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Scedosporium/Lomentospora fungi are increasingly recognized pathogens. As these fungi are resistant to many antifungal agents, early diagnosis is essential for initiating targeted drug therapy. Here, we review the microbiological tools for the detection and diagnosis of invasive scedosporiosis and lomentosporiosis. Of over 10 species, Lomentospora prolificans, Scedosporium apiospermum, S. boydii and S. aurantiacum cause the majority of infections. Definitive diagnosis relies on one or more of visualization, isolation or detection of the fungus from clinical specimens by microscopy techniques, culture and molecular methods such as panfungal PCR or genus-/species-specific multiplex PCR. For isolation from respiratory tract specimens, selective media have shown improved isolation rates. Species identification is achieved by macroscopic and microscopic examination of colonies, but species should be confirmed by ITS with or without β-tubulin gene sequencing or other molecular methods. Matrix-assisted laser desorption ionization-time of flight mass spectrometry databases are improving but may need supplementation by in-house spectra for species identification. Reference broth microdilution methods is preferred for antifungal susceptibility testing. Next-generation sequencing technologies have good potential for characterization of these pathogens. Diagnosis of Scedosporium/Lomentospora infections relies on multiple approaches encompassing both phenotypic- and molecular-based methods.

Aims: We described the presence of Helicobacter pylori (HP) and estimated the prevalence of primary and secondary resistance using molecular detection in gastric biopsies of Ecuadorian patients. Methods and Results: 66.7% (238/357) of the patients demonstrated the presence of HP using CerTest qPCR. Of these, 69.79% (104/149) were without previous HP eradication treatment and 64.42% (134/208) with prior HP eradication treatment. The mutation‐associated resistance rate for clarithromycin was 33.64% (primary resistance) and 32.82% (secondary resistance), whereas that in levofloxacin the primary and secondary resistance was 37.38% and 42%, respectively. For tetracycline and rifabutin, primary and secondary resistance was 0%. Primary and secondary resistance for metronidazole and amoxicillin could not be evaluated by genotypic methods (PCR and sequencing). Conclusions: The analysis of mutations in gyrA, 23S rRNA and 16S rRNA is useful to detect bacterial resistance as a guide for eradication therapy following failure of the first‐line regimen. Significance and Impact of the Study: This study carried out in an Ecuadorian population indicates that the resistance of HP to first‐line antibiotics is high, which may contribute to the high rates of treatment failure, and other treatment alternatives should be considered. [ABSTRACT FROM AUTHOR]

Soybean plants showing symptoms of root rot were collected from fields in Manitoba, Canada, in 2017, to determine the causal agent of the disease. Of 240 isolated strains, including some known as root rot pathogens, five were identified as Fusarium sporotrichioides Sherb. based on morphological characteristics. All five isolates were pathogenic when inoculated on soybean under controlled conditions, and resulted in typical root rot symptoms. In addition, shoot length, root length, and fresh weight were significantly reduced. A PCR-based diagnostic test was developed, which specifically amplified the Trichothecene gene cluster in F. sporotrichioides. The primer SPO1 amplified a 541 bp fragment in fungal isolates originally isolated from commercial soybean crops, and from artificially infected roots of soybean. Trichothecene (TRI) gene expression was detected in infected soybean roots using RT-PCR analysis with TRI gene-specific primers. This is the first report of F. sporotrichioides as one of causal agents of root rot in soybean in Canada. [ABSTRACT FROM AUTHOR]

Objective: To develop N. dimidiatum-specific single PCR-based identification with DNA sequences of nuclear ribosomal internal transcribed spacer (ITS) 1 region primers to facilitate the rapid and accurate detection of N. dimidiatum. Methods: N. dimidiatum-specific PCR primers were designed based on the sequence of the internal transcribed spacer 1 region, which is located between 18S and 5.8S nuclear rDNA. Fungal DNA extracted from common causative species for superficial fungal infection including: 2 strains of N. dimidiatum, 9 species of dermatophyte (DMP) and 25 species of non-dermatophyte (NDM) colonies grown on culture plates were used for PCR analysis. Also, 30 clinical specimens collected from 30 patients clinically diagnosed with fungal nail and feet infection who attended Dermatology clinic Siriraj Hospital during October 2015 to November 2015 were used for PCR assay. Results: Using N. dimidiatum-specific PCR primers, the PCR product was amplified from two standard strains of N. dimidiatum, and there was no amplification from other DMP or NDM species. Regarding sensitivity as lower limit of detection, this PCR method was able to detect 10 pg of N. dimidiatum DNA with ethidium bromide staining and could detect N. dimidiatum in clinical samples. Conclusion: This newly developed N. dimidiatum-specific PCR identification system is rapid, sensitive, and specific. This diagnostic method will facilitate early and accurate diagnosis and accelerate appropriate treatment in patients with N. dimidiatum infection.

Scedosporium/Lomentospora fungi are increasingly recognized pathogens. As these fungi are resistant to many antifungal agents, early diagnosis is essential for initiating targeted drug therapy. Here, we review the microbiological tools for the detection and diagnosis of invasive scedosporiosis and lomentosporiosis. Of over 10 species, Lomentospora prolificans, Scedosporium apiospermum, S. boydii and S. aurantiacum cause the majority of infections. Definitive diagnosis relies on one or more of visualization, isolation or detection of the fungus from clinical specimens by microscopy techniques, culture and molecular methods such as panfungal PCR or genus-/species-specific multiplex PCR. For isolation from respiratory tract specimens, selective media have shown improved isolation rates. Species identification is achieved by macroscopic and microscopic examination of colonies, but species should be confirmed by ITS with or without β-tubulin gene sequencing or other molecular methods. Matrix-assisted laser desorption ionization-time of flight mass spectrometry databases are improving but may need supplementation by in-house spectra for species identification. Reference broth microdilution methods is preferred for antifungal susceptibility testing. Next-generation sequencing technologies have good potential for characterization of these pathogens. Diagnosis of Scedosporium/Lomentospora infections relies on multiple approaches encompassing both phenotypic- and molecular-based methods.

Aims The objective of this work was to design an amplified fragment length polymorphism ( AFLP)-derived specific primer for the detection of Fusarium solani aetiological agent of peanut brown root rot ( PBRR) in plant material and soil. Methods and Results Specific primers for the detection of the pathogen were designed based on an amplified region using AFLPs. The banding patterns by AFLPs showed that isolates from diseased roots were clearly distinguishable from others members of the F. solani species complex. Many bands were specific to F. solani PBRR, one of these fragments was selected and sequenced. Sequence obtained was used to develop specific PCR primers for the identification of pathogen in pure culture and in plant material and soil. Primer pair FS1/ FS2 amplified a single DNA product of 175 bp. Other fungal isolates occurring in soil, included F. solani non- PBRR, were not detected by these specific primers. The assay was effective for the detection of pathogen from diseased root and infected soils. Conclusions The designed primers for F. solani causing PBRR can be used in a PCR diagnostic protocol to rapidly and reliably detect and identify this pathogen. Significance and Impact of the Study These diagnostic PCR primers will aid the detection of F. solani causing PBRR in diseased root and natural infected soils. The method developed could be a helpful tool for epidemiological studies and to avoid the spread of this serious disease in new areas. [ABSTRACT FROM AUTHOR]

Parasite transmission is determined by the rate of contact between a susceptible host and an infective stage and susceptibility to infection given an exposure event. Attempts to measure levels of variation in exposure in natural populations can be especially challenging. The level of exposure to a major class of parasites, trophically transmitted parasites, can be estimated by investigating the host's feeding behaviour. Since the parasites rely on the ingestion of infective intermediate hosts for transmission, the potential for exposure to infection is inherently linked to the definitive host's feeding ecology. Here, we combined epidemiological data and molecular analyses (polymerase chain reaction) of the diet of the definitive host, the white-footed mouse ( Peromyscus leucopus), to investigate temporal and individual heterogeneities in exposure to infection. Our results show that the consumption of cricket intermediate hosts accounted for much of the variation in infection; mice that had consumed crickets were four times more likely to become infected than animals that tested negative for cricket DNA. In particular, pregnant female hosts were three times more likely to consume crickets, which corresponded to a threefold increase in infection compared with nonpregnant females. Interestingly, males in breeding condition had a higher rate of infection even though breeding males were just as likely to test positive for cricket consumption as nonbreeding males. These results suggest that while heterogeneity in host diet served as a strong predictor of exposure risk, differential susceptibility to infection may also play a key role, particularly among male hosts. By combining PCR analyses with epidemiological data, we revealed temporal variation in exposure through prey consumption and identified potentially important individual heterogeneities in parasite transmission. [ABSTRACT FROM AUTHOR]

Abstract: Coccidiosis are the major parasitic diseases in poultry and other domestic animals including the domestic rabbit (Oryctolagus cuniculus). Eleven distinct Eimeria species have been identified in this host, but no PCR-based method has been developed so far for unequivocal species differentiation. In this work, we describe the development of molecular diagnostic assays that allow for the detection and discrimination of the 11 Eimeria species that infect rabbits. We determined the nucleotide sequences of the ITS1 ribosomal DNAs and designed species-specific primers for each species. We performed specificity tests of the assays using heterologous sets of primers and DNA samples, and no cross-specific bands were observed. We obtained a detection limit varying from 500fg to 1pg, which corresponds approximately to 0.8–1.7 sporulated oocysts, respectively. The test reported here showed good reproducibility and presented a consistent sensitivity with three different brands of amplification enzymes. These novel diagnostic assays will permit population surveys to be performed with high sensitivity and specificity, thus contributing to a better understanding of the epidemiology of this important group of coccidian parasites. [Copyright &y& Elsevier]

Using ubiquitous primers which amplify the variable parts of kDNA minicircle of all Leishmania spp, we obtained for Leishmania (viannia) lainsoni a major band of 605 bp (band 1) shared with L. V. braziliensis and a minor 524 bp band (band 2) specific of L. V. lainsoni. The specificity of the two bands was examined through Southern blot hybridization of kDNA PCR obtained from reference strains belonging to L. braziliensis, L. mexicana, L. donovani complexes with L. V. lainsoni species. Band 1 was not specific of L. V. lainsoni since it hybridized with some isolates belonging to L. braziliensis complex. In contrast, band 2 was L. V. lainsoni specific. PCR-based detection followed by hybridization with the new L. V. lainsoni probe (Band 2) and L. V. braziliensis probe (564 bp), was assayed using sample from a pool of 25 females of Lutzomiya nuneztovari anglesi, blood, skin and liver samples of 18 mammals, spinal cords of four mammals and blood and cutaneous ulcers aspirates from 95 patents from Sub Andean region of La Paz, Bolivia. We observed a ositive hybridization of four patients lesions and the pool of L. nuneztovari anglesi with the L. V. lainsoni probe. It is the first time that L. V. lainsoni is observed in a cycle of transmission in Bolivia. PCR products of three patients lesions and the pool of L. nuneztovari anglesi were also hybridized with the specific probe of L. V. braziliensis suggesting mixed infection in this focus. [Copyright &y& Elsevier]

The aim was to determine how often the PCR method is used in different laboratories in Russia. In 2018, we conducted a questionnaire survey in diagnostic laboratories of medical organizations and the Centers of Hygiene and Epidemiology that performed PCR studies to identify microorganisms of the genus Bordetella in all 85 Russian regions. We found that in 2013 the PCR was used in 33 (38.8%) regions, but in 2017 the number of regions increased to 64 (75.3%). During 2013-2017 the study has not been applied in 21 regions. The number of PCR tests performed in the laboratories of medical organizations was significantly different. There has been an increase in the number of tests for the diagnosis of pertussis among people with clinical signs of infection and among contact persons in foci of infection. Compared to the Centers of Hygiene and Epidemiology, in medical organizations the rate of introduction of the PCR was higher. Between 2013 and 2017 the proportion of samples containing DNA B.pertussis decreased, but the proportion of samples containing DNA of other representatives of the genus Bordetella increased. Moreover, in the case of isolation DNA Bordetella spp. clinicians diagnose «Whooping cough, other unspecified organism», since there is no information on the species of the pathogen. Thus, in order to improve the diagnosis of pertussis, it is necessary to optimize PCR tests by including target genes that allow to identify of currently relevant DNAs of different representatives of the genus Bordetella., Competing Interests: The authors declare no conflict of interest.

A comprehensive review of diagnostic techniques for opportunistic systemic mycoses focused on invasive mould disease in immunocompromised patients is presented. We first analysed conventional diagnostic methods, such as microscopy examination, culture and radiology, underlining their limitations, which have led to the development of alternative methods, such as the detection of fungal components. Among these we highlight fungal antigen and DNA quantification, which make it possible to detect infections early and start appropriate treatment. We also briefly review the methods for carrying out susceptibility tests for antifungal drugs, including reference procedures, commercial techniques and their indications. Furthermore, we analyse the recommendations for therapeutic drug monitoring of antifungal agents in body fluids. [ABSTRACT FROM PUBLISHER]