Exome array study did not identify novel variants in Alzheimer's disease

Genetic variants so far identified explain a small fraction of the overall inherited risk of Alzheimer's disease (AD). We aimed to identify novel genetic variants in AD using exome array that contains comprehensive panel. We genotyped 295,988 variants in 1005 subjects (400 AD cases and 605 controls) using Axiom Exome Genotyping Array that contains a pool of variants discovered in over 16 major human exome sequencing initiatives. Logistic regression analysis and the sequence kernel association optimal test were performed. The APOE, APOC1, and TOMM40 showed significant associations with AD in the single variant analysis. However, no significant association of other variants with AD was observed. This exome array study failed to identify novel genetic variants in AD.     

Rapid and accurate detection of rifampin and isoniazid-resistant Mycobacterium tuberculosis using an oligonucleotide array

To rapidly detect rifampin, isoniazid and multidrug resistance in Mycobacterium tuberculosis isolates, a new system (BluePoint MtbDR, Bio Concept Inc., Taichung, Taiwan) including an oligonucleotide array and an automatic reader was evaluated. The array simultaneously identifies M. tuberculosis and predominant mutations in the rpoB, katG and inhA upstream regulatory region (inhA-r) genes. The system was assessed with 324 clinical M. tuberculosis isolates, including 210 multidrug-resistant, 41 rifampin mono-resistant, 34 isoniazid mono-resistant and 39 fully susceptible isolates. The results were compared with those obtained using the GenoType MTBDRplus test, drug-resistant gene sequencing and conventional drug susceptibility testing. The detection limit of the array was 25 pg DNA. The array and the GenoType MTBDRplus test detected 179 (85.2%) and 182 (86.7%) multidrug-resistant M. tuberculosis strains, respectively. The sensitivities of the array for detecting rifampin and isoniazid resistance were 98.4% and 87.7%, respectively, whereas the sensitivities of the GenoType MTBDRplus test for detecting rifampin and isoniazid resistance were 98.8% and 88.9%, respectively. No significant difference was found between the tests with respect to their sensitivities to detect multidrug resistance (p 0.66), rifampin resistance (p 0.69) or isoniazid resistance (p 0.68). The discrepancies were mainly attributed to rare mutations in inhA-r, which were not included in the array. The array can directly reveal transmission-associated mutations, which are useful for epidemiological investigations. The turnaround time of the array test was 6–7 h. This study confirms the feasibility of using this system for rapid and accurate diagnosis of isoniazid and rifampin resistance in M. tuberculosis.     

Species identification and antifungal susceptibility of uncommon blood yeast isolates

Background/PurposeAccurate identification of Candida species is increasingly important in the era of emergence of Candida auris. We aimed to compare the identification performance of two matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) systems (Vitek MS and Bruker biotyper MS) and an oligonucleotide array for uncommon blood yeast isolates and demonstrate the susceptibilities among those isolates.MethodCandida species isolates from blood culture other than Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata, and Candida krusei identified by biochemical methods were collected from multiple hospitals and further identified by an oligonucleotide array based on the internal transcribed spacer-1 (ITS-1) and ITS-2 sequences of the rRNA genes, Vitek MS and Bruker biotyper MS. The minimal inhibitory concentrations (MICs) of these clinical isolates were determined by the Sensititre YeastOne (SYO) system.ResultsAmong 136 isolates, Candida guilliermondii was most common (52, 38.2%), followed by C. lusitaniae (13, 9.6%) and C. haemulonii (12, 8.8%). The oligonucleotide array, Vitek MS and Bruker biotyper MS correctly identified 89.7% (122), 90.4% (123), and 92.6% (126) of these isolates, respectively. Elevated minimal inhibitory concentrations (MICs) of fluconazole were observed for C. haemulonii (MIC₉₀: 256 mg/L), and C. guilliermondii (MIC₉₀: 16 mg/L) with 28.4% of uncommon Candida isolates with MIC ≧ 8 mg/L.ConclusionsFor uncommon Candida species, the unmet need for current databases of two commercial MALDI-TOF MS systems is highlighted, and the oligonucleotide array may serve as a supplement.     

Single exon-resolution targeted chromosomal microarray analysis of known and candidate intellectual disability genes

Intellectual disability affects about 3% of individuals globally, with∼50% idiopathic. We designed an exonic-resolution array targeting all known submicroscopic chromosomal intellectual disability syndrome loci, causative genes for intellectual disability, and potential candidate genes, all genes encoding glutamate receptors and epigenetic regulators. Using this platform, we performed chromosomal microarray analysis on 165 intellectual disability trios (affected child and both normal parents). We identified and independently validated 36 de novo copy-number changes in 32 trios. In all, 67% of the validated events were intragenic, involving only exon 1 (which includes the promoter sequence according to our design), exon 1 and adjacent exons, or one or more exons excluding exon 1. Seventeen of the 36 copy-number variants involve genes known to cause intellectual disability. Eleven of these, including seven intragenic variants, are clearly pathogenic (involving STXBP1, SHANK3 (3 patients), IL1RAPL1, UBE2A, NRXN1, MEF2C, CHD7, 15q24 and 9p24 microdeletion), two are likely pathogenic (PI4KA, DCX), two are unlikely to be pathogenic (GRIK2, FREM2), and two are unclear (ARID1B, 15q22 microdeletion). Twelve individuals with genomic imbalances identified by our array were tested with a clinical microarray, and six had a normal result. We identified de novo copy-number variants within genes not previously implicated in intellectual disability and uncovered pathogenic variation of known intellectual disability genes below the detection limit of standard clinical diagnostic chromosomal microarray analysis.     

Multicenter Evaluation of the BioFire FilmArray Gastrointestinal Panel for Etiologic Diagnosis of Infectious Gastroenteritis

The appropriate treatment and control of infectious gastroenteritis depend on the ability to rapidly detect the wide range of etiologic agents associated with the disease. Clinical laboratories currently utilize an array of different methodologies to test for bacterial, parasitic, and viral causes of gastroenteritis, a strategy that suffers from poor sensitivity, potentially long turnaround times, and complicated ordering practices and workflows. Additionally, there are limited or no testing methods routinely available for most diarrheagenic Escherichia coli strains, astroviruses, and sapoviruses. This study assessed the performance of the FilmArray Gastrointestinal (GI) Panel for the simultaneous detection of 22 different enteric pathogens directly from stool specimens: Campylobacter spp., Clostridium difficile (toxin A/B), Plesiomonas shigelloides, Salmonella spp., Vibrio spp., Vibrio cholerae, Yersinia enterocolitica, enteroaggregative E. coli, enteropathogenic E. coli, enterotoxigenic E. coli, Shiga-like toxin-producing E. coli (stx₁ and stx₂) (including specific detection of E. coli O157), Shigella spp./enteroinvasive E. coli, Cryptosporidium spp., Cyclospora cayetanensis, Entamoeba histolytica, Giardia lamblia, adenovirus F 40/41, astrovirus, norovirus GI/GII, rotavirus A, and sapovirus. Prospectively collected stool specimens (n = 1,556) were evaluated using the BioFire FilmArray GI Panel and tested with conventional stool culture and molecular methods for comparison. The FilmArray GI Panel sensitivity was 100% for 12/22 targets and ≥94.5% for an additional 7/22 targets. For the remaining three targets, sensitivity could not be calculated due to the low prevalences in this study. The FilmArray GI Panel specificity was ≥97.1% for all panel targets. The FilmArray GI Panel provides a comprehensive, rapid, and streamlined alternative to conventional methods for the etiologic diagnosis of infectious gastroenteritis in the laboratory setting. The potential advantages include improved performance parameters, a more extensive menu of pathogens, and a turnaround time of as short as 1 h.     

Craniosynostosis,hydrocephalus, Chiari I malformation and radioulnar synostosis: Probably a new syndrome

We report on clinical and molecular findings of two brothers that both presented with sagittal craniosynostosis, hydrocephalus, Chiari I malformation, blepharophimosis, small low-set ears, hypoplastic philtrum, radioulnar synostosis, kidney malformation, and hypogenitalism. Their father presented mild brachydactyly. Conventional cytogenetic and array CGH screening did not show any chromosomal gains or losses. Furthermore, molecular genetic screening of genes involved in different craniosynostosis syndromes, namely FGFR1, FGFR2, FGFR3, TWIST, RECQL4, and POR genes failed to detect any mutations in genomic DNA. The unique range of clinical manifestations in these two patients and the negative findings of the molecular genetic screening suggest the hypothesis of a previously unrecognized syndrome.     

Application of a microsphere-based array for rapid identification of Acinetobacter spp. with distinct antimicrobial susceptibilities

Acinetobacter spp. have emerged as important nosocomial and multidrug-resistant pathogens in the last decade. A. calcoaceticus, A. baumannii, Acinetobacter genospecies 3, and Acinetobacter genospecies 13TU are genetically closely related and are referred to as the A. calcoaceticus-A. baumannii complex (ACB complex). Distinct Acinetobacter spp. may be associated with differences in antimicrobial susceptibility, so it is important to identify Acinetobacter spp. at the species level. We developed a microsphere-based array that combines an allele-specific primer extension assay and microsphere hybridization for the identification of Acinetobacter spp. This assay can discriminate the 13 different Acinetobacter spp. in less than 8.5 h, and it has high specificity without causing cross-reactivity with 14 other common nosocomial bacterial species. The sensitivity of this assay was 100 A. baumannii cells per ml of blood, and it could discriminate multiple species in various mixture ratios. The developed assay could differentiate clinical Acinetobacter spp. isolates with a 90% identification rate. The antimicrobial susceptibility test showed that A. baumannii isolates were resistant to most antimicrobial agents other than imipenem, while the genospecies 3 and 13TU isolates were more susceptible to most antimicrobial agents, especially ciprofloxacin and ampicillin-sulbactam. These results supported the idea that this assay possibly could be applied to clinical samples and provide accurate species identification, which might be helpful for clinicians when they are treating infections caused by Acinetobacter spp.     

Identification of Legionella Species by Use of an Oligonucleotide Array

The genus Legionella contains a diverse group of motile, asaccharolytic, nutritionally fastidious gram-negative rods. Legionella pneumophila is the most important human pathogen, followed by L. micdadei, L. longbeachae, L. dumoffii, and other rare species. Accurate identification of Legionella spp. other than L. pneumophila is difficult because of biochemical inertness and phenotypic identity of different species. The feasibility of using an oligonucleotide array for identification of 18 species of Legionella was evaluated in this study. The method consisted of PCR amplification of the macrophage infectivity potentiator mip gene, followed by hybridization of the digoxigenin-labeled PCR products to a panel of 30 oligonucleotide probes (16- to 24-mers) immobilized on a nylon membrane. A collection of 144 target strains (strains we aimed to identify) and 50 nontarget strains (44 species) were analyzed by the array. Both test sensitivity (144/144 strains) and specificity (50/50 strains) of the array were 100%. The whole procedure for identification of Legionella species by the array can be finished within a working day, starting from isolated colonies. It was concluded that species identification of clinically relevant Legionella spp. by the array method is very reliable and can be used as an accurate alternative to conventional or other molecular methods for identification of Legionella spp.The genus Legionella currently contains 50 validly named species (http://www.dsmz.de/bactnom/bactname.htm), and among them, 20 have been found to be human pathogens (6, 10). Legionnaires'' disease (LD) is caused mainly by inhalation of aerosols generated from water sources contaminated with Legionella spp. (6, 40). While most species of Legionella are normal environmental flora, many are implicated in opportunistic infections in immunocompromised patients (14). Pulmonary infections caused by Legionella may be subclinical or severe (27), and the fatality rate can approach 50% in immunocompromised patients (49).Legionella pneumophila accounts for about 85 to 90% of cases of LD (6, 26, 49). Other Legionella spp. implicated in human infections include L. micdadei, L. longbeachae, L. dumoffii, and some less encountered species, such as L. anisa, L. bozemanae, L. feeleii, and L. wadsworthii (49). L. pneumophila is normally identified by immunofluorescent-antibody assay. A specific FDA-cleared fluorescein isothiocyanate-labeled monoclonal antibody (Bio-Rad, Hercules, CA) for all serogroups of L. pneumophila and fluorescein isothiocyanate-labeled polyclonal antisera specific for L. pneumophila serogroup 1 (m-TECH, Atlanta, GA) are commercially available (6). Accurate identification of Legionella spp. other than L. pneumophila and L. pneumophila serogroup 1 can be quite difficult due to serological cross-reactivities between serogroups and species, biochemical inertness, and phenotypic identity of different species (6). Legionella isolates which fail to react with L. pneumophila antibodies are recommended to be identified by public health or reference laboratories (6). Antigen detection in urine specimens is also commonly used in hospitals for diagnosing infection caused by L. pneumophila (46).Molecular approaches have been developed to provide more rapid and accurate identification of Legionella spp. These methods include PCR (20, 25, 34), gene probe hybridization (24, 41), restriction fragment length polymorphism analysis (21, 38), and sequence analysis of the rRNA gene (47) and the macrophage infectivity potentiator gene mip (35, 41). Since diagnostic delay may result in increased mortality for patients with LD (15), real-time PCR assay has been a focus of many studies in recent years (5, 13, 14, 17, 19, 34, 36, 41, 48). However, with real-time PCR assay, only L. pneumophila and a very limited number of Legionella spp. can be detected or identified.Recently, DNA array technology has been applied to identify a wide variety of bacteria that are difficult to be differentiated by phenotypic traits or whose identification may take a long time (12, 31, 43). This study aimed to develop an oligonucleotide array based on mip gene sequences to identify 18 species of Legionella that have been found to cause human infections in the literature (10).     

Three de novo losses and one insertion within a pericentric inversion of chromosome 6 in a patient with complete absence of expressive speech and reduced pain perception

A 32-year-old female patient, observed for 30 years because of a distinctive phenotype consisting of a dysmorphic face non-progressive deficit of motor control, lack of speech development, reduced sensitivity to pain, with a known, complex interstitial deletion 6q14 within a de novo pericentric inversion 6p11.2;q15, was re-examined at the molecular level. Applying the Infinium HumanHap300 BeadChip array and BAC-based FISH we found two new non-contiguous microdeletions in addition to the one detected previously by high resolution G-band analysis. A 360 kb loss in band 6p12.3, containing the genes RHAG, CRISP1, 2, and 3, and PGK2, a 1.15 Mb loss in 6p12.2-p12.1, containing the genes PKHD1, IL17, MCM3, EFHC1, and TRAM2 genes, and an 11.9 Mb loss in region 6q14.3-q16.1, reported previously, were mapped on the rearranged chromosome 6. The latter loss contained the central cannabinoid receptor isoform b (CNR1), which may be involved in brain development and function. Since the maternal SNPs were retained this rearrangement of chromosome 6 is most likely of paternal origin.     

Expanding the phenotype of IQSEC2 mutations: truncating mutations in severe intellectual disability

Intellectual disability (ID) is frequent in the general population, with 1 in 50 individuals directly affected worldwide. The multiple etiologies include X-linked ID (XLID). Among syndromic XLID, few syndromes present severe ID associated with postnatal microcephaly and midline stereotypic hand movements. We report on three male patients with ID, midline stereotypic hand movements, hypotonia, hyperkinesia, strabismus, as well as seizures (2/3), and non-inherited and postnatal onset microcephaly (2/3). Using array CGH and exome sequencing we characterised two truncating mutations in IQSEC2, namely two de novo intragenic duplication mapped to the Xp11.22 region and a nonsense mutation in exon 7. We propose that truncating mutations in IQSEC2 are responsible for syndromic severe ID in male patients and should be screened in patients without mutations in MECP2, FOXG1, CDKL5 and MEF2C.     

Gene–gene interaction analysis of personality traits in a Japanese population using an electrochemical DNA array chip analysis

It has been suggested that genes involved in the central dopaminergic pathway may contribute to personality traits. However, the results of association studies for these genes have not been consistent. The present study investigated the relationship between the specific polymorphisms of MAO-A, COMT, DRD2, DRD3 and personality traits in Japanese women using a novel genotyping method involving electrochemical DNA array (ECA) chip analysis. Single marker association analysis for each mutation revealed no significant association between scores for Neuroticism Extraversion Openness-Five Factor Inventory (NEO-FFI) items. Gene–gene interaction analysis showed that a MAO-A 30-bp repeat × COMT (Val158Met) × DRD3 (Ser9Gly) had a marginally significant association with Agreeableness (P = 0.0547). The present results suggest that a combination of polymorphisms of MAO-A, COMT, and DRD3 might affect personality traits in Japanese women.     

Development of a TaqMan Array Card for Acute-Febrile-Illness Outbreak Investigation and Surveillance of Emerging Pathogens,Including Ebola Virus

Acute febrile illness (AFI) is associated with substantial morbidity and mortality worldwide, yet an etiologic agent is often not identified. Convalescent-phase serology is impractical, blood culture is slow, and many pathogens are fastidious or impossible to cultivate. We developed a real-time PCR-based TaqMan array card (TAC) that can test six to eight samples within 2.5 h from sample to results and can simultaneously detect 26 AFI-associated organisms, including 15 viruses (chikungunya, Crimean-Congo hemorrhagic fever [CCHF] virus, dengue, Ebola virus, Bundibugyo virus, Sudan virus, hantaviruses [Hantaan and Seoul], hepatitis E, Marburg, Nipah virus, o''nyong-nyong virus, Rift Valley fever virus, West Nile virus, and yellow fever virus), 8 bacteria (Bartonella spp., Brucella spp., Coxiella burnetii, Leptospira spp., Rickettsia spp., Salmonella enterica and Salmonella enterica serovar Typhi, and Yersinia pestis), and 3 protozoa (Leishmania spp., Plasmodium spp., and Trypanosoma brucei). Two extrinsic controls (phocine herpesvirus 1 and bacteriophage MS2) were included to ensure extraction and amplification efficiency. Analytical validation was performed on spiked specimens for linearity, intra-assay precision, interassay precision, limit of detection, and specificity. The performance of the card on clinical specimens was evaluated with 1,050 blood samples by comparison to the individual real-time PCR assays, and the TAC exhibited an overall 88% (278/315; 95% confidence interval [CI], 84% to 92%) sensitivity and a 99% (5,261/5,326, 98% to 99%) specificity. This TaqMan array card can be used in field settings as a rapid screen for outbreak investigation or for the surveillance of pathogens, including Ebola virus.     

Evaluation of the Vitros ECiQ immunodiagnostic system for detection of anti-Toxoplasma immunoglobulin G and immunoglobulin M antibodies for confirmatory testing for acute Toxoplasma gondii infection in pregnant women

Infection with Toxoplasma gondii is often asymptomatic and, when acquired during pregnancy, may lead to connatal toxoplasmosis in the offspring. The newly introduced Vitros anti-Toxoplasma immunoglobulin G (IgG) and IgM assays, designed for the Vitros ECiQ immunodiagnostic system, a fully automated system based on chemiluminescence, were evaluated as a screening method for the serological detection of acute and chronic Toxoplasma infections in the sera of 719 pregnant women. The combination of the Vitros IgG and IgM assays demonstrated a sensitivity and a specificity of 100% for the successful detection of all acute T. gondii infections by comparison with the Sabin-Feldman dye test as the reference test. The Vitros IgG assay parameter revealed a sensitivity of 95.0%, a specificity of 100.0%, a positive predictive value of 100.0%, a negative predictive value of 86.2%, and an overall agreement of 96.2% by comparison with the dye test. Comparison of the Vitros Toxoplasma IgM assay with the immunosorbent agglutination assay yielded values of 77.1%, 99.0%, 97.7%, 88.5%, and 91.1%, respectively. Subsequent receiver operating characteristic curve analysis for the accurate detection of Toxoplasma IgM in acute (n = 90) and chronic (n = 461) infections demonstrated high sensitivity (92.2%) and specificity (81.6%). The combination of a Toxoplasma-specific IgG assay with specific IgM antibody detection has improved the diagnosis of T. gondii infection by decreasing follow-up testing. Nonetheless, positive Toxoplasma IgM test results during pregnancy necessitate confirmatory testing by a reference laboratory to ensure fast and, above all, accurate test results.     

Production and Application of New Monoclonal Antibodies Specific for a Fecal Helicobacter pylori Antigen

The aim of the present study was to establish monoclonal antibodies that could be used to produce a diagnostic test composed of one kind of monoclonal antibody recognizing a fecal Helicobacter pylori antigen. The need to develop such a test arose from disadvantages of the diagnostic test that uses a polyclonal antibody or plural kinds of monoclonal antibodies, such as the lower specificity for H. pylori antigen and the difficulty of reproduction with consistent quality. Mice were immunized with sonicated cells of the coccoid form of H. pylori, and fecal samples from H. pylori-positive subjects were screened by a direct sandwich enzyme immunoassay (EIA) for antibody production from 32 hybridoma clones. The three stable clones produced antibodies (21G2, 41A5, and 82B9) that reacted with the same soluble antigen. Gel filtration chromatography showed that the molecular masses of the cellular antigen and the fecal antigen were the same, 260 kDa. The antigen was labile in response to sodium dodecyl sulfate and heat treatments. A single-step direct sandwich EIA using a single monoclonal antibody, 21G2, was developed. The EIA could detect the antigen in 41 H. pylori clinical isolates and in fecal samples from seven H. pylori-positive subjects. Several kinds of Helicobacter species (Helicobacter felis, Helicobacter hepaticus, Helicobacter mustelae, and Helicobacter cinaedi) except H. pylori, major bacteria in feces (Campylobacter jejuni, Bacteroides vulgatus, Bifidobacterium breve, Bifidobacterium infantis, and Escherichia coli), and fecal samples from six H. pylori-negative subjects showed negative results. These results indicate that the new monoclonal antibodies and the new specific EIA would be useful as a noninvasive method of diagnosis of H. pylori infection.     

Evaluation of Two Commercial Kits and Arbitrarily Primed PCR for Identification and Differentiation of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, and Haemophilus paraphrophilus

The closely related species Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, and Haemophilus paraphrophilus are common findings in oral microbiota. The aims of this study were to evaluate the applicability of the Rapid NH and API ZYM kits and arbitrarily primed PCR (AP-PCR) in the identification and differentiation of the three species from each other. The material included 62 clinical isolates and three reference strains of A. actinomycetemcomitans representing the 5 serotypes and 18 AP-PCR genotypes. Haemophilus species included 12 clinical isolates and 11 reference strains of H. aphrophilus, H. paraphrophilus, and 5 other species. For the PCR amplification, the oligonucleotide 5′-CAGCACCCAC-3′ was used as a primer. Contrary to the consistent performance of API ZYM, the Rapid NH system was able to identify only 10 of 65 (15%) A. actinomycetemcomitans isolates, whereas all Haemophilus species were correctly identified. The API ZYM test differentiated A. actinomycetemcomitans from H. aphrophilus and H. paraphrophilus by negative β-galactosidase and α-glucosidase reactions and a positive esterase lipase reaction. However, the API ZYM test was unable to differentiate H. aphrophilus from H. paraphrophilus, it also could not differentiate A. actinomycetemcomitans serotypes from each other. Among the H. aphrophilus isolates three AP-PCR genotypes and among H. paraphrophilus isolates only one AP-PCR genotype, distinct from those of A. actinomycetemcomitans, were found. The Rapid NH test showed poor ability to identify clinical isolates of all A. actinomycetemcomitans serotypes. Moreover, AP-PCR genotyping proved to be a rapid method for the species differentiation of A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus.     

30 year experience of index case identification and outcomes of cascade testing in high-risk breast and colorectal cancer predisposition genes

It is 30 years since the first diagnostic cancer predisposition gene (CPG) test in the Manchester Centre for Genomic Medicine (MCGM), providing opportunities for cancer prevention, early detection and targeted treatments in index cases and at-risk family members. Here, we present time trends (1990–2020) of identification of index cases with a germline CPG variant and numbers of subsequent cascade tests, for 15 high-risk breast and gastro-intestinal tract cancer-associated CPGs: BRCA1, BRCA2, PALB2, PTEN, TP53, APC, BMPR1a, CDH1, MLH1, MSH2, MSH6, PMS2, SMAD4, STK11 and MUTYH. We recorded 2082 positive index case diagnostic screening tests, generating 3216 positive and 3140 negative family cascade (non-index) tests. This is equivalent to an average of 3.05 subsequent cascade tests per positive diagnostic index test, with 1.54 positive and 1.51 negative non-index tests per family. The CPGs with the highest numbers of non-index positive cases identified on cascade testing were BRCA1/2 (n = 1999) and the mismatch repair CPGs associated with Lynch Syndrome (n = 731). These data are important for service provision and health economic assessment of CPG diagnostic testing, in terms of cancer prevention and early detection strategies, and identifying those likely to benefit from targeted treatment strategies.Subject terms: Cancer genetics, Genetics research     

Identification of Clinically Important Anaerobic Bacteria by an Oligonucleotide Array

Anaerobic bacteria can cause a wide variety of infections, and some of these infections can be serious. Conventional identification methods based on biochemical tests are often lengthy and can produce inconclusive results. An oligonucleotide array based on the 16S-23S rRNA intergenic spacer (ITS) sequences was developed to identify 28 species of anaerobic bacteria and Veillonella. The method consisted of PCR amplification of the ITS regions with universal primers, followed by hybridization of the digoxigenin-labeled PCR products to a panel of 35 oligonucleotide probes (17- to 30-mers) immobilized on a nylon membrane. The performance of the array was determined by testing 310 target strains (strains which we aimed to identify), including 122 reference strains and 188 clinical isolates. In addition, 98 nontarget strains were used for specificity testing. The sensitivity and the specificity of the array for the identification of pure cultures were 99.7 and 97.1%, respectively. The array was further assessed for its ability to detect anaerobic bacteria in 49 clinical specimens. Two species (Finegoldia magna and Bacteroides vulgatus) were detected in two specimens by the array, and the results were in accordance with those obtained by culture. The whole procedure of array hybridization took about 8 h, starting with the isolated colonies. The array can be used as an accurate alternative to conventional methods for the identification of clinically important anaerobes.Anaerobic bacteria are important human pathogens, and infections caused by these bacteria can be serious and life-threatening (6). A recent report from the Mayo Clinic (Rochester, MN) revealed an overall increase in the incidence of anaerobic bacteremias of 74% from 2001 to 2004 compared to that from 1993 to 1996 (20), although the same trend was not found in community hospitals or in an European countries (2, 11). The commonly isolated anaerobic bacteria are the members of the Bacteroides fragilis group and Peptostreptococcus, Clostridium, and Fusobacterium species (3, 6, 20).Most clinical laboratories use differential biochemical tests for the identification of anaerobic microorganisms (35). However, Simmon et al. (31) found that 24% of the isolates of anaerobic bacteria recovered from blood cultures were misidentified and that 10% isolates were not identified to the species level by phenotypic characteristics. A rapid commercial kit, the Rapid ID 32A kit (bioMérieux, Marcy l''Etoile, France), was evaluated for its ability to identify strains in the Bacteroides fragilis group. The results showed that only 78.4% of the strains were correctly identified to the species level without supplemental tests (15). The success of the Rapid ID 32A system for species identification varied with different taxa (10), and a low identification rate (50%) was observed for fusobacteria (16). Veillonella isolates are relatively easily identified to the genus level, but the differentiation of Veillonella isolates at the species level remains difficult and inconclusive due to the lack of discriminatory tests (14). In recent years, increasing antimicrobial resistance for some anaerobic bacteria (1, 13, 33) were noted, especially for species in the B. fragilis group (40). The rapid identification of anaerobic bacteria and the administration of appropriate antimicrobials play crucial roles in preventing mortality and morbidity in patients (6).Molecular methods have emerged as accurate alternatives for the identification of anaerobic bacteria (21, 22, 34, 36). Approximately 9% isolates of bacteremic anaerobes could not be identified to the species level by 16S rRNA gene sequencing, although all isolates were correctly assigned to the genus level (31). Other molecular identification methods targeting the rRNA operon include PCR (32), real-time PCR (26), PCR-restriction fragment length polymorphism analysis (39), and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (37).The intergenic spacer (ITS) region separating the 16S and 23S rRNA genes has been suggested to be a good candidate for use for the identification of aerobic and anaerobic bacteria (8, 19, 42). Moreover, the DNA array technology has been applied to the identification of a variety of microorganisms (12, 17, 41). The aim of the study described here was to develop an oligonucleotide array based on the ITS sequences to identify 28 clinically important species of anaerobes and Veillonella.     

Chemical characterization and acaricide potential of essential oil from aerial parts of Tagetes patula L. (Asteraceae) against engorged adult females of Rhipicephalus sanguineus (Latreille, 1806)

Rhipicephalus sanguineus, commonly known as the brown dog tick, is one of the most widely distributed species of tick. In dogs, it can cause anemia and provide the transmission of pathogenic microorganisms such as Babesia canis, Ehrlichia canis, Hepatozoon canis, Anaplasma platys, and Mycoplasma haemocanis. To man, it can transmit the intracellular parasites Rickettsia rickettsii and Rickettsia conorii, the causative agents of the Rocky Mountain spotted fever in the Americas and Mediterranean and spotted fever in Europe and North Africa. Its control is performed by applying synthetic formulations composed of pyrethroids; however, continued use of these products results in environmental damage and acquisition of resistance. Alternatively, studies with botanical insecticides have been increasingly recurrent. Therefore, this study aimed to test the efficacy of essential oil of Tagetes patula, a ruderal species widely described in the literature for its insecticidal properties, in engorged females of R. sanguineus by the adults immersion test (AIT) and impregnated paper disk test (IPDT). The essential oil used, through gas chromatography coupled to mass spectrometry, revealed the presence of 55 compounds, being the 4-vinyl guaiacol and gamma terpinene the majority ones. The AIT compared to the IPDT was more efficient in inhibiting oviposition of tick; however, the eggs laid by the females submitted to saturated atmosphere with essential oil, from IPDT, not hatched, interrupted their development cycle. Besides being a pioneer work, the results presented here contributes to new researches, aiming the incorporation of essential oil in an acaricide for use in the environment.     

Accurate molecular diagnosis of phenylketonuria and tetrahydrobiopterin-deficient hyperphenylalaninemias using high-throughput targeted sequencing

Genetic diagnostics of phenylketonuria (PKU) and tetrahydrobiopterin (BH4) deficient hyperphenylalaninemia (BH4DH) rely on methods that scan for known mutations or on laborious molecular tools that use Sanger sequencing. We have implemented a novel and much more efficient strategy based on high-throughput multiplex-targeted resequencing of four genes (PAH, GCH1, PTS, and QDPR) that, when affected by loss-of-function mutations, cause PKU and BH4DH. We have validated this approach in a cohort of 95 samples with the previously known PAH, GCH1, PTS, and QDPR mutations and one control sample. Pooled barcoded DNA libraries were enriched using a custom NimbleGen SeqCap EZ Choice array and sequenced using a HiSeq2000 sequencer. The combination of several robust bioinformatics tools allowed us to detect all known pathogenic mutations (point mutations, short insertions/deletions, and large genomic rearrangements) in the 95 samples, without detecting spurious calls in these genes in the control sample. We then used the same capture assay in a discovery cohort of 11 uncharacterized HPA patients using a MiSeq sequencer. In addition, we report the precise characterization of the breakpoints of four genomic rearrangements in PAH, including a novel deletion of 899 bp in intron 3. Our study is a proof-of-principle that high-throughput-targeted resequencing is ready to substitute classical molecular methods to perform differential genetic diagnosis of hyperphenylalaninemias, allowing the establishment of specifically tailored treatments a few days after birth.     

In vivo and in situ evaluation of a wireless magnetoelastic sensor array for plastic biliary stent monitoring

This paper presents the in vivo and in situ evaluation of a system that wirelessly monitors the accumulation of biliary sludge in a plastic biliary stent. The sensing element, located within the stent, is a passive array of magnetoelastic resonators that is queried by a wireless electromagnetic signal. The in vivo and in situ testing uses commercially-available plastic biliary stents, each enhanced with an array of ribbon sensors (formed from Metglas? 2826 MB). The sensor array is approximately 70 mm long and contains individual resonators that are 1 mm in width and have lengths of 10 mm, 14 mm, and 20 mm. The array is anchored into the 2.8 mm inner-diameter stent using a thermal staking technique. For the in situ testing, an instrumented stent is placed in various locations within the abdominal cavity of a female domestic swine carcass to evaluate the wireless range of the system; these results show that a wireless signal can be obtained from a range of at least 7.5 cm from a sensor array covered in bile. The in vivo testing includes the endoscopic implantation of an instrumented stent into the bile duct of a swine. After implantation, the swine was housed for a period of 4 weeks, during which the animal showed no ill effects and followed the expected growth curve from 29 kg to 42 kg. At the conclusion of the in vivo test, the animal was euthanized, and the instrumented stent explanted and examined. The results presented in this paper indicate that the monitoring system does not adversely affect the health of the animal and can feasibly provide sufficient wireless range after implantation.