Sensitive and specific detection of Trypanosoma cruzi DNA in clinical specimens using a multi-target real-time PCR approach

Background: The laboratory diagnosis of Chagas disease is challenging because the usefulness of different diagnostic tests will depend on the stage of the disease. Serology is the preferred method for patients in the chronic phase, whereas PCR can be successfully used to diagnose acute and congenital cases. Here we present data using a combination of three TaqMan PCR assays to detect T. cruzi DNA in clinical specimens. Methods/Principal Findings: Included in the analysis were DNA extracted from 320 EDTA blood specimens, 18 heart tissue specimens, 6 umbilical cord blood specimens, 2 skin tissue specimens and 3 CSF specimens. For the blood specimens both whole blood and buffy coat fraction were analyzed. The specimens were from patients living in the USA, with suspected exposure to T. cruzi through organ transplantation, contact with triatomine bugs or laboratory accidents, and from immunosuppressed patients with suspected Chagas disease reactivation. Real-time PCR was successfully used to diagnose acute and Chagas disease reactivation in 20 patients, including one case of organ-transmitted infection and one congenital case. Analysis of buffy coat fractions of EDTA blood led to faster diagnosis in six of these patients compared to whole blood analysis. The three real-time PCR assays produced identical results for 94% of the specimens. The major reason for discrepant results was variable sensitivity among the assays, but two of the real-time PCR assays also produced four false positive results. Conclusions/Significance: These data strongly indicate that at least two PCR assays with different performances should be combined to increase the accuracy. This evaluation also highlights the benefit of extracting DNA from the blood specimen's buffy coat to increase the sensitivity of PCR analysis.Fil: Qvarnstrom, Yvonne. Centers for Disease Control and Prevention; Estados UnidosFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Veron, Vincent. Centre hospitalier Andrée-Rosemon; Guayana FrancesaFil: Aznar, Christine. Centre hospitalier Andrée-Rosemon; Guayana FrancesaFil: Steurer, Francis. Centers for Disease Control and Prevention; Estados UnidosFil: da Silva, Alexandre J.. Centers for Disease Control and Prevention; Estados Unido

Lower risk for cardiovascular mortality for patients with root filled teeth in a Finnish population

AimTo investigate the relationship of radiographic evidence of root filled teeth to cardiovascular outcomes.MethodologyBaseline data for 506 subjects including 256 angiographically verified heart disease patients and 250 matched cardiologically healthy controls participating in the Kuopio Oral Health and Heart study were collected in 1995â 1996. Cardiovascular disease (CVD) mortalities were accrued until 31 May 2015 and appended to the baseline data. Mortality status data were obtained from the Finnish National Death Register where all mortality cases and the causes of death are compiled for all Finnish citizens. Of the 506 participants, 473 subjects who had no missing values in the predictor, outcome or confounding factors were included in the analyses to assess the relationship of radiographic evidence of root filled teeth with prevalent coronary artery disease (CAD) cross sectionally and also with CVD mortality longitudinally. Multivariable logistic regression was used for the crossâ sectional part and proportional hazard regression analyses for the longitudinal part of the study were used adjusting for age, sex, smoking, edentulism, diabetes, hypertension, total/HDL cholesterol ratio and income. Additionally, whether this association was independent of periodontitis, and a systemic marker of inflammation, serum Câ reactive protein (CRP) was examined.ResultsHaving â ¥1 root filled teeth was associated with 84% lower odds of prevalent CAD with Odds Ratio (OR) = 0.16, 95% confidence interval (CI) 0.09â 0.28, P < 0.0001. The OR for edentulism was 1.32 (CI: 0.73â 2.38), P = 0.36, suggesting a nonsignificant increase in risk. Prospectively, having at least one root filled teeth was associated with a 49% lower risk of CVD mortality (hazard ratio [HR] = 0.51, CI = 0.27â 0.97, P = 0.04) whilst edentulism was associated with nonsignificantly increased risk for CVD mortality: HR = 1.25 (CI: 0.65â 2.42), P = 0.36. Adjustment for periodontitis or serum CRP levels changed the OR or HR slightly but the associations remained significant.ConclusionsHaving â ¥1 root filled teeth was associated with significantly lower odds for prevalent CAD cross sectionally and lower risk of cardiovascular mortality prospectively. These reduced associations with CVD were independent of periodontitis or serum CRP levels.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/139934/1/iej12772_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/139934/2/iej12772.pd

Variations in gene organization and DNA uptake signal sequence in the folP region between commensal and pathogenic Neisseria species

BACKGROUND: Horizontal gene transfer is an important source of genetic variation among Neisseria species and has contributed to the spread of resistance to penicillin and sulfonamide drugs in the pathogen Neisseria meningitidis. Sulfonamide resistance in Neisseria meningitidis is mediated by altered chromosomal folP genes. At least some folP alleles conferring resistance have been horizontally acquired from other species, presumably from commensal Neisseriae. In this work, the DNA sequence surrounding folP in commensal Neisseria species was determined and compared to corresponding regions in pathogenic Neisseriae, in order to elucidate the potential for inter-species DNA transfer within this region. RESULTS: The upstream region of folP displayed differences in gene order between species, including an insertion of a complete Correia element in Neisseria lactamica and an inversion of a larger genomic segment in Neisseria sicca, Neisseria subflava and Neisseria mucosa. The latter species also had DNA uptake signal sequences (DUS) in this region that were one base different from the DUS in pathogenic Neisseriae. Another interesting finding was evidence of a horizontal transfer event from Neisseria lactamica or Neisseria cinerea that introduced a novel folP allele to the meningococcal population. CONCLUSION: Genetic recombination events immediately upstream of folP and horizontal transfer have resulted in sequence differences in the folP region between the Neisseria species. This variability could be a consequence of the selective pressure on this region exerted by the use of sulfonamide drugs

Adaptive coloration in pied flycatchers (Ficedula hypoleuca)-The devil is in the detail

Understanding the origin and persistence of phenotypic variation within and among populations is a major goal in evolutionary biology. However, the eagerness to find unadulterated explanatory models in combination with difficulties in publishing replicated studies may lead to severe underestimations of the complexity of selection patterns acting in nature. One striking example is variation in plumage coloration in birds, where the default adaptive explanation often is that brightly colored individuals signal superior quality across environmental conditions and therefore always should be favored by directional mate choice. Here, we review studies on the proximate determination and adaptive function of coloration traits in male pied flycatchers (Ficedula hypoleuca). From numerous studies, we can conclude that the dark male color phenotype is adapted to a typical northern climate and functions as a dominance signal in male-male competition over nesting sites, and that the browner phenotypes are favored by relaxed intraspecific competition with more dominant male collared flycatchers (Ficedula albicollis) in areas where the two species co-occur. However, the role of avoidance of hybridization in driving character displacement in plumage between these two species may not be as important as initially thought. The direction of female choice on male coloration in pied flycatchers is not simply as opposite in direction in sympatry and allopatry as traditionally expected, but varies also in relation to additional contexts such as climate variation. While some of the heterogeneity in the observed relationships between coloration and fitness probably indicate type 1 errors, we strongly argue that environmental heterogeneity and context-dependent selection play important roles in explaining plumage color variation in this species, which probably also is the case in many other species studied in less detail.Peer reviewe

Number of teeth, C‐reactive protein, fibrinogen and cardiovascular mortality: a 15‐year follow‐up study in a Finnish cohort

Aim To test whether the number of teeth, an inverse proxy for composite oral infection scores is associated with better survival. Materials and Methods The Kuopio Oral Health and Heart study initiated a case–control study in 1995–1996 consisting of 256 consecutive coronary artery disease patients and 250 age and gender‐matched controls. We appended the mortality data and formulated a longitudinal study. By May 31st, 2011, 124 mortalities had occurred and 80 of which were of cardiovascular origin. Using Cox proportional hazards models, we assessed the association of the teeth group (Teethgrp) – consisting of 10 teeth – with cardiovascular and all‐cause mortality after 15.8 years of median follow‐up. Results In multivariate models, with the edentulous state as reference, one level increase in Teethgrp was associated with significantly increased survival from cardiovascular disease (CVD) mortality with a Hazard Ratio (HR) 0.73, p ‐value = 0.02 but not with all‐cause mortality (HR = 0.87, p  = 0.13). The findings were not mediated by C‐reactive protein (CRP) levels ≥3 mg/L or by median fibrinogen levels, but were mediated by CRP levels >5 mg/L. Conclusion Each increment of 10 teeth from the edentulous state was associated with a 27% improved CVD survival, independent of low‐grade systemic inflammation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102655/1/jcpe12192.pd

Novel insights on the genetic population structure of human-infecting Cyclospora spp. and evidence for rapid subtype selection among isolates from the USA.

Human-infecting Cyclospora was recently characterized as three species, two of which (C. cayetanensis and C. ashfordi) are currently responsible for all known human infections in the USA, yet much remains unknown about the genetic structure within these two species. Here, we investigate Cyclospora genotyping data from 2018 through 2022 to ascertain if there are temporal patterns in the genetic structure of Cyclospora parasites that cause infections in US residents from year to year. First, we investigate three levels of genetic characterization: species, subpopulation, and strain, to elucidate annual trends in Cyclospora infections. Next, we determine if shifts in genetic diversity can be linked to any of the eight loci used in our Cyclospora genotyping approach. We observed fluctuations in the abundance of Cyclospora types at the species and subpopulation levels, but no significant temporal trends were identified; however, we found recurrent and sporadic strains within both C. ashfordi and C. cayetanensis. We also uncovered major shifts in the mitochondrial genotypes in both species, where there was a universal increase in abundance of a specific mitochondrial genotype that was relatively abundant in 2018 but reached near fixation (was observed in over 96% of isolates) in C. ashfordi by 2022. Similarly, this allele jumped from 29% to 82% relative abundance of isolates belonging to C. cayetanensis. Overall, our analysis uncovers previously unknown temporal-genetic patterns in US Cyclospora types from 2018 through 2022 and is an important step to presenting a clearer picture of the factors influencing cyclosporiasis outbreaks in the USA

Assortative mating in an ecological context:Effects of mate choice errors and relative species abundance on the frequency and asymmetry of hybridization

The frequency and asymmetry of mixed-species mating set the initial stage for the ecological and evolutionary implications of hybridization. How such patterns of mixed-species mating, in turn, are influenced by the combination of mate choice errors and relative species abundance remains largely unknown. We develop a mathematical model that generates predictions for how relative species abundances and mate choice errors affect hybridization patterns. When mate choice errors are small (5%), the highest hybridization frequency occurs when species occur in equal proportions. Furthermore, females of the less abundant species are overrepresented in mixed-species matings. We compare our theoretical predictions with empirical data on naturally hybridizing Ficedula flycatchers and find that hybridization is highest when the two species occur in equal abundance, implying rather high mate choice errors. We discuss ecological and evolutionary implications of our findings and encourage future work on hybrid zone dynamics that take demographic aspects, such as relative species abundance, into account

Application of a universal parasite diagnostic test to biological specimens collected from animals.

A previously described universal parasite diagnostic (nUPDx) based on PCR amplification of the 18S rDNA and deep-amplicon sequencing, can detect human blood parasites with a sensitivity comparable to real-time PCR. To date, the efficacy of this assay has only been assessed on human blood. This study assessed the utility of nUPDx for the detection of parasitic infections in animals using blood, tissues, and other biological sample types from mammals, birds, and reptiles, known to be infected with helminth, apicomplexan, or pentastomid parasites (confirmed by microscopy or PCR), as well as negative samples. nUPDx confirmed apicomplexan and/or nematode infections in 24 of 32 parasite-positive mammals, while also identifying several undetected coinfections. nUPDx detected infections in 6 of 13 positive bird and 1 of 2 positive reptile samples. When applied to 10 whole parasite specimens (worms and arthropods), nUPDx identified all to the genus or family level, and detected one incorrect identification made by morphology. Babesia sp. infections were detected in 5 of the 13 samples that were negative by other diagnostic approaches. While nUPDx did not detect PCR/microscopy-confirmed trichomonads or amoebae in cloacal swabs/tissue from 8 birds and 2 reptiles due to primer template mismatches, 4 previously undetected apicomplexans were detected in these samples. Future efforts to improve the utility of the assay should focus on validation against a larger panel of tissue types and animal species. Overall, nUPDx shows promise for use in both veterinary diagnostics and wildlife surveillance, especially because species-specific PCRs can miss unknown or unexpected pathogens

The mitochondrial genome of Angiostrongylus mackerrasae as a basis for molecular, epidemiological and population genetic studies

BACKGROUND: Angiostrongylus mackerrasae is a metastrongyloid nematode endemic to Australia, where it infects the native bush rat, Rattus fuscipes. This lungworm has an identical life cycle to that of Angiostrongylus cantonensis, a leading cause of eosinophilic meningitis in humans. The ability of A. mackerrasae to infect non-rodent hosts, specifically the black flying fox, raises concerns as to its zoonotic potential. To date, data on the taxonomy, epidemiology and population genetics of A. mackerrasae are unknown. Here, we describe the mitochondrial (mt) genome of A. mackerrasae with the aim of starting to address these knowledge gaps. METHODS: The complete mitochondrial (mt) genome of A. mackerrasae was amplified from a single morphologically identified adult worm, by long-PCR in two overlapping amplicons (8 kb and 10 kb). The amplicons were sequenced using the MiSeq Illumina platform and annotated using an in-house pipeline. Amino acid sequences inferred from individual protein coding genes of the mt genomes were concatenated and then subjected to phylogenetic analysis using Bayesian inference. RESULTS: The mt genome of A. mackerrasae is 13,640 bp in size and contains 12 protein coding genes (cox1-3, nad1-6, nad4L, atp6 and cob), and two ribosomal RNA (rRNA) and 22 transfer RNA (tRNA) genes. CONCLUSIONS: The mt genome of A. mackerrasae has similar characteristics to those of other Angiostrongylus species. Sequence comparisons reveal that A. mackerrasae is closely related to A. cantonensis and the two sibling species may have recently diverged compared with all other species in the genus with a highly specific host selection. This mt genome will provide a source of genetic markers for explorations of the epidemiology, biology and population genetics of A. mackerrasae