PCR-based Method Research Articles

Development of a clinical microarray system for genetic analysis screening

ObjectivesResearch on the relationship between diseases and genes and the advancement of genetic analysis technologies have made genetic testing in medical care possible. There are various methods for genetic testing, including PCR-based methods and next-generation sequencing; however, screening tests in clinical laboratories are becoming more diverse; therefore, novel measurement systems and equipment are required to meet the needs of each situation. In this study, we aimed to develop a novel microarray-based genetic analysis system that uses a Peltier element to overcome the issues of conventional microarrays, such as the long measurement time and high cost. MethodsWe constructed a microarray system to detect the UDP-glucuronosyltransferase gene polymorphisms UGT1A1*6 and UGT1A1*28 in patients eligible for irinotecan hydrochloride treatment for use in clinical laboratories. To evaluate the performance of the system, the hybridization temperature and reaction time were determined, and the results were compared with those obtained using a conventional hybridization oven. ResultsThe hybridization temperature reached its target in 1/27th of the time required by the conventional system. We assessed 111 human clinical samples and found that our results agreed with those obtained using existing methods. The total time for the newly developed device was reduced by 85 min compared to that for existing methods, as the automated DNA microarray eliminates the time that existing methods spend on manual operation. ConclusionsThe surface treatment technology used in our system enables high-density and strong DNA fixation, allowing the construction of a measurement system suitable for clinical applications.

Usefulness of Molecular Methods for Helicobacter pylori Detection in Pediatric Patients and Their Correlation with Histopathological Sydney Classification

Helicobacter pylori infections, as one of the most prevalent among humans, are generally acquired during childhood, and are one of the main causes of chronic gastritis and peptic ulcer disease. A bacterial culture from a gastric biopsy is the gold standard and is the only method that has 100% specificity. However, its sensitivity varies, depending on experience of the laboratory staff, applied culture media, specimen transport conditions, biopsy site, and quality of the sample. The same factors compromise all invasive methods and a culture-based H. pylori infection diagnostic, as well as a recent intake of antibiotics, bismuth-containing compounds, and proton pump inhibitors. Molecular methods have been used for clinical microbiology investigation since the beginning of the 21st century. However, their usefulness for H. pylori infections diagnosis remains unclear, especially in pediatric patients. The aim of the study was to assess the incidence of H. pylori infections in a group of 104 pediatric patients and to compare the results of the PCR test with the corresponding histopathological investigation effects. Among the biopsy samples collected from 104 children, 44 (42.3%) were positive in PCR, while 43 (41.3%) and 39 (37.5%) presented histologically-confirmed signs of inflammation and H. pylori colonization, respectively. Moreover, the mean grades of the parameters of the histopathological examination were higher in the group of PCR-positive samples. The compatibility of both research methods was confirmed, emphasizing the usefulness of molecular methods for detecting H. pylori infections in pediatric patients. Considering that the PCR-based method gives reliable results and is less time-consuming and costly, it is worth discussing this method as a new standard in the diagnosis of H. pylori infections, at least among pediatric patients, for which culture-based diagnostics is not sufficient or histopathological examination is negative, while inflammation signs are observed macroscopically.

Development of a duplex qPCR for the differentiation of a live attenuatedEscherichia coliaroA mutant vaccine strain from field isolates in chickens.

Avian pathogenic Escherichia coli (APEC) can cause colibacillosis in poultry, characterised by localised or systemic infections. Colibacillosis is considered one of the leading causes of economic losses in the poultry industry due to reduced performance, increased mortality, treatment costs and carcass condemnations. A live attenuated Escherichia coli O78 aroA gene mutant is widely used to prevent disease. However, no effective strategies to differentiate the vaccine strain from field strains are available, hampering follow-up of vaccination campaigns. In the current study, we report a PCR-based method to simultaneously detect the vaccine strain by targeting the vaccine-specific mutation in the aroA gene, as well as the wild type E. coli strains by targeting the xanQ gene. The specificity of this PCR was evaluated using 123 E. coli isolates, form which 5 WT aroA auxotrophic strains (WT strains with a natural aroA deficiency), as well as 7 non-Escherichia isolates. The PCR showed 100% sensitivity of the xanQ primers for E. coli detection and 100% sensitivity of the ΔaroA primers for the vaccine strain. In order to allow quantification of the vaccine strain in complex samples containing many different E. coli strains and other related organisms, such as chicken faeces, a probe-based duplex qPCR was developed. The limit of detection (LOD) of this duplex qPCR method was 8.4*103 copies/g faeces. The specificity of the duplex qPCR was confirmed by determining both the vaccine strain levels, and the total E. coli load in intestinal digesta from both vaccinated and non-vaccinated birds. E. coli could be detected in both vaccinated and non-vaccinated birds. The duplex qPCR was specific for the vaccine strain as this strain was detected in all vaccinated birds, whereas no signal was detected in non-vaccinated birds. The duplex qPCR is helpful in monitoring colonization and shedding of the vaccine strain.

Spoligotyping of the Mycobacterium tuberculosis complex using on-Chip PCR.

The aim of this study was to develop a rapid PCR-based method for spoligotyping of mycobacteria in the microarray format and to compare it to conventional spoligotyping by hybridization. The method employs the On-Chip PCR technique with primers specific for 43 spacers that separate direct repeats (DRs) in the DR region of mycobacterial DNA. The primers were immobilized on gel-based microarrays, and PCR was performed directly on the chips. The PCR fluorescence images were acquired and processed using a portable fluorescence analyzer equipped with dedicated software. Analysis takes 1.5-2 hours and can be carried out on clinical samples without additional handling. The analytical sensitivity of the method was 103 copies of target DNA. The spoligotyping results of 51 samples produced by the proposed method and by conventional reverse hybridization approach were in full concordance. High throughput capacity, computerized data analysis, compact equipment, and reliable results make the On-Chip PCR an attractive alternative to intra- and interspecific spoligotyping of Mycobacterium tuberculosis complex bacteria.

Wastewater surveillance of antibiotic-resistant bacterial pathogens: A systematic review.

Infectious diseases caused by antibiotic-resistant bacterial (ARB) pathogens are a serious threat to human and animal health. The active surveillance of ARB using an integrated one-health approach can help to reduce the emergence and spread of ARB, reduce the associated economic impact, and guide antimicrobial stewardship programs. Wastewater surveillance (WWS) of ARB provides composite samples for a total population, with easy access to the mixed community microbiome. This concept is emerging rapidly, but the clinical utility, sensitivity, and uniformity of WWS of ARB remain poorly understood especially in relation to clinical evidence in sewershed communities. Here, we systematically searched the literature to identify studies that have compared findings from WWS of ARB and antibiotic resistance genes (ARG) with clinical evidence in parallel, thereby evaluating how likely WWS of ARB and ARG can relate to the clinical cases in communities. Initially, 2,235 articles were obtained using the primary search keywords, and 1,219 articles remained after de-duplication. Among these, 35 articles fulfilled the search criteria, and an additional 13 relevant articles were included by searching references in the primary literature. Among the 48 included papers, 34 studies used a culture-based method, followed by 11 metagenomics, and three PCR-based methods. A total of 28 out of 48 included studies were conducted at the single sewershed level, eight studies involved several countries, seven studies were conducted at national or regional scales, and five at hospital levels. Our review revealed that the performance of WWS of ARB pathogens has been evaluated more frequently for Escherichia coli, Enterococcus spp., and other members of the family Enterobacteriaceae, but has not been uniformly tested for all ARB pathogens. Many wastewater-based ARB studies comparing the findings with clinical evidence were conducted to evaluate the public health risk but not to relate with clinical evidence and to evaluate the performance of WWS of ARB. Indeed, relating WWS of ARB with clinical evidence in a sewershed is not straightforward, as the source of ARB in wastewater cannot be only from symptomatic human individuals but can also be from asymptomatic carriers as well as from animal sources. Further, the varying fates of each bacterial species and ARG within the sewerage make the aim of connecting WWS of ARB with clinical evidence more complicated. Therefore, future studies evaluating the performance of many AMR pathogens and their genes for WWS one by one can make the process simpler and the interpretation of results easier.

Two species-specific TaqMan-based quantitative polymerase chain reaction assays for the detection in soil of Paenibacillus polymyxa inocula.

The increasingly widespread use of beneficial microbial inocula in agriculture gives rise to two primary needs: i) the assessment of the environmental risk, i.e. their impact on local soil microbiome and soil properties; ii) being able to track them and monitor their persistence and fate to both optimize their formulation and application method. In previous years, PCR-based methods have detected bacterial or fungal bioinoculant at the species or strain level. However, the selective detection, quantification, and monitoring of target microbial species in a complex ecosystem such as soil require that the tests possess high specificity and sensitivity. The work proposes a quantitative real-time PCR detection method using TaqMan chemistry, showing high specificity and sensitivity for the Paenibacillus polymyxa K16 strain. The primer and probe sets were designed using the polymyxin gene cluster targeting pmxC and pmxE sequences. Validation tests showed that these assays allowed a discriminant and specific detection of P. polymyxa K16 in soil. The TaqMan-assay developed could thus ensure the necessary level of discrimination required by commercial and regulatory purposes to detect and monitor the bioinoculant in soil.

Mycoplasma testing: regulatory guidance and strategies for cGMP cell and gene therapy manufacturing

Mycoplasma, the smallest known free-living, self-replicating microorganisms, are potential contaminants of mammalian cell cultures, including biotherapeutic manufacturing cell cultures. As such, these cultures must be tested and are required to be free of Mycoplasma. Regulatory guidance globally for Mycoplasma testing has evolved, from strictly requiring culture-based testing methods to now accepting rapid molecular methods, including real-time quantitative PCR-based methods. This poster details a highly sensitive, specific, and robust method for rapid Mycoplasma detection for lot-release and in-process testing

How, where and when to screen for porcine cytomegalovirus (PCMV) in donor pigs for xenotransplantation

Porcine cytomegalovirus (PCMV), that is actually a porcine roseolovirus (PRV), is a common herpesvirus in domestic pigs and wild boars. In xenotransplantation, PCMV/PRV has been shown to significantly reduce the survival time of pig kidneys and hearts in preclinical trials with different non-human primates. Furthermore, PCMV/PRV has been transmitted in the first pig to human heart xenotransplantation and contributed to the death of the patient. Although transmitted to the recipient, there is no evidence that PCMV/PRV can infect primate cells including human cells. PCMV/PRV is closely related to the human herpesviruses 6 and 7, and only distantly related to the human CMV (HCMV). Antiviral drugs used for the treatment of HCMV are less effective against PCMV/PRV. However, there are well described strategies to eliminate the virus from pig facilities. In order to detect the virus and to eliminate it, highly sensitive detection methods and the knowledge of how, where and when to screen the donor pigs is required. Here, a comparative testing of organs from pigs of different ages using polymerase chain reaction (PCR)-based and immunological methods was performed. Testing young piglets, PCMV/PRV was detected effectively by PCR in blood, bronchoalveolar lavage fluid, tonsils and heart. In adult animals, detection by PCR was not successful in most cases, because the virus load was below the detection limit or the virus was in its latent stage. Therefore, detection of antibodies against selected recombinant proteins corresponding to epitopes detected by nearly all infected animals in a Western blot assay is advantageous. By contrast, immunological testing is not beneficial in young animals as piglets might have PCMV/PRV-specific antibodies obtained from their infected mother via the colostrum. Using a thoughtful combination of PCR-based and immunological methods, detection of PCMV/PRV in donor pigs for xenotransplantation is feasible and a controlled elimination of the virus by early weaning or other methods is possible.

Evaluation of a quantitative PCR-based method for chimerism analysis of Japanese donor/recipient pairs.

Chimerism analysis is a surrogate indicator of graft rejection or relapse after allogeneic hematopoietic stem cell transplantation (HSCT). Although short tandem repeat PCR (STR-PCR) is the usual method, limited sensitivity and technical variability are matters of concern. Quantitative PCR-based methods to detect single nucleotide polymorphisms (SNP-qPCR) are more sensitive, but their informativity and quantitative accuracy are highly variable. For accurate and sensitive chimerism analysis, a set of KMR kits (GenDx, Utrecht, Netherlands), based on detection of insertions/deletions (indels) by qPCR, have been developed. Here, we investigated informativity and validated the accuracy of KMR kits in Japanese donor/recipient pairs and virtual samples of DNA mixtures representative of Japanese genetic diversity. We found that at least one recipient-specific marker among 39 KMR-kit markers was informative in all of 65 Japanese donor/recipient pairs. Moreover, the percentage of recipient chimerism estimated by KMRtrack correlated well with ratios of mixed DNA in virtual samples and with the percentage of chimerism in HSCT recipients estimated by STR-PCR/in-house SNP-qPCR. Moreover, KMRtrack showed better sensitivity with high specificity when compared to STR-PCR to detect recipient chimerism. Chimerism analysis with KMR kits can be a standardized, sensitive, and highly informative method to evaluate the graft status of HSCT recipients.

MicroRNA Profiling Using a PCR-Based Method.

MicroRNAs (miRNAs) are small non-coding RNA molecules involved in the post-transcriptional regulation of specific mRNA targets, thus possibly controlling many biological processes. The miRNA profiling analysis can contribute to understanding several signaling pathways, as biomarkers for molecular diagnostic, as well as potential to be used as therapeutic targets. The miRNAs expression can be analyzed by quantitative reverse transcription PCR (RT-qPCR), microarrays, and RNA sequencing. The RT-qPCR method is sensitive and specific and has a lower cost when compared to other techniques as microarrays and RNA sequencing. Therefore, the protocol presented in this chapter describes step by step all the details to perform miRNA analysis using primer-based RT-qPCR.

Phenotypic Assessment of Clinical Escherichia coli Isolates as an Indicator for Uropathogenic Potential.

For women in the United States, urinary tract infections (UTIs) are the most frequent diagnosis in emergency departments, comprising 21.3% of total visits. Uropathogenic Escherichia coli (UPEC) causes ~80% of uncomplicated UTIs. To combat this public health issue, it is vital to characterize UPEC strains as well as to differentiate them from commensal strains to reduce the overuse of antibiotics. It has been challenging to determine a consistent genetic signature that clearly distinguishes UPEC from other E. coli strains. Therefore, we examined whether phenotypic data could be predictive of uropathogenic potential. We screened 13 clinical strains of UPEC, isolated from cases of uncomplicated UTI in young otherwise healthy women, in a series of microbiological phenotypic assays using UPEC prototype strain CFT073 and nonpathogenic E. coli strain MG1655 K-12 as controls. Phenotypes included adherence, iron acquisition, biofilm formation, human serum resistance, motility, and stress resistance. By use of a well-established experimental mouse model of UTI, these data were able to predict the severity of the bacterial burden in both the urine and bladders. Multiple linear regression using three different phenotypic assays, i.e., growth in minimal medium, siderophore production, and type 1 fimbrial expression, was predictive of bladder colonization (adjusted R2 = 0.6411). Growth in ex vivo human urine, hemagglutination of red blood cells, and motility modeled urine colonization (adjusted R2 = 0.4821). These results showcase the utility of phenotypic characterization to predict the severity of infection that these strains may cause. We predict that these methods will also be applicable to other complex, genetically redundant, pathogens. IMPORTANCE Urinary tract infections are the second leading infectious disease worldwide, occurring in over half of the female population during their lifetime. Most infections are caused by uropathogenic Escherichia coli (UPEC) strains. These strains can establish a reservoir in the gut, in which they do not cause disease but, upon introduction to the urinary tract, can infect the host and elicit pathogenesis. Clinically, it would be beneficial to screen patient E. coli strains to understand their pathogenic potential, which may lead to the administration of prophylactic antibiotic treatment for those with increased risk. Others have proposed the use of PCR-based genetic screening methods to detect UPEC strains and differentiate them from other E. coli pathotypes; however, this method has not yielded a consistent uropathogenic genetic signature. Here, we used phenotypic characteristics such as growth rate, siderophore production, and expression of fimbriae to better predict uropathogenic potential.

Higher Daily Air Temperature Is Associated with Shorter Leukocyte Telomere Length: KORA F3 and KORA F4.

Higher air temperature is associated with increased age-related morbidity and mortality. To date, short-term effects of air temperature on leukocyte telomere length have not been investigated in an adult population. We aimed to examine the short-term associations between air temperature and leukocyte telomere length in an adult population-based setting, including two independent cohorts. This population-based study involved 5864 participants from the KORA F3 (2004-2005) and F4 (2006-2008) cohort studies conducted in Augsburg, Germany. Leukocyte telomere length was assessed by a quantitative PCR-based method. We estimated air temperature at each participant's residential address through a highly resolved spatiotemporal model. We conducted cohort-specific generalized additive models to explore the short-term effects of air temperature on leukocyte telomere length at lags 0-1, 2-6, 0-6, and 0-13 days separately and pooled the estimates by fixed-effects meta-analysis. Our study found that between individuals, an interquartile range (IQR) increase in daily air temperature was associated with shorter leukocyte telomere length at lags 0-1, 2-6, 0-6, and 0-13 days (%change: -2.96 [-4.46; -1.43], -2.79 [-4.49; -1.07], -4.18 [-6.08; -2.25], and -6.69 [-9.04; -4.27], respectively). This meta-analysis of two cohort studies showed that between individuals, higher daily air temperature was associated with shorter leukocyte telomere length.

Non-cyanobacterial diazotrophs: Global diversity, distribution, ecophysiology, and activity in marine waters.

Biological dinitrogen (N2) fixation supplies nitrogen to the oceans, supporting primary productivity, and is carried out by some bacteria and archaea referred to as diazotrophs. Cyanobacteria are conventionally considered to be the major contributors to marine N2 fixation, but non-cyanobacterial diazotrophs (NCDs) have been shown to be distributed throughout ocean ecosystems. However, the biogeochemical significance of marine NCDs has not been demonstrated. This review synthesizes multiple datasets, drawing from cultivation-independent molecular techniques and data from extensive oceanic expeditions, to provide a comprehensive view into the diversity, biogeography, ecophysiology, and activity of marine NCDs. A NCD nifH gene catalog was compiled containing sequences from both PCR-based and PCR-free methods, identifying taxa for future studies. NCD abundances from a novel database of NCD nifH-based abundances were colocalized with environmental data, unveiling distinct distributions and environmental drivers of individual taxa. Mechanisms that NCDs may use to fuel and regulate N2 fixation in response to oxygen and fixed nitrogen availability are discussed, based on a metabolic analysis of recently available Tara Oceans expedition data. The integration of multiple datasets provides a new perspective that enhances understanding of the biology, ecology, and biogeography of marine NCDs and provides tools and directions for future research.

Interference reduction isothermal nucleic acid amplification strategy for COVID-19 variant detection

Common reference methods for COVID-19 variant diagnosis include viral sequencing and PCR-based methods. However, sequencing is tedious, expensive, and time-consuming, while PCR-based methods have high risk of insensitive detection in variant-prone regions and are susceptible to potential background signal interference in biological samples. Here, we report a loop-mediated interference reduction isothermal nucleic acid amplification (LM-IR-INA) strategy for highly sensitive single-base mutation detection in viral variants. This strategy exploits the advantages of nicking endonuclease-mediated isothermal amplification, luminescent iridium(III) probes, and time-resolved emission spectroscopy (TRES). Using the LM-IR-INA strategy, we established a luminescence platform for diagnosing COVID-19 D796Y single-base substitution detection with a detection limit of 2.01 × 105 copies/μL in a linear range of 6.01 × 105 to 3.76 × 108 copies/μL and an excellent specificity with a variant/wild-type ratio of significantly less than 0.0625%. The developed TRES-based method was also successfully applied to detect D796Y single-base substitution sequence in complicated biological samples, including throat and blood, and was a superior to steady-state technique. LM-IR-INA was also demonstrated for detecting the single-base substitution D614G as well as the multiple-base mutation H69/V70del without mutual interference, indicating that this approach has the potential to be used as a universal viral variant detection strategy.

An Improved Recombinase Polymerase Amplification Coupled with Lateral Flow Assay for Rapid Field Detection of 'Candidatus Liberibacter asiaticus'.

Huanglongbing (HLB) is a destructive citrus disease that affects citrus production worldwide. 'Candidatus Liberibacter asiaticus' (CLas), a phloem-limited bacterium, is the associated causal agent of HLB. The current standard for detection of CLas is real-time quantitative polymerase chain reaction (qPCR) using either the CLas 16S rRNA gene or the ribonucleotide reductase (RNR) gene-specific primers/probe. qPCR requires well-equipped laboratories and trained personnel, which is not convenient for rapid field detection of CLas-infected trees. Recombinase polymerase amplification (RPA) assay is a fast, portable alternative to PCR-based diagnostic methods. In this study, an RPA assay was developed to detect CLas in crude citrus extracts utilizing isothermal amplification, without the need for DNA purification. Primers were designed to amplify a region of the CLas RNR gene, and a fluorescent labeled probe allowed for detection of the amplicon in real-time within 8 mins at 39°C. The assay was specific to CLas, and the sensitivity was comparable to qPCR, with a detection limit cycle threshold of 34. Additionally, the RPA assay was combined with a lateral flow device for a point-of-use assay that is field deployable. Both assays were 100% accurate in detecting CLas in fresh citrus crude extracts from leaf midribs and roots from five California strains of CLas tested in the Contained Research Facility in Davis, California. This assay will be important for distinguishing CLas-infected trees in California from those infected by other pathogens that cause similar disease symptoms and can help control HLB spread.

A PCR-based method for the diagnosis of Enterobius vermicularis in stool samples, specifically designed for clinical application.

Enterobius vermicularis (E. vermicularis) is a nematode that infects up to 200 million people worldwide, despite effective medications being available. Conventional diagnostic tests are hindered by low sensitivity and poor patient compliance. Furthermore, no biomolecular techniques are available for clinical application. The aim of this study was to develop a procedure specifically designed for clinical application to detect E. vermicularis by means of PCR. Two subject groups were taken into account: a group of 27 infected patients and a control group of 27 healthy subjects. A nested-PCR was performed on fecal samples to detect E. vermicularis. Due to the intrinsic difficulties of the fecal matrix, several countermeasures were adopted to ensure the efficient performance of the method: (a) a large amount of feces for the extraction process (20 g instead of 200 mg); (b) a combination of chemical and physical treatments to grind the fecal matrix; (c) an additional purification process for the negative samples after the first nested-PCR; and (d) the selection of a very specific target region for the PCR. Due to the lack of overlap with other organisms, a sequence of the 5S ribosomal DNA (rDNA) spacer region including the tract SL1 was chosen to design appropriate external and internal primers. The first nested-PCR detected E.vermicularis in 19/27 samples from infected patients. After further purification, 5/8 of the negative samples resulted positive at the second PCR. Conversely, all the samples from healthy controls resulted negative to both PCRs. Sensitivity and specificity of the method were, respectively, 88.9% and 100%. The results prove the high diagnostic accuracy of the proposed method, addressing and overcoming the challenges posed by both conventional tests and PCR-based approaches. Therefore, the method can be proposed for clinical application.

Sequence-based detection and typing procedures for Burkholderia mallei: Assessment and prospects.

Although glanders has been eradicated in most of the developed world, the disease still persists in various countries such as Brazil, India, Pakistan, Bangladesh, Nepal, Iran, Bahrain, UAE and Turkey. It is one of the notifiable diseases listed by the World Organization for Animal Health. Occurrence of glanders imposes restriction on equestrian events and restricts equine movement, thus causing economic losses to equine industry. The genetic diversity and global distribution of the causing agent, Burkholderia (B.) mallei, have not been assessed in detail and are complicated by the high clonality of this organism. Among the identification and typing methods, PCR-based methods for distinguishing B. mallei from its close relative B. pseudomallei as well as genotyping using tandem repeat regions (MLVA) are established. The advent and continuous advancement of the sequencing techniques and the reconstruction of closed genomes enable the development of genome guided epidemiological tools. For achieving a higher genomic resolution, genotyping methods based on whole genome sequencing data can be employed, like genome-wide single nucleotide polymorphisms. One of the limitations in obtaining complete genomic sequences for further molecular characterization of B. mallei is its high GC content. In this review, we aim to provide an overview of the widely used detection and typing methods for B. mallei and illustrate gaps that still require development. The genomic features of Burkholderia, their high homology and clonality will be first described from a comparative genomics perspective. Then, the commonly used molecular detection (PCR systems) and typing systems (e.g., multilocus sequence typing, variable number of tandem repeat analysis) will be presented and put in perspective with recently developed genomic methods. Also, the increasing availability of B. mallei genomic sequences and evolution of the sequencing methods offers exciting prospects for further refinement of B. mallei typing, that could overcome the difficulties presently encountered with this particular bacterium.

What Can Haemosporidian Lineages Found in Culicoides Biting Midges Tell Us about Their Feeding Preferences?

Haemoproteus (Parahaemoproteus) parasites are transmitted by Culicoides biting midges. However, the natural vectors of only six of the almost 180 recognized Haemoproteus species have been identified. The aim of this study was to investigate wild biting midges naturally infected with Haemoproteus and to understand the interaction network between Culicoides and Haemoproteus in Europe. Culicoides were collected with UV light traps from different sites in Lithuania. Parous females were morphologically identified based on their wings and heads. PCR-based methods were used to detect the Haemoproteus DNA, and salivary gland preparations were analyzed for the presence of sporozoites. Of the 580 Culicoides analyzed, 5.9% were positive for Haemoproteus DNA, and sporozoites were found in two of 11 sampled biting midge species: Culicoides kibunensis and Culicoides segnis. The interaction network revealed that C. kibunensis and C. segnis are frequently associated with several Haemoproteus lineages. On the other hand, some Haemoproteus lineages were found to interact with only one Culicoides species. This was the first report of C. segnis being a competent vector for H. minutus TURDUS2, H. asymmetricus TUPHI01, H. majoris PHSIB1, and H. fringillae CCF3; and of C. kibunensis being a competent vector for H. belopolskyi HIICT1. Culicoides segnis and C. kibunensis are both important vectors of Haemoproteus parasites.

A Survey of Methicillin-Resistance Staphylococcus aureus Isolated from Patients Hospitalized in the ICU, NICU and Surgical Departments of Tehran University of Medical Sciences Hospitals

Background and Aim: Staphylococcus aureus is an important pathogen that causes various infections. The presence of some pathogenic factors in this bacterium helps the bacteria to survive in response to the immune system and antimicrobial treatments. Efforts to treat infections caused by these strains have resulted in resistance to most antibiotics, especially methicillin and vancomycin resistance, during recent years. Therefore, the consequences of infections caused by this bacterium can be serious. In this study we aimed to survey methicillin- resistance Staphylococcus aureus isolated from patients hospitalized in the ICU, NICU and surgical departments of Tehran University of Medical Sciences hospitals. Material and Method: 430 clinical samples of the patients were collected and identified as Staphylococcus aureus by phenotype and biotype tests. MRSA isolates were first screened by showing resistance to cefoxitin by disc diffusion method and finally confirmed by examining the mecA gene by PCR. The microbial resistance pattern of MRSA was also measured by disk diffusion method. Investigating the presence of a vancomycin resistance gene in isolated MRSA occurred by PCR. Results: Out of 430 clinical samples, 117 (27.20%) Staphylococcus aureus were isolated of which 42 (35.89%) were identified as MRSA. The most MRSA obtained from the ICU department with 25 cases (59.52%). The majority of patients infected with MRSA belonged to the age group of 40-65 years. The mecA gene was detected in all MRSA. The entire MRSA was 100% sensitive to vancomycin and linezolid, and 100% resistance to cloxacillin. Molecular detection of vanA gene in isolated MRSA showed 2 (4.76%) of them contained vanA gene. Conclusion: The high prevalence of MRSA and its antibiotic resistance pattern to vancomycin and other antibiotic families can lead to treatment failures, uncontrolled nosocomial infections, and mortality. Although the PCR-based detection method is superior to other conventional techniques, a combination of these methods can provide diagnostic accuracy.

Myeloma immunoglobulin rearrangement and translocation detection through targeted capture sequencing.

Multiple myeloma is a plasma cell neoplasm characterized by clonal immunoglobulin V(D)J signatures and oncogenic immunoglobulin gene translocations. Additional subclonal genomic changes are acquired with myeloma progression and therapeutic selection. PCR-based methods to detect V(D)J rearrangements can have biases introduced by highly multiplexed reactions and primers undermined by somatic hypermutation, and are not readily extended to include mutation detection. Here, we report a hybrid-capture approach (CapIG-seq) targeting the 3' and 5' ends of the V and J segments of all immunoglobulin loci that enable the efficient detection of V(D)J rearrangements. We also included baits for oncogenic translocations and mutation detection. We demonstrate complete concordance with matched whole-genome sequencing and/or PCR clonotyping of 24 cell lines and report the clonal sequences for 41 uncharacterized cell lines. We also demonstrate the application to patient specimens, including 29 bone marrow and 39 cell-free DNA samples. CapIG-seq shows concordance between bone marrow and cfDNA blood samples (both contemporaneous and follow-up) with regard to the somatic variant, V(D)J, and translocation detection. CapIG-seq is a novel, efficient approach to examining genomic alterations in myeloma.