Multilocus Polymerase Chain Reaction Research Articles

Different OXA-Carbapenemases in Genetically Unrelated Klebsiella pneumoniae Strains Isolated in a North Italian Hospital During Multidrug Resistance Screening.

Klebsiella pneumoniae is one of the main opportunistic pathogens that cause a broad spectrum of diseases with increasingly frequent acquisition of resistance to antibiotics, namely carbapenems. This study focused on the characterization of 23 OXA-48-like carbapenemase-producing K. pneumoniae isolates using phenotypic and molecular tests. Phenotypic determination of the presence of β-lactamases was performed using the extended-spectrum beta-lactamase (ESBL) NP test, and phenotypic determination of the presence of carbapenemase was based on the Carba NP test. Antimicrobial susceptibility tests were performed to assess the resistance against carbapenems. Molecular characterization of ESBL genes and carbapenemase genes (blaOXA-48, blaKPC, blaVIM, and blaNDM) was performed using polymerase chain reaction (PCR) techniques. In addition, K. pneumoniae strains were analyzed for their relatedness using multilocus sequence typing PCR analysis based on the Institut Pasteur protocol, which produces allelic profiles that contain their evolutionary and geographic pattern. Following further Sanger sequencing of the blaOXA-48 genes, no genetic mutations were found. Some OXA-48-producing K. pneumoniae isolates coharbored blaKPC, blaNDM, and blaVIM genes, which encode other carbapenemases that can hydrolyze carbapenem antibiotics. The final part of the study focused on the characterization of the plasmid profiles of all isolates to better understand the spreading of the IncL/M blaOXA-48 plasmid gene. The plasmid profile also revealed other incompatibility groups, suggesting that other plasmid genes are spreading in K. pneumoniae isolates, which can coharbor and spread different carbapenemases simultaneously.

Development and study of the performance of a reagent kit for the identification of <i>Vibrio cholerae</i> and <i>Vibrio parahaemolyticus</i> by multilocus allele-specific polymerase chain reaction in clinical trials

BACKGROUND: Cholera belongs to the group of infections, and the fight against it is regulated by the WHO International Health Regulations (2005). To date, several test systems have been developed for the polymerase chain reaction (PCR) detection of Vibrio cholerae strains. However, no PCR test systems have been registered for detecting V. parahaemolyticus DNA in clinical material in Russia.
 AIM: To conduct clinical trials and evaluate the performance of the Reagent kit for identification of V. cholerae and V. parahaemolyticus by multilocus allele-specific PCR (kit of reagents Vibrio-screen FL) for the state registration of the specified medical device.
 MATERIALS AND METHODS: The study presents the results of the clinical trials for the reagent kit Vibrio-screen FL that has been developed to optimize the means and methods of identification and differentiation of pathogens of dangerous infections based on data on the diversity of genomes. Clinical trials were conducted at the Head Center of Hygiene and Epidemiology of the FMBA of Russia (license from 02.12.2013 No. ФС-99-01-008342; permission of the Ministry of Health from 16.05.2013 No. 300n) in accordance with the program of clinical trials No. 20.ПМКИ-042/10.20 from 23.09.2020 (Clinical Trial Certificate No. 01.AK-042/10.20 dated 12.10.2020). The clinical trials of the developed reagent kit Vibrio-screen FL used clinical strains of microorganisms of the genus Vibrio and other microorganism species as target analytes. Bacteriological method and multilocus PCR with hybridization-fluorescence detection were used.
 RESULTS: The reagent kit Vibrio-screen FL is a medical device for professional use in clinical laboratory diagnostics. The analytical sensitivity of the reagent kit Vibrio-screen FL was at least 106 copies of the SecY gene when determining belonging to the genus Vibrio; to the species V. cholerae, at least 106 copies of the hlyA gene; and to the species V. parahaemolyticus, at least 106 copies of the vppC gene. When determining the analytical specificity, no non-specific reactions with DNA samples of other microorganism species were found. A medical device for in vitro diagnostics A reagent kit for the identification of V. cholerae and V. parahaemolyticus by multilocus allele-specific PCR (Vibrio-screen FL) meets the requirements of the intended use, which allows it to be used for the differentiation of V. cholerae and V. parahaemolyticus strains isolated from a cultured clinical samples by multilocus allele-specific polymerase chain reaction with hybridization-fluorescent detection. The registration certificate No. РЗН 2021/13360 from 08.02.2021 was received. By the order of Roszdravnadzordated 08.02.2021 No. 1041, a reagent kit Vibrio-screen FL by the Rostov-on-Don Anti-Plague Institute of Rospotrebnadzor was admitted to distribution on the territory of the Russian Federation.
 CONCLUSIONS: The reagent kit Vibrio-screen FL is a high-quality, effective, and safe diagnostic medical device for in vitro diagnostics with sufficient diagnostic sensitivity and specificity to identify specific fragments of Vibrio spp., V. cholerae, and V. parahaemolyticus nucleic acids. These allows it to be used in clinical laboratory diagnostics for epidemiological analysis and registration of diseases caused by pathogenic vibrions.

Correlation of TcII discrete typing units with severe chronic Chagas cardiomyopathy in patients from various Brazilian geographic regions.

Chagas disease (ChD) is caused by Trypanosoma cruzi. The genetic structure of the species is divided into seven distinct genetic groups, TcI to TcVI, and Tcbat, which have shown differences in terms of geographic distribution, biological properties, and susceptibility to drugs. However, the association between genetic variability and clinical forms of ChD has not yet been fully elucidated. The predominance of TcII and TcVI discrete typing units (DTUs) (genetic groups) is known to occur in several Brazilian regions and is associated with both the domestic and the wild cycles of ChD. Thus, this study aimed to verify the genotypes of the parasites present in 330 patients with chronic Chagas cardiomyopathy (CCC) from different Brazilian states attended at the Clinical Hospital of the Ribeirão Preto Medical School and to assess the existence of a correlation between the clinical forms with the main cardiovascular risk factors and the genetics of the parasite. All patients with CCC were clinically evaluated through anamnesis, physical examination, biochemical tests, 12-lead electrocardiogram, echocardiogram and chest X-ray. Peripheral blood (5 mL) was collected in guanidine/ethylenediaminetetraacetic acid from each patient for DNA extraction and real-time polymerase chain reaction (PCR) for Chagas disease and genotyping of the parasite in the 7 DTUs. Parasite genotyping was performed using conventional multilocus PCR. Samples of only 175 patients were positive after amplification of the specific genes contained in the T. cruzi genotyping criteria. TcII (64/175), TcVI (9/175), and TcI (3/175) DTUs were predominant, followed by TcII/TcV/TcVI (74/175), and TcII/TcVI (23/175). The TcIII and TcIV DTU´s was detected in only one sample of CCC patients. Our data corroborate previous findings, indicating the predominance of the TcII genotype in patients with CCC of Brazilian origin. Moreover, this study pioneered disclosing a direct correlation between the TcII DTU and severe CCC.

Infection in Orthopaedics.

Infection in orthopaedic trauma patients is a common problem associated with significant financial and psychosocial costs, and increased morbidity. This review outlines technologies to diagnose and prevent orthopaedic infection, examines implant-related infection and its management, and discusses the treatment of post-traumatic osteomyelitis. The gold standard for diagnosing infection has a number of disadvantages, and thus new technologies to diagnose infection are being explored, including multilocus polymerase chain reaction with electrospray ionization-mass spectrometry and optical imaging. Numerous strategies have been employed to prevent orthopaedic infection, including use of antibiotic-impregnated implant coatings and cement; however, further research is required to optimize these technologies. Biofilm formation on orthopaedic implants is attributed to the glycocalyx-mediated surface mode of bacterial growth and is usually treated through a secondary surgery involving irrigation, debridement and the appropriate use of antibiotics, or complete removal of the infected implant. Research into the treatment of post-traumatic osteomyelitis has focused on developing an optimal local antibiotic delivery vehicle, such as antibiotic-impregnated polymethylmethacrylate (PMMA) cement beads or bioabsorbable bone substitute (BBS) delivery systems. As these new technologies to diagnose, prevent and treat orthopaedic infection advance, the incidence of infection will decrease and patient care will be optimized.

My-Forensic-Loci-queries (MyFLq) framework for analysis of forensic STR data generated by massive parallel sequencing

Forensic scientists are currently investigating how to transition from capillary electrophoresis (CE) to massive parallel sequencing (MPS) for analysis of forensic DNA profiles. MPS offers several advantages over CE such as virtually unlimited multiplexy of loci, combining both short tandem repeat (STR) and single nucleotide polymorphism (SNP) loci, small amplicons without constraints of size separation, more discrimination power, deep mixture resolution and sample multiplexing. We present our bioinformatic framework My-Forensic-Loci-queries (MyFLq) for analysis of MPS forensic data. For allele calling, the framework uses a MySQL reference allele database with automatically determined regions of interest (ROIs) by a generic maximal flanking algorithm which makes it possible to use any STR or SNP forensic locus. Python scripts were designed to automatically make allele calls starting from raw MPS data. We also present a method to assess the usefulness and overall performance of a forensic locus with respect to MPS, as well as methods to estimate whether an unknown allele, which sequence is not present in the MySQL database, is in fact a new allele or a sequencing error. The MyFLq framework was applied to an Illumina MiSeq dataset of a forensic Illumina amplicon library, generated from multilocus STR polymerase chain reaction (PCR) on both single contributor samples and multiple person DNA mixtures. Although the multilocus PCR was not yet optimized for MPS in terms of amplicon length or locus selection, the results show excellent results for most loci. The results show a high signal-to-noise ratio, correct allele calls, and a low limit of detection for minor DNA contributors in mixed DNA samples. Technically, forensic MPS affords great promise for routine implementation in forensic genomics. The method is also applicable to adjacent disciplines such as molecular autopsy in legal medicine and in mitochondrial DNA research.

Assessment of genetic diversity, genetic relationship and bottleneck using microsatellites in some native Turkish goat breeds

Genetic diversity, genetic relationship and bottleneck were evaluated in 5 native Turkish goat breeds (Angora, Kilis, Honamli, Hair and Norduz goat breeds) using 20 microsatellite markers. In order to investigate the genetic characterization of these goat breeds, this study was carried out with 20 different microsatellite markers in 4 different multi-locus polymerase chain reaction (PCR) systems. Twenty microsatellite loci analyses revealed that the average number of alleles per locus (15.65 allele/locus) and levels of heterozygosity (0.5192–0.9400) were fairly high. The calculated overall FIS value for all populations was 0.03656±0.033 and it was not significant. All the populations were in the Hardy–Weinberg equilibrium. According to FST values, a medium level of genetic diversity was found between the Angora goat breed and other breeds. Among the other breeds, genetic diversity was low and this diversity was statistically significant. All loci were polymorphic in all populations. Results of various analyses, such as allelic variation analysis, heterozygosity analysis, F statistics, STRUCTURE test and factorial correspondence analysis, indicated that the Angora goat breed is different than the other goat breeds. Furthermore, analysis showed that the other native goat breeds could not be distinguished from each other; these breeds were grouped together. Non-significant heterozygote excess on the basis of the TPM model, as revealed from the Wilcoxon signed-rank tests, along with a normal ‘L’-shaped distribution of mode-shift test, indicated no bottleneck in Angora, Kilis, Honamli, Hair and Norduz goat populations. The results obtained from the analysis of 20 microsatellite loci indicated that goat breeds other than the Angora goat breed cannot be genetically distinguished from each other.

Toxoplasma gondii infection in sentinel and free-range chickens from Argentina

This study aimed at isolating and genotyping Toxoplasma gondii from serologically positive free-range chickens from Argentina, and to evaluate the use of sentinel animals during a short time period of exposure to determine environmental contamination with T. gondii oocysts. Two groups of chickens on six farms were compared in this study: (i) young, 2–3 month-old broiler-type chickens reared as sentinel animals on the farms and (ii) adult chickens reared on the same farms for more than one year. Seroconversion rates of 7.0% or 5.7% were observed in sentinel broiler chickens reared for a period of 74 days (January–April 2010) or 88 days (August–November 2010) respectively, as shown by a T. gondii specific immunofluorescent antibody test. Fifty-three percent (17 of 32) of adult chickens were positive and showed higher titres than sentinel animals. Isolation of T. gondii from tissues (brain and heart) of serologically positive chickens was achieved from six of seven free-range adult birds with IFAT titres of 200 and higher. The isolated parasites were analysed by multi-locus polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP). The isolated T. gondii showed three different genotypes: two genotypes consisted in atypical allele combinations, and the remaining genotype had exclusively clonal type II alleles. All isolates obtained at a single farm, corresponded to the same genotype. The T. gondii genotypes observed are identical to those described in cats, dogs, chickens and capybaras elsewhere in South America. Two isolates, which showed different allele combinations in PCR-RFLP, were characterized in a mouse virulence assay. While one isolate showed a low virulence a second isolate was of intermediate virulence to mice.

Bilateral retinochoroiditis caused by an atypical strain of Toxoplasma gondii

Background:A 53-year-old man presented with an acute bilateral posterior uveitis with extensive necrotising retinochoroiditis but without chorioretinal scarring. A thorough workup did not reveal any underlying disease. The possibilities of...

Usefulness of multilocus polymerase chain reaction followed by electrospray ionization mass spectrometry to identify a diverse panel of bacterial isolates

Polymerase chain reaction electrospray ionization mass spectrometry (PCR/ESI-MS) was tested for its ability to accurately identify a blinded panel of 156 diverse bacterial isolates, mostly human and/or animal pathogens. Here, 142/156 (91%) isolates were correctly identified to the genus level and 115/156 (74%) were correctly identified to the species level. Only 9% were misidentified. This study shows that multilocus PCR/ESI-MS has the potential to be a useful technique for identifying a broad range of bacteria.

Genotypic Evolution of Acinetobacter baumannii Strains in an Outbreak Associated With War Trauma

The Walter Reed Army Medical Center has experienced an influx of traumatically injured patients either infected or colonized with Acinetobacter baumannii. Using multilocus polymerase chain reaction (PCR) and mass spectrometry to genotype isolates, we found an atypical and evolving strain distribution, distinct from those found at nonmilitary hospitals in the United States.

Persistence and Spatial Autocorrelation of Clones ofErysiphe necatorOverwintering as Mycelium in Dormant Buds in an Isolated Vineyard in Northern Italy

The population structure of the grape powdery mildew fungus, Erysiphe necator (formerly Uncinula necator), has been hypothesized to vary from being clonal to highly diverse and recombining. We report here on the structure of an E. necator population sampled during a 4-year period from an isolated vineyard in northern Italy (Voghera, Pavia Province). We obtained 54 isolates of E. necator that overwintered asexually as mycelium in grapevine buds and caused severe symptoms on the emerging shoots, known as flag shoots. All isolates were genotyped for mating type, four multilocus polymerase chain reaction (PCR)-based markers (a total of 64 loci were scored), and two single-copy loci designed to identify genetic subgroups in E. necator. All isolates had the same mating type and single-locus alleles that correlate to isolates from flag shoots in other areas. Only 2 of the 64 loci scored from multilocus markers were polymorphic; 46 of the 54 isolates had the same multilocus haplotype. Seven isolates had a second haplotype that was recovered over 3 years, and only a single isolate was found with a third haplotype. Both variant haplotypes differed from the main clonal haplotype by single loci. Spatial autocorrelation analyses showed that vines with flag shoots were not aggregated within years, but they were aggregated between consecutive years. These results demonstrate that this subpopulation of E. necator on flag shoots is composed of a single clonal lineage that has persisted for at least 4 years. We speculate that the lack of diversity in the flag shoot subpopulation in this vineyard is the result of restricted immigration from surrounding areas and genetic drift operating through founder effects and periodic bottlenecks. We propose a model that integrates epidemiology and population genetics to explain the variation observed in genetic structure of E. necator flag shoot subpopulations from different vineyards or viticultural regions.

Multilocus Polymerase Chain Reaction Restriction Fragment–Length Polymorphism Genotyping ofTrypanosoma cruzi(Chagas Disease): Taxonomic and Clinical Applications

Trypanosoma cruzi, the agent of Chagas disease, is subdivided into 6 discrete typing units (DTUs); their identification is important to understand clinical pleomorphism and track sylvatic DTUs that might (re-)invade domestic foci of the disease and jeopardize the running control programs. The genetic polymorphism of 12 loci was analyzed by multilocus polymerase chain reaction restriction fragment--length polymorphism (PCR-RFLP) analysis (MLP analysis) in a sample representative of the diversity within T. cruzi. We paid particular attention to genes involved in host-parasite relationships, because these may be prone to polymorphism as an adaptive answer to the immune selective pressure. The results of MLP analysis were shown to agree with the current multilocus enzyme electrophoresis- and random amplified polymorphic DNA-based classification of T. cruzi in 6 DTUs, thereby providing a taxonomic validation of our method. Our data supported hypotheses of genetic recombination within T. cruzi. We demonstrated direct applicability of PCR-RFLP analysis to blood of mammal hosts and intestine content of vector insects. Domestic DTUs were encountered in wild animals, and, reciprocally, sylvatic DTUs were encountered in humans, raising questions about changes of transmission patterns. MLP analysis represents a new alternative to existing molecular methods for T. cruzi typing. It might offer an invaluable support to clinical and epidemiological studies and to control programs.

Genetic Polymorphisms and Cerebral Palsy in Very Preterm Infants

In the present study, we examine whether selected genetic polymorphisms contribute to the development of cerebral palsy (CP) in very preterm infants. Subjects were 96 singleton infants with later-diagnosed CP and 119 control children, white non-Hispanic (n for CP=74, controls=88) or white Hispanic (CP=22, controls=31), born <32 wk gestation. Presence of CP was identified through state service agencies, with review of medical records. DNA extracted from archived neonatal blood was genotyped using multi-locus polymerase chain reaction amplification and immobilized sequence-specific oligonucleotide probes. Single nucleotide polymorphisms (SNPs) showing evidence of association with development of CP were endothelial nitric oxide synthase (eNOS) A(-922)G, factor 7 (F7) arg353gln and del(-323)10bp-ins, and lymphotoxin A (LTA) thr26asn. In white non-Hispanic children, beta-2 adrenergic receptor gln27glu was associated with CP risk; in Hispanic children, plasminogen activator inhibitor-1 (PAI-1) 4G(-675)5G and G11053T were associated with risk of CP. In a logistic regression considering these SNPs simultaneously in non-Hispanics, an association with CP was observed for heterozygotes of eNOS -922 (OR 3.0, CI 1.4-6.4), F7 (OR 2.7, CI 1.1-6.5), LTA (OR 2.1, CI 1.0-4.6), and PAI-1 (OR 3.2, CI 1.2-8.7). Factor 5, Factor 2, methylene tetrahydrofolate reductase, tumor necrosis factor-alpha, and other SNPs tested were not significantly associated with CP risk. We conclude that further study of genetic factors that may influence susceptibility to CP in very preterm infants is warranted.

Polymerase chain reaction amplification of the RuBisCo small subunit genes and their novel application to plant tissue identification

Degenerate polymerase chain reaction (PCR) primers and specific PCR conditions have been developed for the selective amplification of a polymorphic region of ribulose-1,5-bisphosphate carboxylase (RuBisCo) small subunit (ssu) genes. Reliable amplification has been achieved for genes from more than 20 plant species from a variety of taxonomic groups. Analysis of Nicotiana species provides strong evidence that the test loci are polymorphic at the interspecies level but show little polymorphic variation at the intraspecies level. This specific multilocus PCR approach provides a powerful counterpart to random amplification of polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) analysis for the identification of plant tissues.

Pathogenicity of a Species of Phomopsis Causing a Shoot Blight on Peach in Georgia and Evaluation of Possible Infection Courts.

A species of Phomopsis was isolated consistently from blighted shoots of peach trees in Georgia and Alabama. Isolates of the fungus caused characteristic cankers on current season's shoots of Babygold-7 peach trees when wounded or nonwounded dormant buds, breaking buds, and natural or artificially-created leaf scars were inoculated with conidia of the fungus. Mean canker length for the isolates was 35 mm 30 days after inoculation. No disease developed on control trees. There was no significant difference between the canker length on inoculated wounded dormant buds (59 mm) and nonwounded breaking buds (54 mm). However, cankers from both sites of inoculation were significantly longer than those resulting from inoculated artificially-created leaf scars (33 mm), natural leaf scars (33 mm), and non-wounded dormant buds (30 mm). There was no significant difference in virulence among the 5 isolates of Phomopsis sp. tested, and multi-locus DNA fingerprint analysis resulted in a similarity coefficient of 0.94 among the isolates. Additionally, results of the internal transcribed spacer (ITS) regions sequence comparisons for the isolates were consistent with the multi-locus polymerase chain reaction profiles, and the nucleotide sequences of the ITS region were identical for all 5 isolates. This is the first documentation of the pathogenicity of a Phomopsis sp. associated with shoot blight of peach in Georgia. Rapid development of disease in breaking buds indicates that they may be the primary site of invasion by the fungus. Natural leaf scars and dormant buds may also serve as possible infection courts.