Monitoring of Bordetella isolates circulating in Saint Petersburg, Russia between 2001 and 2009

Bordetella isolates in the Saint Petersburg region have been monitored since 1998. Over the past ten years, concomitant with the increase in pertussis whole-cell vaccine coverage, the incidence of whooping cough has decreased. However, this decrease exists only for Bordetella pertussis infections, as the incidence of Bordetella parapertussis confirmed cases has remained stable, suggesting that pertussis-vaccine-induced immunity is not protective against parapertussis, as expected. B. pertussis and B. parapertussis clinical isolates were analyzed using serotyping, immunoblotting, pulsed-field gel electrophoresis of chromosomal DNA (after digestion with XbaI) and sequencing of virulence genes. The bacterial population is now similar to that observed in other European countries.     

Functional and structural studies on different forms of the adenylate cyclase toxin of Bordetella pertussis

A comparison was made of the cytotoxic activity and secondary structural features of four recombinant forms of adenylate cyclase toxin (CyaA). These forms were fully functional CyaA, CyaA lacking adenylate cyclase enzymatic activity (CyaA*), and non-acylated forms of these toxins, proCyaA and proCyaA*. At a toxin concentration>1 microg/ml, CyaA* was as cytotoxic towards J774.2 cells as CyaA and mediated cell killing at a faster rate than CyaA. At concentrations<0.5 microg/ml, CyaA* was less cytotoxic than CyaA and, at <0.1 microg/ml of CyaA*, no activity was detected. CyaA, but not CyaA*, was able to induce caspase 3/7 activity, a measure of apoptosis. ProCyaA and proCyaA* had no detectable cytotoxic or apoptotic activity. CyaA caused 50% inhibition of the zymosan-stimulated oxidative burst at 0.003 microg/ml, whereas a approximately 500-fold greater toxin concentration of CyaA* or proCyaA was needed for 50% inhibition. ProCyaA* was inactive. CyaA is a calcium-binding protein and far UV circular dichroism (CD), near UV CD and fluorescence spectra analyses showed that all the forms of CyaA had similar overall structures at different calcium concentrations up to 5.0 mM. At 7.5 mM CaCl2, the far UV spectrum of CyaA altered significantly, indicating a change in secondary structure associated with high beta-sheet content or a beta-aggregated state, whereas the spectrum of CyaA* showed only a slight alteration at this calcium concentration. Near UV CD and fluorescence studies were consistent with a rearrangement of secondary structural elements in the presence of CaCl2 for all CyaA forms. There was a marked dependence on protein concentration of the far UV spectra of these CyaA forms, implying an interaction between individual molecules at higher protein concentrations.     

Protective activity of Bordetella adenylate cyclase-hemolysin against bacterial colonization

Bordetella pertussis synthesizes several factors. It has been suggested that one of these factors, the adenylate cyclase-hemolysin (AC-Hly), directly penetrates target cells and impairs their normal functions by elevating intracellular cAMP. In the present study, we show that active immunization with purified B. pertussis AC-Hly or AC (a fragment of the AC-Hly molecule carrying only the adenylate cyclase activity but no toxin activity in vitro) protects mice against B. pertussis intranasal infection. Immunization with AC-Hly or AC significantly shortens the period of bacterial colonization of the mouse respiratory tract. Furthermore, B. parapertussis AC-Hly or AC are also protective antigens against B. parapertussis colonization; their protective activities are equivalent to that of the whole-cell vaccine. These results suggest that AC-Hly may play an important role in Bordetella pathogenesis, in a murine model. If this factor plays a similar role in the human disease, its use as a protective antigen could reduce not only the incidence of the disease, but also the asymptomatic human reservoir by limiting bacterial carriage.     

Extensive molecular genetic survey of Taiwanese patients with amyotrophic lateral sclerosis

Identification of genetic mutations has been of burgeoning importance in amyotrophic lateral sclerosis (ALS) in recent years. The aim of this study was to determine the frequency and spectrum of mutations in major ALS-causing genes in a Taiwanese ALS cohort of Han Chinese origin. Mutational analyses of the SOD1, TARDBP, FUS, OPTN, VCP, UBQLN2, SQSTM1, PFN1, HNRNPA1, and HNRNPA2B1 genes were carried out by direct sequencing in 161 unrelated patients with ALS, including 30 with familial ALS (FALS) and 131 with sporadic ALS (SALS). The CAG repeat size in ATXN2 and the GGGGCC repeat expansion in C9ORF72 of the patients were also investigated. Mutations were identified in 33 patients (20.5%, 33/161), including 22 with FALS and 11 with SALS. Mutations were identified most frequently in SOD1 (7.5%). Three mutations are novel, including SOD1 p.G10A, SOD1 p.D83N, and OPTN p.L494W. These findings broaden the spectrum of ALS-causing mutations and are indispensable for designing optimal strategies of mutational analysis and genetic counseling of ALS for patients of Chinese origin.     

Cholesterol-rich domains are involved in Bordetella pertussis phagocytosis and intracellular survival in neutrophils

Bordetella pertussis-specific antibodies protect against whooping cough by facilitating host defense mechanisms such as phagocytosis. However, the mechanism involved in the phagocytosis of the bacteria under non-opsonic conditions is still poorly characterized. We report here that B. pertussis binding and internalization is cholesterol dependent. Furthermore, we found cholesterol to be implicated in B. pertussis survival upon interaction with human neutrophils. Pre-treatment of PMN with cholesterol sequestering drugs like nystatin or methyl-β-cyclodextrin (MβCD) resulted in a drastic decrease of uptake of non-opsonized B. pertussis. Conversely, phagocytosis of opsonized bacteria was not affected by these drugs, showing that cholesterol depletion affects neither the viability of PMN nor the route of entry of opsonized B. pertussis. Additionally, intracellular survival rate of non-opsonized bacteria was significantly decreased in cholesterol-depleted PMN. Accordingly, confocal laser microscopy studies showed that non-opsonized B. pertussis co-localized with lysosomal markers only in cholesterol-depleted PMN but not in normal PMN. Our results indicate that B. pertussis docks to molecules that eventually prevent cellular bactericidal activity.     

Triplex real‐time PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis

Xu Y, Xu Y, Hou Q, Yang R, Zhang S. Triplex real‐time PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis. APMIS 2010; 118: 685–91. A triplex real‐time PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis was developed. Three targets were used for amplification in a single tube: the insertion sequence IS481 and the pertussis toxin promoter region (ptxP) for B. pertussis, and the insertion sequence IS1001 for B. parapertussis. The performance of this PCR assay was evaluated in parallel in three single‐target real‐time PCR assays using DNA extracted from B. pertussis and B. parapertussis reference strains and nasopharyngeal swabs taken from 105 patients who had been coughing for more than 7 days. The minimum detection limit of the triplex PCR was one to five colony‐forming units (CFU) of B. pertussis and 1 CFU of B. parapertussis per reaction, and the coefficients of both intra‐ and inter‐assay variation were less than 7%. Results were available within 4 h. Of the 105 nasopharyngeal samples, seven were culture positive and 23 were PCR positive for B. pertussis. All culture‐positive samples were also PCR positive. Our single‐tube triplex real‐time PCR assay proved to be sensitive, specific and suitable for simultaneous detection and discrimination of B. pertussis and B. parapertussis.     

Development of a real-time PCR for the identification of Bordetella pertussis and Bordetella parapertussis

This study describes a real-time PCR assay for the detection and identification of Bordetella pertussis and Bordetella parapertussis. The assay is based on amplification of a fragment from the repeat sequence regions IS481 and IS1001 found in B. pertussis and B. parapertussis, respectively, with subsequent species identification by melting curve analysis using SYBR Green chemistry. Discrimination between the two species was straightforward, as the corresponding melting points showed a significant difference of 7 degrees C. The assay was evaluated first with reference strains and retrospective human clinical samples, and then prospectively with 132 human clinical specimens received between March 2003 and December 2005. The assay allowed the rapid detection of 22 positive clinical samples, of which 15, including one fatal case, were not identified by standard culture techniques. The new assay was sensitive and specific, and can be implemented easily using any real-time PCR apparatus.     

Recent Trends of Antigenic Variation in Bordetella pertussis Isolates in Korea

Pertussis is a representative vaccine-preventable disease. However, there have been recent outbreaks in countries where even higher vaccination against the disease. One reason is the emergence of antigenic variants, which are different to vaccine type. In Korea, reported cases have rapidly increased since 2009. Therefore, we analyzed genotype of strains isolated in 2011-2012 by multilocus sequence typing method. As expected, the genotype profiles of tested genes dramatically changed. The major sequence type changed from ST1 to ST2, and new sequence type (ST8) appeared. In the minimum spanning tree, recent isolates belonging to the ACC-I-ST3 subgroup were detected that were composed of ST2, ST3, and ST6. In particular, the ST2 frequency increased to 81%. The novel ST8 was linked to the increased frequency of ST2. In addition, toxic strains carrying the ptxP3 promoter type were confirmed. This ptxP3 type emerged from 2009 and its frequency had increased to 100% in 2012. Based on these results, it can be inferred that the genotypic changes in the currently circulating strains are strongly associated with the recent increasing of pertussis in Korea. Therefore, the surveillance system should be strengthened, and genetic characterization of the isolates should be expanded to the whole genome sequence level.     

Interactions between Bordetella pertussis and the complement inhibitor factor H

Bordetella pertussis causes whooping cough in humans, a highly contagious disease of the upper respiratory tract. An increase in cases of whooping cough in adolescents and adults in many countries has been reported, despite high immunization rates in children. To efficiently colonize the host the bacteria have to resist complement, the first defence line of innate immunity. B. pertussis has previously been shown to bind the classical pathway inhibitors C4b-binding protein and C1-inhibitor being thereby able to escape the classical pathway of complement. In this study recent clinical isolates of B. pertussis and B. parapertussis were found to survive alternative pathway attack in fresh non-immune serum better than the reference B. pertussis strain, Tohama I. By using adsorption assays, flow cytometry and a radioligand binding assay we observed that both B. pertussis and B. parapertussis bound the alternative pathway inhibitor factor H (FH) from normal human serum. The surface attached FH maintained its complement regulatory activity and promoted factor I-mediated cleavage of C3b. The main binding region was located to the C-terminal part of FH, into short consensus repeat domains 19-20. In contrast, the avian pathogen B. avium did not bind FH and was sensitive to the alternative pathway of human complement. In conclusion, the human pathogens B. pertussis and B. parapertussis are able to evade the alternative complement pathway by surface acquisition of the host complement regulator FH.     

Development of an in vitro exposure model for investigating the biological effects of therapeutic aerosols on human cells from the respiratory tract

Respiratory diseases like asthma or COPD are gaining more and more importance worldwide due to an increased exposure of humans to inhalable compounds such as cigarette smoke, diesel exhaust or other forms of environmental pollution. Therefore, a high impact on national health systems is expected, meaning long-term treatment, with periodic examinations accompanied by high costs. Although a number of efficient drugs for these disease patterns, like Tiotropium (antimuscarinic), Salmetron (β-antagonist) or corticosteroids, are already available, a great deal of effort has to be put into the development of new drugs and therapy concepts. In this context, in vitro methods may be useful to establish more efficient prescreening procedures to analyze, for example, the toxicity of new compounds during the research and development process. These studies should aim to achieve a better selection of substances relevant for further development and a final reduction in the number of animal experiments. Therefore, we established an in vitro exposure device that allows the analysis of inhalable compounds for their pharmacological and toxicological effects. This CULTEX^® device is composed of an exposure entity representing the in vivo respiratory air compartment and a basal feeding compartment representing the subepithelium. Both compartments are connected by porous transwells on which cells form an epithelium-like cell layer. We have used this system for exposing human lung cells directly to liquid aerosols and present the first data with regard to aerosolized model substances.     

Prevalence,identification by a DNA microarray-based assay of human and food isolates Listeria spp. from Tunisia

Objectives

We aimed at evaluating the prevalence of Listeria species isolated from food samples and characterizing food and human cases isolates.

Material and methods

Between 2005 and 2007, one hundred food samples collected in the markets of Tunis were analysed in our study. Five strains of Listeria monocytogenes responsible for human listeriosis isolated in hospital of Tunis were included. Multiplex PCR serogrouping and pulsed field gel electrophoresis (PFGE) applying the enzyme AscI and ApaI were used for the characterization of isolates of L. monocytogenes. We have developed a rapid microarray-based assay to a reliable discrimination of species within the Listeria genus.

Results

The prevalence of Listeria spp. in food samples was estimated at 14% by using classical biochemical identification. Two samples were assigned to L. monocytogenes and 12 to L. innocua. DNA microarray allowed unambiguous identification of Listeria species. Our results obtained by microarray-based assay were in accordance with the biochemical identification. The two food L. monocytogenes isolates were assigned to the PCR serogroup IIa (serovar 1/2a). Whereas human L. monocytogenes isolates were of PCR serogroup IVb, (serovars 4b). These isolates present a high similarity in PFGE. Food L. monocytogenes isolates were classified into two different pulsotypes. These pulsotypes were different from that of the five strains responsible for the human cases.

Conclusion

We confirmed the presence of Listeria spp. in variety of food samples in Tunis. Increased food and clinical surveillance must be taken into consideration in Tunisia to identify putative infections sources.     

Comparison of the Sensititre YeastOne dilution method with the Clinical Laboratory Standards Institute (CLSI) M27-A3 microbroth dilution reference method for determining MIC of eight antifungal agents on 102 yeast strains

The Clinical Laboratory Standards Institute ([CLSI] formerly NCCLS) reference broth microdilution testing method (protocol M27-A3) was compared with a commercially available methods (Sensititre YeastOne^®) by testing two quality control strains and 102 isolates of Candida sp. and Cryptococcus sp. against fluconazole, itraconazole, ketoconazole, posaconazole, voriconazole, flucytosin, amphotericin B and caspofungin. Minimal inhibitory concentrations (MIC) endpoints were determined after 24 h of incubation for Sensititre YeastOne^® and after 24 and 48 h for CLSI microdilution method. Essential agreements between methods vary from 70.6 to 92.2%. Categorical agreements vary from 94.1% for 5FC to 72.6% for AMB. Sensititre YeastOne^® reading appears to be useful for avoiding very major errors and this confirms the interest of this method for evaluating new antifungals activity in vitro.     

Distribution of Human papillomavirus load in clinical specimens

The information about the range and distribution of Human papillomavirus load in clinical specimens is important for the design of accurate clinical tests. The amount of Human papillomavirus in cervical specimens was estimated using the digene HC2 HPV DNA Test^® (QIAGEN). This semi-quantitative assay is based on linear signal amplification with an analytical limit-of-detection of approximately 2500 virus copies per assay and 3-4 log dynamic range. The dynamic range of the assay was extended by a serial dilution strategy. Two large sets of positive specimens (n = 501 and 569) were analyzed and 9-11% of specimens was estimated to contain more than 7 × 10⁷ copies of virus. The viral load was also assessed for an assortment of specimens with known cytology diagnoses (n = 9435) and histological diagnoses (n = 2056). The percentage of specimens with more than 7 × 10⁷ copies of virus was estimated to be 0.89 for normal cells, 4.2 for atypical cells (unknown significance), 14.31 for cells of low-grade lesions and 22.24 for cells of high-grade lesions. The viral load increased with disease severity, but its broad distribution may not support its use as a disease biomarker. This information is important for assay design and automation, where cross-reactivity and sample-to-sample contamination must be addressed rigorously.     

A single step multiplex PCR for identification of six diarrheagenic E. coli pathotypes and Salmonella

E. coli is generally a commensal but includes some highly pathogenic strains carrying additional genes in plasmids and/or the chromosome. Based on these genes the pathogenic strains are divided into pathotypes including enteropathogenic (EPEC), enterohemorrhagic (EHEC), enterotoxigenic (ETEC), enteroaggregative (EAEC), enteroinvasive (EIEC) and diffusely adherent (DAEC) E. coli. Here, previously developed multiplex PCR strategies for these strains were integrated into one single step multiplex that differentiates all these E. coli pathotypes, usually based on multiple characteristic PCR products. This multiplex PCR works reliably for colony PCR. Two additional markers were added: one to detect most Enterobacteriacea, which acts as a positive control for successful PCR, and one to distinguish Salmonella. The multiplex correctly classified a set of 45 reference strains by colony PCR and 71 (45 + 26) strains by in silico PCR. It was then used to interrogate 44 clinical strains from bovine hosts resulting in detection of EAEC and DAEC determinants.     

Validation of a high-throughput real-time polymerase chain reaction assay for the detection of capripoxviral DNA

Capripoxviruses, which are endemic in much of Africa and Asia, are the aetiological agents of economically devastating poxviral diseases in cattle, sheep and goats. The aim of this study was to validate a high-throughput real-time PCR assay for routine diagnostic use in a capripoxvirus reference laboratory. The performance of two previously published real-time PCR methods were compared using commercially available reagents including the amplification kits recommended in the original publication. Furthermore, both manual and robotic extraction methods used to prepare template nucleic acid were evaluated using samples collected from experimentally infected animals. The optimised assay had an analytical sensitivity of at least 63 target DNA copies per reaction, displayed a greater diagnostic sensitivity compared to conventional gel-based PCR, detected capripoxviruses isolated from outbreaks around the world and did not amplify DNA from related viruses in the genera Orthopoxvirus or Parapoxvirus. The high-throughput robotic DNA extraction procedure did not adversely affect the sensitivity of the assay compared to manual preparation of PCR templates. This laboratory-based assay provides a rapid and robust method to detect capripoxviruses following suspicion of disease in endemic or disease-free countries.     

Optimization of adenovirus 40 and 41 recovery from tap water using small disk filters

Currently, the U.S. Environmental Protection Agency's Information Collection Rule (ICR) for the primary concentration of viruses from drinking and surface waters uses the 1MDS filter, but a more cost effective option, the NanoCeram^® filter, has been shown to recover comparable levels of enterovirus and norovirus from both matrices. In order to achieve the highest viral recoveries, filtration methods require the identification of optimal concentration conditions that are unique for each virus type. This study evaluated the effectiveness of 1MDS and NanoCeram filters in recovering adenovirus (AdV) 40 and 41 from tap water, and optimized two secondary concentration procedures the celite and organic flocculation method. Adjustments in pH were made to both virus elution solutions and sample matrices to determine which resulted in higher virus recovery. Samples were analyzed by quantitative PCR (qPCR) and Most Probable Number (MPN) techniques and AdV recoveries were determined by comparing levels of virus in sample concentrates to that in the initial input. The recovery of adenovirus was highest for samples in unconditioned tap water (pH 8) using the 1MDS filter and celite for secondary concentration. Elution buffer containing 0.1% sodium polyphosphate at pH 10.0 was determined to be most effective overall for both AdV types. Under these conditions, the average recovery for AdV40 and 41 was 49% and 60%, respectively. By optimizing secondary elution steps, AdV recovery from tap water could be improved at least two-fold compared to the currently used methodology. Identification of the optimal concentration conditions for human AdV (HAdV) is important for timely and sensitive detection of these viruses from both surface and drinking waters.     

Reticular angiomatoid "malignant" fibrous histiocytoma--a case report with cytogenetics and molecular genetic analyses

Angiomatoid "malignant" fibrous histiocytoma is a rare sarcoma of low malignant potential that occurs most commonly in the extremities of children and young adults. Herein, we present a case of angiomatoid malignant fibrous histiocytoma with unusual histologic features arising in the mediastinum of an 80-year-old man. The tumor exhibited a reticular growth pattern and myxoid stroma. The tumor cells expressed epithelial membrane antigen and desmin. Cytogenetic analysis revealed the translocation t(2;22)(q33;q12). Molecular genetic analysis confirmed the rearrangement of the EWSR1 locus and the presence of the EWSR1/CREB1 fusion. This report expands the clinicopathologic spectrum of angiomatoid malignant fibrous histiocytoma and underscores the value of integrating morphologic, immunophenotypic, and molecular findings in the identification of its unusual morphologic variants.     

Characterization of recombinant Bordetella pertussis adenylate cyclase toxins carrying passenger proteins

Bordetella pertussis secretes a calmodulin-activated adenylate cyclase toxin, CyaA, that is able to deliver its N-terminal catalytic domain (400 amino acid residues) into the cytosol of eukaryotic target cells, directly through the cytoplasmic membrane. We have previously shown that CyaA can be used as a vehicle to deliver CD8⁺ T-cell epitopes, inserted within the catalytic domain of the toxin, into antigen-presenting cells and can trigger specific class I-restricted cytotoxic T-cell (CTL) responses in vivo. To explore the tolerance of CyaA to insertion of polypeptides of larger size, we constructed and characterized different recombinant CyaA toxins with protein inserts of 87 to 206 amino acids in length. Several of these recombinant CyaA toxins were found to be invasive. Furthermore, we showed that the unfolding of the passenger protein is a prerequisite for the translocation of the recombinant toxins into eukaryotic cells. Our results highlight the remarkable tolerance of the CyaA toxin and suggest that CyaA might be used to deliver proteins into eukaryotic cells.