addition, we review features and characteristics of M. abscessus infections in recipients of SOT reported in the literature from 1992 to 2008 and summarize some selected therapeutic concerns and issues related to treatment.

Validation of five-colony pool analysis using multiplex real-time PCR for detection of diarrheagenic Escherichia coli.

Five Escherichia coli colonies/patient were studied to evaluate the reliability of a multiplex real-time PCR assay for detection of diarrheagenic Escherichia coli groups, using a pool of five colonies rather than individual colonies. Sensitivity and specificity were 98% and 100%, respectively, at a fifth of the cost of the individual colony analysis.

A case of subcutaneous Mycoleptodiscus indicus infection in a liver transplant recipient successfully treated with antifungal therapy.

Mycoleptodiscus indicus, a dematiaceous mold, occurs on the leaves of a number of different host plants and has been only recently described as a cause of human infection. Immunosuppressed individuals are at risk for developing infections with opportunistic fungal pathogens, which are a major cause of morbidity and mortality in this population. In addition, the treatment of infections caused by these fungi is frequently challenging. We report a case of M. indicus subcutaneous infection in a 51-year-old man with human immunodeficiency virus and hepatitis C co-infection, who had a liver transplant. He developed skin nodules with a sporotrichoid lymphangitic distribution. Histopathology demonstrated unusual fungal elements with angioinvasion. Mycology cultures isolated a dematiaceous mold with the characteristic curved hyaline conidia of M. indicus. Initial treatment involved a combination of amphotericin B lipid complex and voriconazole, followed by monotherapy with voriconazole. The subcutaneous lesions resolved completely after 4 months of antifungal therapy.

Antimicrobial activity of UMFix tissue fixative.

AIMS: The aim of this study was to determine the antimicrobial effects of UMFix, an alcohol based tissue fixative, on various microorganisms. The UMFix solution was compared with 10% neutral buffered formalin. METHODS: Standard methods to determine microorganism colony counts were performed after exposure of the microorganisms to UMFix and 10% neutral buffered formalin. RESULTS: After a short exposure, UMFix rapidly killed vegetative bacteria, yeasts, moulds, and viruses. Bacterial spores were resistant to killing by UMFix. All organisms were killed by the 10% neutral buffered formalin preparation. CONCLUSIONS: UMFix was microbicidal for vegetative bacteria, yeasts, and aspergillus species after a short exposure, although it was not active against spore forming bacillus species. The methanol content of the fixative was responsible for the killing effect of this fixative. No killing was seen when polyethylene glycol was used alone.

Multidrug-resistant tuberculous meningitis in patients with AIDS.

We present clinical manifestations, bacteriologic characteristics, and outcomes for eight patients with multidrug-resistant (MDR) tuberculous meningitis and AIDS. All developed meningitis as a terminal complication of previously diagnosed MDR-TB despite anti-tuberculosis therapy. Seven patients presented with fever, five with headache, four with altered mentation, two with focal deficits and one with seizures. CSF examination revealed pleocytosis, hypoglychorrhachia and elevated protein. Mycobacterium tuberculosis resistant to at least isoniazid and rifampin was isolated from all patients. Intracerebral mass lesions were detected in three patients, hydrocephalus in three, meningeal enhancement in five, and infarcts in two. Seven patients died 1-16 weeks after the diagnosis of meningitis; the eighth was lost to follow-up. MDR tuberculous meningitis is a difficult-to-treat infection with a high fatality rate.

Cerebral phaeohyphomycosis due to a novel species: report of a case and review of the literature.

Cerebral phaeohyphomycosis is a rare disease caused by dematiaceous (darkly pigmented) fungi. Cladophialophora species are highly neurotropic, and Cladophialophora bantiana (synonym=Xylohypha bantiana or C. trichoides) is the most commonly identified agent. Most reported cases of cerebral phaeohyphomycosis have occurred in immunocompetent patients; however, some case reports and experimental data have suggested that cellular immune deficiency is a risk factor. We report a case of pulmonary and cerebral phaeohyphomycosis in a cardiac transplant patient due to a newly identified species of Cladophialophora. Optimal management includes both antifungal therapy and surgery.

Tumor necrosis factor alpha increases human cerebral endothelial cell Gb3 and sensitivity to Shiga toxin.

Hemolytic uremic syndrome (HUS) is associated with intestinal infection by enterohemorrhagic Escherichia coli strains that produce Shiga toxins. Globotriaosylceramide (Gb3) is the functional receptor for Shiga toxin, and tumor necrosis factor alpha (TNF-alpha) upregulates Gb3 in both human macrovascular umbilical vein endothelial cells and human microvascular brain endothelial cells. TNF-alpha treatment enhanced Shiga toxin binding and sensitivity to toxin. This upregulation was specific for Gb3 species containing normal fatty acids (NFA). Central nervous system (CNS) pathology in HUS could involve cytokine-stimulated elevation of endothelial NFA-Gb3 levels. Differential expression of Gb3 species may be a critical determinant of Shiga toxin toxicity and of CNS involvement in HUS.

Rapid and specific detection of the Mycobacterium tuberculosis complex using fluorogenic probes andreal-time PCR.

A rapid and sensitive strategy for the specific identification of Mycobacterium tuberculosis (TB) was designed and evaluated using crude mycobacterial lysates. The speed of real-time polymerase chain reaction (PCR) was combined with the sensitivity of fluorogenic probes to confirm the presence of mycobacteria as well as specifically identify the presence of members of the mycobacteria tuberculosis complex (MTC) in a single-tube assay. Oligonucleotides were designed to amplify the internal transcribed spacer (ITS) from several mycobacterial species. Specific fluorogenic probes were included in the PCR reaction for the identification of TB as well as Mycobacterium bovia and Mycobacterium africanum in bacterial lysates. The combination of TB-specific fluorogenic probes with real-time PCR formed an approach determined to be fast (less than 40 min), sensitive (less than 800 copies of DNA) and reliable for the specific detection of the MTC. Our data demonstrate the use of real-time PCR and fluorogenic probes in a rapid and sensitive assay to distinguish members of the MTC from other mycobacterial species.

Human milk lipids bind Shiga toxin.

Hemolytic uremic syndrome, a serious complication of Shiga toxin-associated diarrhea, is rare before 6 months of age. Immunologic and nonimmunologic factors present in human milk may partially explain this observation. In prior studies, we have demonstrated that human milk contains Gb3, the receptor for the B subunit of Shiga toxin, and also contains secretory IgA (sIgA) against the toxin. We therefore sought to determine the relative importance of milk glycolipid and toxin-specific sIgA in toxin binding. We studied two populations that differed in their frequency of exposure to Shiga toxin. Human milk samples obtained from healthy donors from Boston and Buenos Aires were separated by centrifugation into aqueous (antibody enriched) and cream (glycosphingolipid enriched) fractions. An emulsion of equal volumes of aqueous phase or cream layer of each sample and purified Shiga toxin was incubated, and the amount of free toxin present in each was determined by enzyme immunoassay. The cream layers bound 85%+/-2 (mean +/- SE) (Argentina milk samples) and 86%+/-1 (Boston milk samples) of Shiga toxin. In contrast, the soluble fraction in samples from Buenos Aires, a population expected to frequently have antibodies to Shiga toxin, bound more toxin (48%+/-2) than did this fraction in samples from Boston, an area where toxin exposure is infrequent (30%+/-3) (P < 0.0001). Toxin-binding lipids present in human milk are biologically active and may contribute to the putative protective effect of human milk. In a population frequently exposed to Shiga toxins (Argentina), protection may be due to both immune (sIgA), and nonimmune (lipid) factors present in human milk. In a population infrequently exposed to Shiga toxins, cream fraction-associated glycolipids represent the major toxin binding activity in human milk.

Protective role of human lactoferrin against invasion of Shigella flexneri M90T.

Lactoferrin is an iron-binding protein found in human mucosal secretions such as milk. A variety of functions have been ascribed to this protein, it appears to contribute to antimicrobial host defense. Still its overall physiological role remains to be defined. We sought to study the role of recombinant human lactoferrin (rhLf) in Shigella infection. Invasion of epithelial cells is essential to the development of bacillary dysentery. Shigella flexneri 5 M90T, a virulent strain, was evaluated in the classic HeLa cell invasion model, in immunoblots, and by transmission electron microscopy, immunofluorescence, and deconvolved microscopy Bacteria not exposed to rhLf were used as controls. We found that rhLf decreased significantly the invasiveness of S. flexneri 5 M90T in a HeLa cell model. The immunoblot data showed that invasion plasmid antigen B (IpaB) was released from the bacteria during incubation with rhLf. Lactoferrin treatment did not directly dissociate the complex of IpaB and IpaC (IpaBC) once the complex had been formed. Furthermore, ferric iron had no effect on release of IpaB. Electron microscopy of rhLf-treated bacteria suggested a reduction in vacuolization of the HeLa cell cytoplasm and decreased number of bacteria within HeLa cells. At 40,000 x magnification the few rhLf-treated Shigella that invaded exhibited a dense ring completely surrounding them. Immunofluorescence and deconvolved microscopy suggested that rhLf-treated bacteria were completely surrounded by a thick layer of actin. The fact that two cell surface functions (invasion and actin-mediated movement) were deranged suggests that rhLf disrupts the integrity of the bacterial outer membrane in which virulence proteins are anchored. The mechanism by which rhLf impairs Shigella invasiveness may be relevant to other enteropathogens that share similar virulence strategies.

Evaluation of the LiPA MYCOBACTERIA assay for identification of mycobacterial species from BACTEC 12B bottles.

The LiPA MYCOBACTERIA (Innogenetics NV, Ghent, Belgium) assay was used to identify mycobacterial isolates using culture fluid from positive BACTEC 12B bottles. The LiPA method involves reverse hybridization of a biotinylated mycobacterial PCR fragment, a 16 to 23S rRNA spacer region, to oligonucleotide probes arranged in lines on a membrane strip, with detection via biotin-streptavidin coupling by a colorimetric system. This system identifies Mycobacterium species and differentiates M. tuberculosis complex, M. avium-M. intracellulare complex, and the following mycobacterial species: M. avium, M. intracellulare, M. kansasii, M. chelonae group, M. gordonae, M. xenopi, and M. scrofulaceum. The mycobacteria were identified in the laboratory by a series of tests, including the Roche AMPLICOR Mycobacterium tuberculosis (MTB) test, the Gen-Probe ACCUPROBE, and a PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 65-kDa heat shock protein gene. The LiPA MYCOBACTERIA assay detected 60 mycobacterium isolates from 59 patients. There was complete agreement between LiPA and the laboratory identification tests for 26 M. tuberculosis complex, 9 M. avium, 3 M. intracellulare complex, 3 M. kansasii, 4 M. gordonae, and 5 M. chelonae group (all were M. abscessus) isolates. Three patient samples were LiPA positive for M. avium-M. intracellulare complex, and all were identified as M. intracellulare by the PCR-RFLP analysis. Seven additional mycobacterial species were LiPA positive for Mycobacterium spp. (six were M. fortuitum, and one was M. szulgai). The LiPA MYCOBACTERIA assay was easy to perform, and the interpretation of the positive bands was clear-cut. Following PCR amplification and gel electrophoresis, the LiPA assay was completed within 3 h.

Diarrheogenic Escherichia coli.

The diarrheogenic E coli are currently difficult to diagnose and treat. For physicians in the United States, they are primarily a concern in children returning from international travel. The exception to this generalization is STEC, which, because of the low inoculum, ease of transmission, and serious consequences, are important pathogens in the United States.

Steady state hemodynamic and energetic characterization of the Penn State/3M Health Care Total Artificial Heart.

Total Artificial Heart (TAH) development at Penn State University and 3M Health Care has progressed from design improvements and manufacturing documentation to in vitro and in vivo testing to characterize the system's hemodynamic response and energetic performance. The TAH system is completely implantable and intended for use as an alternative to transplantation. It includes a dual pusher plate pump and rollerscrew actuator, welded electronics and battery assembly, transcutaneous energy transmission system, telemetry, and a compliance chamber. In vitro testing was conducted on a Penn State mock circulatory loop with glycerol/water solution at body temperature. Tests were performed to characterize the preload and afterload response, left atrial pressure control, and power consumption. A sensitive preload response was demonstrated with left atrial pressure safely maintained at less than 15 mm Hg for flow rates up to 7.5 L/min. Variations in aortic pressure and pulmonary vascular resistance were found to have minimal effects on the preload sensitivity and left atrial pressure control. In vivo testing of the completely implanted system in its final configuration was carried out in two acute studies using implanted temperature sensors mounted on the electronics, motor, and energy transmission coil in contact with adjacent tissue. The mean temperature at the device-tissue interface was less than 4 degrees C above core temperature.

Shiga toxin 1 elicits diverse biologic responses in mesangial cells.

BACKGROUND: Shiga toxin 1 (Stx1) is a causative agent in hemolytic uremic syndrome (HUS). Its receptor, the glycosphingolipid globotriaosylceramide (Gb3), is expressed on cultured human endothelial and mesangial cells. Mesangial cell injury in HUS ranges from mild cellular edema to severe mesangiolysis and eventual glomerulosclerosis. We hypothesized that, in addition to endothelial cells, mesangial cells are targets of Stx1. METHODS: Human mesangial cells were exposed to Stx1. Protein synthesis was measured using [35S]-methionine/cysteine. Cell viability was measured as the lysosomal uptake of Neutral Red. Monocyte chemotactic peptide (MCP-1) mRNA and protein were analyzed by Northern blotting and ELISA. RESULTS: Stx1 (0.25 to 2500 ng/ml) resulted in a dose-dependent inhibition of protein synthesis. This effect of Stx1 was potentiated by preincubation of the cells with interleukin-1alpha (IL-1alpha; 2 ng/ml) or tumor necrosis-alpha (TNF-alpha; 500 U/ml). Stx1 had little effect on mesangial cell viability during the first 24 hours of exposure to Stx1. However, prolonged incubation with Stx1 for 48 and 72 hours resulted in a 68% and 80% decrease in cell-viability, respectively. Stx1 elicited a dose and time dependent increase in the levels of MCP-1 mRNA, an effect that was potentiated by preincubation with IL-1alpha. CONCLUSION: These data indicate that mesangial cells are susceptible to the effects of Stx1 in vitro. Stx1 exerts a spectrum of biologic effects on mesangial cells ranging from activation of chemokine genes to a lethal toxic injury. Immunoinflammatory cytokines potentiate the effects of Stx1. Thus, glomerular pathology in HUS may also result from a direct effect of Stx1 on mesangial cells.