Genomic Comparative of Pseudomonas aeruginosa Small Colony Variant, Mucoid and Non-mucoid Phenotypes Obtained from a Patient with Cystic Fibrosis During Respiratory Exacerbations.

Pseudomonas aeruginosa, the most prevalent opportunistic pathogen in chronic obstructive pulmonary disease, associated with high morbidity and mortality in patients with cystic fibrosis (CF), is practically impossible to be eradicated from the airways in chronicity. Its extraordinary genomic plasticity is possibly associated with high antimicrobial resistance, virulence factors, and its phenotypic diversity. The occurrence of P. aeruginosa isolates promoting airway infection, showing mucoid, non-mucoid, and small colony variant (SCV) phenotypes, was observed simultaneously, in the present study, in sputum cultures obtained from a male CF young patient with chronic pulmonary infection for over a decade. The isolates belonged to a new ST (2744) were obtained in two moments of exacerbation of the respiratory disease, in which he was hospitalized. Genetic background and phenotypic analysis indicated that the isolates exhibited multi- and pan-antimicrobial resistant profiles, as well as non-susceptible to polymyxin and predominantly hypermutable (HPM) phenotypes. Whole genome sequencing showed variations in genome sizes, coding sequences and their determinants of resistance and virulence. The annotated genomes were compared for antimicrobial resistance, hypermutability, and SCV characteristics. We highlight the lack of reported genetic determinants of SCV emergence and HPM phenotypes, which can be explained in part due to the very short time between collections of isolates. To the best of our knowledge, this is the first report of genome sequencing of P. aeruginosa SCV from a CF patient in Brazil.

Genetic determinants of antimicrobial resistance in polymyxin B resistant Pseudomonas aeruginosa isolated from airways of patients with cystic fibrosis.

Pseudomonas aeruginosa is the main pathogen associated with pulmonary exacerbation in patients with cystic fibrosis (CF). CF is a multisystemic genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene, which mainly affects pulmonary function. P. aeruginosa isolated from individuals with CF in Brazil is not commonly associated with multidrug resistance (MDR), especially when compared to global occurrence, where the presence of epidemic clones, capable of expressing resistance to several drugs, is often reported. Due to the recent observations of MDR isolates of P. aeruginosa in our centers, combined with these characteristics, whole-genome sequencing was employed for analyses related to antimicrobial resistance, plasmid identification, search for phages, and characterization of CF clones. All isolates in this study were polymyxin B resistant, exhibiting diverse mutations and reduced susceptibility to carbapenems. Alterations in mexZ can result in the overexpression of the MexXY efflux pump. Mutations in oprD, pmrB, parS, gyrA and parC may confer reduced susceptibility to antimicrobials by affecting permeability, as observed in phenotypic tests. The phage findings led to the assumption of horizontal genetic transfer, implicating dissemination between P. aeruginosa isolates. New sequence types were described, and none of the isolates showed an association with epidemic CF clones. Analysis of the genetic context of P. aeruginosa resistance to polymyxin B allowed us to understand the different mechanisms of resistance to antimicrobials, in addition to subsidizing the understanding of possible relationships with epidemic strains that circulate among individuals with CF observed in other countries.

[Analysis of a differentiated resuscitation room activation at a national trauma center]. / Analyse einer differenzierten Schockraumalarmierung an einem überregionalen Traumazentrum.

BACKGROUND: In order to continue to efficiently provide both personnel-intensive and resource-intensive care to severely injured patients, some hospitals have introduced individually differentiated systems for resuscitation room treatment. The aim of this study was to evaluate the concept of the A and B classifications in terms of practicability, indications, and potential complications at a national trauma center in Bavaria. METHODS: In a retrospective study, data from resuscitation room trauma patients in the year 2020 were collected. The assignment to A and B was made by the prehospital emergency physician. Parameters such as the injury severity score (ISS), Glasgow outcome scale (GOS), upgrade rate, and the indication criteria according to the S3 guidelines were recorded. Statistical data comparisons were made using t­tests, χ2-tests, or Mann-Whitney U­tests. RESULTS: A total of 879 resuscitation room treatments (A 473, B 406) met the inclusion criteria. It was found that 94.5% of resuscitation room A cases had physician accompaniment, compared to 48% in resuscitation room B assignments. In addition to significantly lower ISS scores (4.1 vs. 13.9), 29.8% of B patients did not meet the treatment criteria defined in the S3 guidelines. With a low upgrade rate of 4.9%, 98% of B patients had a GOS score of 4 or 5. CONCLUSION: The presented categorization is an effective and safe way to manage the increasing number of resuscitation room alerts in a resource-optimized manner.

In ovo delivery of oregano essential oil activated xenobiotic detoxification and lipid metabolism at hatch in broiler chickens.

In ovo interventions are used to improve embryonic development and robustness of chicks. The objective of this study was to identify the optimal dose for in ovo delivery of oregano essential oil (OEO), and to investigate metabolic impacts. Broiler chickens Ross 308 fertile eggs were injected with 7 levels of OEO (0, 5, 10, 20, 30, 40, and 50 µL) into the amniotic fluid at embryonic d 17.5 (E17.5) (n = 48). Chick quality was measured by navel score (P < 0.05) and/or hatchability rates (P < 0.01) were significantly decreased at doses at or above 10 or 20 µL/egg, respectively, indicating potential toxicity. However, no effects were observed at the 5 µL/egg, suggesting that compensatory mechanisms were effective to maintain homeostasis in the developing embryo. To pursue a better understanding of these mechanisms, transcriptomic analyses of the jejunum were performed comparing the control injected with saline and the group injected with 5 µL of OEO. The transcriptomic analyses identified that 167 genes were upregulated and 90 were downregulated in the 5 µL OEO compared to the control group injected with saline (P < 0.01). Functional analyses of the differentially expressed genes (DEG) showed that metabolic pathways related to the epoxygenase cytochrome P450 pathway associated with xenobiotic catabolic processes were significantly upregulated (P < 0.05). In addition, long-chain fatty acid metabolism associated with ATP binding transporters was also upregulated in the OEO treated group (P < 0.05). The results indicated that low doses of OEO in ovo have the potential to increase lipid metabolism in late stages (E17.5) of embryonic development. In conclusion, in ovo delivery of 5 µL OEO did not show any negative impact on hatchability and chick quality. OEO elevated expression of key enzymes and receptors involved in detoxification pathways and lipid metabolism in the jejunum of hatchling broiler chicks.

Nanoscaled RIM clustering at presynaptic active zones revealed by endogenous tagging.

Chemical synaptic transmission involves neurotransmitter release from presynaptic active zones (AZs). The AZ protein Rab-3-interacting molecule (RIM) is important for normal Ca2+-triggered release. However, its precise localization within AZs of the glutamatergic neuromuscular junctions of Drosophila melanogaster remains elusive. We used CRISPR/Cas9-assisted genome engineering of the rim locus to incorporate small epitope tags for targeted super-resolution imaging. A V5-tag, derived from simian virus 5, and an HA-tag, derived from human influenza virus, were N-terminally fused to the RIM Zinc finger. Whereas both variants are expressed in co-localization with the core AZ scaffold Bruchpilot, electrophysiological characterization reveals that AP-evoked synaptic release is disturbed in rimV5-Znf but not in rimHA-Znf In addition, rimHA-Znf synapses show intact presynaptic homeostatic potentiation. Combining super-resolution localization microscopy and hierarchical clustering, we detect ∼10 RIMHA-Znf subclusters with ∼13 nm diameter per AZ that are compacted and increased in numbers in presynaptic homeostatic potentiation.

Pre-operative Colonization by Staphylococcus aureus and Cephalosporin Non-susceptible Bacteria in Patients with Proximal Femoral Fractures / Colonização pré-operatória por Staphylococcus aureus e as bactérias não suscetíveis à cefalosporina, em pacientes com fratura proximal do fêmur

Abstract Objective To estimate the frequency of Staphylococcus aureus and cephalosporin nonsusceptible bacteria colonization in patients with proximal femoral fracture during preoperative hospitalization. Methods Prevalence and incidence assessment in 63 hospitalized patients over 1 year. The median time of pretreatment hospitalization was 12 days. Samples were collected from the nostrils, groin skin and anal mucosa during the pretreatment hospitalization and were tested by the disc-diffusion technique. Results The hospital colonization incidence and the prevalence of positive results were 14.3 and 44.4% for S. aureus; 3.2 and 6.4% for meticillin-resistant S. aureus; 28.6 and 85.7% for meticillin-resistant coagulase-negative Staphylococcus; 28.6 and 61.9% for cefazolin nonsusceptible Enterobacteriaceae (KFNSE); and 20.6 and 28.6% for cefuroxime nonsusceptible Enterobacteriaceae (CXNSE). In addition, factors such as to the duration of the pretreatment hospitalization period, being non-walker before fracture, antimicrobial use, American Society of Anesthesiologists (ASA) 4 surgical risk, and previous hospitalization, were related to an increase in the incidence of hospital acquisition and prevalence of colonization by the evaluated strains. The prevalence of colonization by KFNSE was three times higher than by CXNSE on admission, and twice as high at the time of fracture treatment. Conclusion There was a high incidence of hospital colonization and prevalence of colonization by all strains studied, which may guide the indication of prophylactic measures for infection.

Resumo Objetivo Estimar a frequência da colonização por Staphylococcus aureus e as bactérias não suscetíveis à cefalosporina, em pacientes com fratura proximal do fêmur durante a internação pré-operatória. Métodos Avaliação da prevalência e incidência em 63 pacientes hospitalizados ao longo de um ano. O tempo médio de internação pré-tratamento foi de 12 dias. As amostras foram coletadas das narinas, pele da virilha e mucosa anal, durante a internação prévia ao tratamento e testadas pela técnica de disco-difusão. Resultados A incidência da colonização hospitalar e a prevalência de resultados positivos foram de 14,3% e 44,4% para Staphylococcus aureus; 3,2% e 6,4% para S. aureus resistente à meticilina; 28,6% e 85,7% para Staphylococcus coagulase-negativo resistente à meticilina; 28,6% e 61,9% para Enterobacteriaceae não suscetível à cefazolina (KFNSE); e 20,6% e 28,6% para Enterobacteriaceae não suscetível à cefuroxima (CXNSE). Além da duração do período de internação pré-tratamento, os pacientes não deambularam previamente à ocorrência da fratura e nem fizeram uso de antimicrobiano. Além disso, a duração do período de internação pré-tratamento cirúrgico, ser não-deambulador antes da fratura, uso de antimicrobianos, risco cirúrgico IV pela American Society of Anesthesiologists (ASA) e internação anterior, estiveram relacionados a um aumento na incidência de aquisição hospitalar e prevalência de colonização pelas cepas avaliadas. A prevalência de colonização pela KFNSE foi três vezes maior do que pela CXNSE na admissão e duas vezes maior no momento do tratamento da fratura. Conclusão Observou-se uma alta incidência da colonização hospitalar e prevalência da colonização por todas as cepas estudadas, o que pode orientar a indicação de medidas profiláticas contra a infecção.

Side-by-side comparison of parent vs. technician-collected respiratory swabs in low-income, multilingual, urban communities in the United States.

BACKGROUND: Home-based swabbing has not been widely used. The objective of this analysis was to compare respiratory swabs collected by mothers of 7-12-year-olds living in low-income, multilingual communities in the United States with technician collected swabs. METHODS: Retrospective data analysis of respiratory samples collected at home by mothers compared to technicians. Anterior nasal and throat specimens collected using flocked swabs were combined in dry tubes. Test was done using TaqMan array cards for viral and bacterial pathogens. Cycle threshold (Ct) values of ribonuclease P (RNP) gene were used to assess specimen quality. Ct < 40 was interpreted as a positive result. Concordance of pathogen yield from mother versus technician collected swabs were analyzed using Cohen's Kappa coefficients. Correlation analysis, paired t-test, and Wilcoxon signed-rank test for paired samples were used for RNP Ct values. RESULTS: We enrolled 36 households in Cincinnati (African American) and 44 (predominately Chinese or Latino) in Boston. In Cincinnati, eight of 32 (25%) mothers did not finish high school, and 11 (34%) had finished high school only. In Boston, 13 of 44 (30%) mothers had less than a high school diploma, 23 (52%) had finished high school only. Mother versus technician paired swabs (n = 62) had similar pathogen yield (paired t-test and Wilcoxon signed rank test p-values = 0.62 and 0.63, respectively; 95% confidence interval of the difference between the two measurements = - 0.45-0.75). Median Ct value for RNP was 22.6 (interquartile range, IQR = 2.04) for mother-collected and 22.4 (IQR = 2.39) for technician-collected swabs (p = 0.62). Agreement on pathogen yield between samples collected by mothers vs. technicians was higher for viruses than for bacterial pathogens, with high concordance for rhinovirus/enterovirus, human metapneumovirus, and adenovirus (Cohen's kappa coefficients ≥80%, p < 0.0001). For bacterial pathogens, concordance was lower to moderate, except for Chlamydia pneumoniae, for which kappa coefficient indicated perfect agreement. CONCLUSION: Mothers with a range of education levels from low-income communities were able to swab their children equally well as technicians. Home-swabbing using dry tubes, and less invasive collection procedures, could enhance respiratory disease surveillance.

Changes in Influenza and Other Respiratory Virus Activity During the COVID-19 Pandemic - United States, 2020-2021.

The COVID-19 pandemic and subsequent implementation of nonpharmaceutical interventions (e.g., cessation of global travel, mask use, physical distancing, and staying home) reduced transmission of some viral respiratory pathogens (1). In the United States, influenza activity decreased in March 2020, was historically low through the summer of 2020 (2), and remained low during October 2020-May 2021 (<0.4% of respiratory specimens with positive test results for each week of the season). Circulation of other respiratory pathogens, including respiratory syncytial virus (RSV), common human coronaviruses (HCoVs) types OC43, NL63, 229E, and HKU1, and parainfluenza viruses (PIVs) types 1-4 also decreased in early 2020 and did not increase until spring 2021. Human metapneumovirus (HMPV) circulation decreased in March 2020 and remained low through May 2021. Respiratory adenovirus (RAdV) circulated at lower levels throughout 2020 and as of early May 2021. Rhinovirus and enterovirus (RV/EV) circulation decreased in March 2020, remained low until May 2020, and then increased to near prepandemic seasonal levels. Circulation of respiratory viruses could resume at prepandemic levels after COVID-19 mitigation practices become less stringent. Clinicians should be aware of increases in some respiratory virus activity and remain vigilant for off-season increases. In addition to the use of everyday preventive actions, fall influenza vaccination campaigns are an important component of prevention as COVID-19 mitigation measures are relaxed and schools and workplaces resume in-person activities.

Efficiency Improvements and Discovery of New Substrates for a SARS-CoV-2 Main Protease FRET Assay.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has a huge impact on the world. Although several vaccines have recently reached the market, the development of specific antiviral drugs against SARS-CoV-2 is an important additional strategy in fighting the pandemic. One of the most promising pharmacological targets is the viral main protease (Mpro). Here, we present an optimized biochemical assay procedure for SARS-CoV-2 Mpro. We have comprehensively investigated the influence of different buffer components and conditions on the assay performance and characterized Förster resonance energy transfer (FRET) substrates with a preference for 2-Abz/Tyr(3-NO2) FRET pairs. The substrates 2-AbzSAVLQSGTyr(3-NO2)R-OH, a truncated version of the established DABCYL/EDANS FRET substrate, and 2-AbzVVTLQSGTyr(3-NO2)R-OH are promising candidates for screening and inhibitor characterization. In the latter substrate, the incorporation of Val at position P5 improved the catalytic efficiency. Based on the obtained results, we present here a reproducible, reliable assay protocol using highly affordable buffer components.

Carbapenem-Resistant Pseudomonas aeruginosa in Chronic Lung Infection: Current Resistance Profile and Hypermutability in Patients with Cystic Fibrosis.

Pseudomonas aeruginosa is associated with chronic and progressive lung disease and is closely related to increased morbidity and mortality in cystic fibrosis (CF) patients. Hypermutable (HPM) P. aeruginosa isolates have been described in these patients and are usually associated with antibiotic resistance. This study aimed to investigate the occurrence of carbapenem resistance and hypermutable phenotype in 179 P. aeruginosa isolates from 8 chronically CF patients assisted at two reference centers in Rio de Janeiro, Brazil. Using disk diffusion test, non-susceptible (NS) rates higher than 40% were observed for imipenem, amikacin, and gentamicin. A total of 79 isolates (44.1%), 71 (39.6%), and 8 (4.4%) were classified as carbapenem-resistant (CR resistance to at least one carbapenem), multidrug-resistant (MDR), and extensively drug-resistant (XDR), respectively. Minimal inhibitory concentration was determined for 79 CR P. aeruginosa and showed the following variations: 4 and 128 µg/mL to imipenem, 4 and 64 µg/mL to meropenem, and 4 and ≥ 32 µg/mL to doripenem. We have found only four (2.23%) HPM isolates from 4 patients. Analyzing the genetic relationship among the HPM isolates, 3 pulsed-field gel electrophoresis/pulsotypes (D, M, and J) were observed. Only M pulsotype was recovered from two patients in different years. Polymerase chain reaction screening for blaGES, blaIMP, blaKPC, blaNDM, blaOXA-48, blaSPM, and blaVIM genes was performed for all CR isolates and none of them were positive. Our results demonstrate a high occurrence of CR and MDR P. aeruginosa of CF patients follow-up in both centers studied, while the presence of HPM is still unusual.

Active surveillance for acute respiratory infections among pediatric long-term care facility staff.

BACKGROUND: Transmission of respiratory viruses between staff and residents of pediatric long-term care facilities (pLTCFs) can occur. We assessed the feasibility of using text or email messages to perform surveillance for acute respiratory infections (ARIs) among staff. METHODS: From December 7, 2016 to May 7, 2017, 50 staff participants from 2 pLTCFs received weekly text or email requests to report the presence or absence of ARI symptoms. Those who fulfilled the ARI case definition (≥2 symptoms) had respiratory specimens collected to detect viruses by reverse transcriptase polymerase chain reaction assays. Pre- and postsurveillance respiratory specimens were collected to assess subclinical viral shedding. RESULTS: The response rate to weekly electronic messages was 93%. Twenty-one ARIs reported from 20 (40%) participants fulfilled the case definition. Respiratory viruses were detected in 29% (5/17) of specimens collected at symptom onset (influenza B, respiratory syncytial virus, coronavirus [CoV] 229E, rhinovirus [RV], and dual detection of CoV OC43 and bocavirus). Four participants had positive presurveillance (4 RV), and 6 had positive postsurveillance specimens (3 RV, 2 CoV NL63, and 1 adenovirus). CONCLUSIONS: Electronic messaging to conduct ARI surveillance among pLTCF staff was feasible.

Peptide-ß-lactam Inhibitors of Dengue and West Nile Virus NS2B-NS3 Protease Display Two Distinct Binding Modes.

The ß-lactam ring represents a valuable moiety that can induce covalent binding of an inhibitor to its target. In this study, we explored di- and tripeptides with ß-lactam electrophilic warheads as inhibitors of dengue and West Nile virus NS2B-NS3 protease. Tripeptides with a (3S)-ß-lactam moiety displayed the highest activity, with IC50 and EC50 values in the lower micromolar range in biochemical and cellular assays. The activity against dengue protease was in general higher than against West Nile virus protease. The compounds were inactive against the off-targets thrombin and trypsin. Liquid chromatography-mass spectrometry experiments revealed that tripeptide-ß-lactam inhibitors bind to the protease in two distinct binding modes. Only one binding mode leads to a covalent, but reversible, interaction of the ß-lactam ring with the catalytic serine, followed by release of the inhibitor with opened ß-lactam ring. The other binding mode leads to the cleavage of the peptide backbone. This observation provides the first experimental evidence that benzyloxyphenylglycine in flaviviral protease inhibitors is positioned in the prime site of the enzyme.

Local Autograft and Strong Pulvertaft Fixation Technique for Reconstruction of a Rerupture of the Extensor Hallucis Longus Tendon: A Case Report.

There are different treatment options for extensor hallucis longus injuries. For primary repair, the end-to-end suture is recommended. The treatment of reruptures or tendon defects is challenging, and a wide range of procedures have been used in this regard, including primary and secondary repairs with and without auto- and allografts. To overcome the disadvantages of second-site morbidity and to achieve high primary stability, we demonstrate a technique using a local tendon graft in combination with a strong Pulvertaft suture technique in a case of rerupture of the extensor hallucis longus tendon.

De Novo Discovery of Nonstandard Macrocyclic Peptides as Noncompetitive Inhibitors of the Zika Virus NS2B-NS3 Protease.

The Zika virus presents a major public health concern due to severe fetal neurological disorders associated with infections in pregnant women. In addition to vaccine development, the discovery of selective antiviral drugs is essential to combat future epidemic Zika virus outbreaks. The Zika virus NS2B-NS3 protease, which performs replication-critical cleavages of the viral polyprotein, is a promising drug target. We report the first macrocyclic peptide-based inhibitors of the NS2B-NS3 protease, discovered de novo through in vitro display screening of a genetically reprogrammed library including noncanonical residues. Six compounds were selected, resynthesized, and isolated, all of which displayed affinities in the low nanomolar concentration range. Five compounds showed significant protease inhibition. Two of these were validated as hits with submicromolar inhibition constants and selectivity toward Zika over the related proteases from dengue and West Nile viruses. The compounds were characterized as noncompetitive inhibitors, suggesting allosteric inhibition.

Fragile X syndrome: An overview and update of the FMR1 gene.

Fragile X syndrome (FXS) is the most common cause of inherited intellectual disability and the leading form of the monogenic cause of autism. Fragile X mental retardation type 1 (FMR1) gene premutation is the first single-gene cause of primary ovarian failure (Fragile X-associated primary ovarian insufficiency [FXPOI]) and one of the most common causes of ataxia (fragile X-associated tremor/ataxia syndrome [FXTAS]), multiple additional phenotypes such as fibromyalgia, hypothyroidism, migraine headaches, sleep disturbances, sleep apnea, restless legs syndrome, central pain syndrome, neuropathy and neuropsychiatric alterations has been described. Clinical involvement in men and women carrying the FMR1 premutation currently constitutes a real health problem in the society that should be taken into account. It is important to highlight that while in FXS there is a loss-of-function of the FMR1 gene, in premutation associated disorders there is a gain of FMR1 mRNA function. To date, the tremendous progress achieved in the understanding of the pathophysiology of FXS, has led to the development of several targeted therapies aimed at preventing or improving the neurological manifestations of the disease. This review is an update of the diseases associated with the FMR1 gene.

Relation between acute and long-term cognitive decline after surgery: Influence of metabolic syndrome.

INTRODUCTION: The relationship between persistent postoperative cognitive decline and the more common acute variety remains unknown; using data acquired in preclinical studies of postoperative cognitive decline we attempted to characterize this relationship. METHODS: Low capacity runner (LCR) rats, which have all the features of the metabolic syndrome, were compared postoperatively with high capacity runner (HCR) rats for memory, assessed by trace fear conditioning (TFC) on the 7th postoperative day, and learning and memory (probe trial [PT]) assessed by the Morris water-maze (MWM) at 3 months postoperatively. Rate of learning (AL) data from the MWM test, were estimated by non-linear mixed effects modeling. The individual rat's TFC result at postoperative day (POD) 7 was correlated with its AL and PT from the MWM data sets at postoperative day POD 90. RESULTS: A single exponential decay model best described AL in the MWM with LCR and surgery (LCR-SURG) being the only significant covariates; first order AL rate constant was 0.07 s(-1) in LCR-SURG and 0.16s(-1) in the remaining groups (p<0.05). TFC was significantly correlated with both AL (R=0.74; p<0.0001) and PT (R=0.49; p<0.01). CONCLUSION: Severity of memory decline at 1 week after surgery presaged long-lasting deteriorations in learning and memory.

Prevalence of human papillomavirus infection in pregnant versus non-pregnant women in Brazil.

PURPOSE: To investigate the prevalence of human papillomavirus (HPV) in cervical samples of pregnant and non-pregnant women in South-Brazil. METHODS: A prospective study of 91 pregnant and 92 non-pregnant women with no previous history of cervical dysplasia or cancer was carried out. Cervical samples for HPV testing and cytology were collected in each trimester of pregnancy and in the puerperium for pregnant women and at matched intervals for the non-pregnant women. All samples were analyzed through PCR with consensus primers GP5+/GP6+. Genotyping was performed using specific primers. To control for confounding factors, the analysis of multivariate logistic regression was applied. The measure of odds ratio (OR) and the 95 % confidence interval (95 % CI) were used. The level of statistical significance was set at 5 % (P ≤ 0.05). RESULTS: HPV DNA was detected in 23/91 (25.3 %) cervical samples from the pregnant women and in 12/92 (13 %) cervical samples from non-pregnant women (P = 0.035). There was a significant association among cervical HPV infection and young age, number of lifetime sexual partners, and the presence of abnormal cervical cytology. HPV16 and HPV18 were the viral types more frequently detected. Out of the 23 HPV-positive pregnant women, 17 (73.9 %) had normal cervical cytology. CONCLUSION: Our results suggest a higher prevalence of HPV infection in pregnant vs. non-pregnant women. This finding may be related to the relative immunosuppression observed in pregnant women, outlining the importance of the appropriate monitoring of the viral infection in this specific population.

High incidence of large deletions in the PMS2 gene in Spanish Lynch syndrome families.

Lynch syndrome (LS) is caused by germline mutations in one of the four mismatch repair (MMR) genes. Defects in this pathway lead to microsatellite instability (MSI) in DNA tumors, which constitutes the molecular hallmark of this disease. Selection of patients for genetic testing in LS is usually based on fulfillment of diagnostic clinical criteria (i.e. Amsterdam criteria or the revised Bethesda guidelines). However, following these criteria PMS2 mutations have probably been underestimated as their penetrances appear to be lower than those of the other MMR genes. The use of universal MMR study-based strategies, using MSI testing and immunohistochemical (IHC) staining, is being one proposed alternative. Besides, germline mutation detection in PMS2 is complicated by the presence of highly homologous pseudogenes. Nevertheless, specific amplification of PMS2 by long-range polymerase chain reaction (PCR) and the improvement of the analysis of large deletions/duplications by multiplex ligation-dependent probe amplification (MLPA) overcome this difficulty. By using both approaches, we analyzed 19 PMS2-suspected carriers who have been selected by clinical or universal strategies and found five large deletions and one frameshift mutation in PMS2 in six patients (31%). Owing to the high incidence of large deletions found in our cohort, we recommend MLPA analysis as the first-line method for searching germline mutations in PMS2.

Field evaluation of TaqMan Array Card (TAC) for the simultaneous detection of multiple respiratory viruses in children with acute respiratory infection.

BACKGROUND: Multipathogen reverse transcription real-time PCR (RT-qPCR) platforms have proven useful in surveillance for acute respiratory illness (ARI) and study of respiratory outbreaks of unknown etiology. The TaqMan(®) Array Card (TAC, Life Technologies™), can simultaneously test 7 clinical specimens for up to 21 individual pathogens (depending on arrangement of controls and use of duplicate wells) by arrayed singleplex RT-qPCR on a single assay card, using minimal amounts of clinical specimens. A previous study described the development of TAC for the detection of respiratory viral and bacterial pathogens; the assay was evaluated against well-characterized analytical materials and a limited collection of human clinical specimens. OBJECTIVES: We wished to compare TAC assay performance against standard individual RT-qPCR assays for respiratory viral detection, focusing on 10 viruses (adenovirus, human metapneumovirus, human parainfluenza viruses 1-4, influenza viruses A and B, respiratory syncytial virus, and rhinovirus) from a larger collection of human specimens. STUDY DESIGN: We used specimens from 942 children with ARI enrolled systematically in a population-based, ARI surveillance study (New Vaccine Surveillance Network, NVSN). RESULTS: Compared with standard individual RT-qPCR assays, the sensitivity of TAC for the targeted viruses ranged from 54% to 95% (54%, 56%, and 75% for adenovirus, human parainfluenza viruses-1 and -2, respectively, and 82%-95% for the other viruses). Assay specificity was 99%, and coefficients of variation for virus controls ranged from 1.5% to 4.5%. CONCLUSION: The TAC assay should prove useful for multipathogen studies and rapid outbreak response.