scholarly journals Rapid and real-time identification of fungi up to species level with long amplicon Nanopore sequencing from clinical samples

2020 ◽  
Author(s):  
Sara D’Andreano ◽  
Anna Cuscó ◽  
Olga Francino
Keyword(s):  
Real Time ◽  
Clinical Signs ◽  
Amplicon Sequencing ◽  
Species Level ◽  
Clinical Samples ◽  
Microsporum Canis ◽  
Mock Community ◽  
Candida Spp ◽  
Taxonomic Assignment ◽  
Malassezia Pachydermatis

Abstract The availability of long-read technologies, like Oxford Nanopore Technologies, provides the opportunity to sequence longer fragments of the fungal ribosomal operon, up to 6 Kb (18S-ITS1-5.8S-ITS2-28S), and to improve the taxonomy assignment of the communities up to species level and in real-time. We assess the applicability for taxonomic assignment of amplicons targeting a 3.5 Kb region (V3 18S-ITS1-5.8S-ITS2-28S D2) and a 6 Kb region (V1 18S-ITS1-5.8S-ITS2-28S D12) with the What's in my pot (WIMP) classifier. We used the ZymoBIOMICSTM mock community and different microbiological fungal cultures as positive controls. Long amplicon sequencing correctly identified Saccharomyces cerevisiae and Cryptococcus neoformans from the mock community and Malassezia pachydermatis, Microsporum canis, and Aspergillus fumigatus from the microbiological cultures. Besides, we identified Rhodotorula graminis in a culture mislabeled as Candida spp. We applied the same approach to external otitis in dogs. Malassezia was the dominant fungal genus in dogs' ear skin, whereas M. pachydermatis was the main species in the healthy sample. Conversely, we identified a higher representation of M. globosa and M. sympodialis in otitis affected samples. We demonstrate the suitability of long ribosomal amplicons to characterize the fungal community of complex samples, either healthy or with clinical signs of infection.

scholarly journals Rapid and real-time identification of fungi up to the species level with long amplicon Nanopore sequencing from clinical samples

2020 ◽  
Author(s):  
Sara D’Andreano ◽  
Anna Cuscó ◽  
Olga Francino
Keyword(s):  
Real Time ◽  
Clinical Signs ◽  
Amplicon Sequencing ◽  
Species Level ◽  
Clinical Samples ◽  
Microsporum Canis ◽  
Mock Community ◽  
Malassezia Pachydermatis ◽  
Long Read ◽  
Real Time Identification

ABSTRACTThe availability of long-read technologies, like Oxford Nanopore Technologies, provides the opportunity to sequence longer fragments of the fungal ribosomal operon, up to 6 Kb (18S-ITS1-5.8S-ITS2-28S), and to improve the taxonomy assignment of the communities up to the species level and in real-time. We assess the taxonomy skills of amplicons targeting a 3.5 Kb region (V3 18S-ITS1-5.8S-ITS2-28S D2) and a 6 Kb region (V1 18S-ITS1-5.8S-ITS2-28S D12) with the What’s in my pot (WIMP) classifier. We used the ZymoBIOMICS™ mock community and different microbiological fungal cultures as positive controls. Long amplicon sequencing correctly identified Saccharomyces cerevisiae and Cryptococcus neoformans from the mock community and Malassezia pachydermatis, Microsporum canis, and Aspergillus fumigatus from the microbiological cultures. Besides, we identified Rhodotorula graminis in a culture mislabeled as Candida spp.We applied the same approach to external otitis in dogs. Malassezia was the dominant fungal genus in dogs’ ear skin, whereas M. pachydermatis was the main species in the healthy sample. Conversely, we identified a higher representation of M. globosa and M. sympodialis in otitis affected samples. We demonstrate the suitability of long ribosomal amplicons to characterize the fungal community of complex samples, either healthy or with clinical signs of infection.

scholarly journals Real-time loop-mediated isothermal amplification for rapid detection of Enterocytozoon hepatopenaei

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5993 ◽  
Author(s):  
Shao-Xin Cai ◽  
Fan-De Kong ◽  
Shu-Fei Xu ◽  
Cui-Luan Yao
Keyword(s):  
Real Time ◽  
Clinical Signs ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Lamp Method ◽  
The Real ◽  
Loop Mediated Isothermal Amplification ◽  
Enterocytozoon Hepatopenaei ◽  
Time Loop

Background Enterocytozoon hepatopenaei (EHP) is a newly emerged microsporidian parasite that causes retarded shrimp growth in many countries. But there are no effective approaches to control this disease to date. The EHP could be an immune risk factor for increased dissemination of other diseases. Further, EHP infection involves the absence of obvious clinical signs and it is difficult to identify the pathogen through visual examination, increasing the risk of disease dissemination. It is urgent and necessary to develop a specific, rapid and sensitive EHP-infected shrimp diagnostic method to detect this parasite. In the present study, we developed and evaluated a rapid real-time loop-mediated isothermal amplification (real-time LAMP) for detection of EHP. Methods A rapid and efficient real-time LAMP method for the detection of EHP has been developed. Newly emerged EHP pathogens in China were collected and used as the sample, and three sets of specificity and sensitivity primers were designed. Three other aquatic pathogens were used as templates to test the specificity of the real-time LAMP assay. Also, we compared the real-time LAMP with the conventional LAMP by the serial dilutions of EHP DNA and their amplification curves. Application of real-time LAMP was carried out with clinical samples. Results Positive products were amplified only from EHP, but not from other tested species, EHP was detected from the clinical samples, suggesting a high specificity of this method. The final results of this assay were available within less than 45 min, and the initial amplification curve was observed at about 6 min. We found that the amplification with an exponential of sixfold dilutions of EHP DNA demonstrated a specific positive signal by the real-time LAMP, but not for the LAMP amplicons from the visual inspection. The real-time LAMP amplification curves demonstrated a higher slope than the conventional LAMP. Discussion In this study, pathogen virulence impacts have been increased in aquaculture and continuous observation was predominantly focused on EHP. The present study confirmed that the real-time LAMP assay is a promising and convenient method for the rapid identification of EHP in less time and cost. Its application greatly aids in the detection, surveillance, and prevention of EHP.

scholarly journals Mycotic zoonoses risk from clipping instruments in Pet Shops in Sinop – MT

2021 ◽  
Vol 14 (10) ◽  
Author(s):  
Rayssa Tuanna Schmidt Balbinot ◽  
Dâmaris Carolini Teodoro Alves Costa ◽  
Neocimar Saraiva Correia ◽  
Fabiana Cristina Donofrio
Keyword(s):  
Filamentous Fungi ◽  
Microsporum Canis ◽  
Candida Spp ◽  
Malassezia Pachydermatis ◽  
Pathogenic Potential ◽  
Daily Lives ◽  
Sabouraud Agar ◽  
Urease Test ◽  
Close Relationship ◽  
The City

Pets have conquered the daily lives of families worldwide and because they have a close relationship with humans, they can transmit mycotic zoonoses such as dermatophytosis, malassezioses and candidosis. Studies show that fungi with pathogenic potential have already been granted in clipping instruments and bath articles in veterinary clinics and in Pet Shops. In the absence of data in the city of Sinop - MT, this study aimed to isolate fungi with pathogenic potential in clipping instruments used in the routine of Pet Shops and to identify etiological agents capable of causing mycotic zoonoses. Samples were carried out in 18 clipping instruments, without cleaning, from 10 Pet Shops (A, B, C, D, E, F, G, H, I, J) in the city of Sinop - MT with swabs and sterile carpets square and then seeded in Sabouraud agar with 0.05% chloramphenicol and mycobiotic agar, incubated at room temperature (25°C) for 30 - 45 days. The dermatophytes and non-dermatophyte filamentous fungi were identified using the microculture technique and urease test and as yeasts using a urease test, zymogram, auxanogram, germ tube and corn meal agar with polysorbate 80. Of the samples from 18 clipping instruments, 15 were positive for at least one fungal genus. The yeast that showed a high prevalence of isolation in clipping instruments from the studied Pet Shops (B, C, D, E, H and J) was Malassezia pachydermatis (86.7%), followed by the genus Candida spp. (C and D; 26.7%), filamentous fungi of the genus Aspergillus spp. (A; 13.3%), Microsporum canis (B; 13.3%) and Trichosporon spp. (J, 6.7%). Our results demonstrate that Pet Shops treat a large number of animals daily in a single environment, which may amplify the risk of spreading zoonoses, as an asymptomatic or sick carrier animal can potentially transmit the microorganism to other animals inside the store and thus to a large number of new pet owners. There is a need to reinforce the commercial requirements specialized in bathing and grooming in the city of Sinop - MT on the good practices of cleaning and disinfection of the elements used and the environment, eliminating or eliminating the risk of contracting mycotic zoonoses.

scholarly journals Co-detection of the measles vaccine and wild-type virus by real-time PCR: public health laboratory protocol

2021 ◽  
Vol 3 (11) ◽  
Author(s):  
Kamelia R. Stanoeva ◽  
Robert H. G. Kohl ◽  
Rogier Bodewes
Keyword(s):  
Public Health ◽  
Real Time ◽  
Measles Virus ◽  
Clinical Signs ◽  
Control Measures ◽  
Wild Type Virus ◽  
Clinical Samples ◽  
Wild Type ◽  
Characteristic Analysis ◽  
Genotype A

In rare cases vaccination with the measles virus vaccine genotype A (MeVA) may cause a vaccine reaction with clinical signs similar to infection with wild-type measles virus (MeVwt). Rapid differentiation between MeVA and MeVwt infection is important for taking adequate public health measures. Recently, a few MeVA real-time reverse-transcription quantitative PCR methods (RT-qPCRs) were described that can distinguish between MeVA and MeVwt. However, detection of MeVA does in theory not exclude infection with MeVwt. In the present study, we established a protocol for determination of co-infections with MeVA and MeVwt. To this end, MeVA RT-qPCRs were used in combination with the routine measles virus (MeV) RT-qPCR, and the results suggested that the differences between the RT-qPCR Ct values (delta Ct, ∆Ct) could be used as criteria. Subsequently, we tested samples from vaccine-associated measles cases that were confirmed by genotyping. In addition, experimental mixtures of MeVA and MeVwt were tested in different concentrations. All tested MeVA clinical samples had ∆Ct ≤3.6. The results of experimental mixtures showed a mean ∆Ct ≤2.8 for genotype A alone and >3.2 when combined with either genotype B3 or D8. The results of a receiver operator characteristic analysis indicated that the optimum ∆Ct for use as a cut-off value was 3.5, while with ∆Ct values of 2.9 and 3.7 sensitivity and specificity were respectively 1.00. Thus, ∆Ct could be used to exclude the presence of MeVwt if MeVA is detected and ∆Ct is <2.9, while ∆Ct >3.7 were highly suggestive of co-infection and ≥2.9 ∆Ct <3.7 warranted additional confirmation, such as next-generation sequencing. This RT-qPCR-based protocol could be used for the exclusion of infection with MeVwt in cases with vaccine-associated measles reaction, crucial for the timely implementation of public health prevention and control measures.

scholarly journals Porcine Gammaherpesviruses in Italian Commercial Swine Population: Frequent but Harmless

Pathogens ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 47
Author(s):  
Giovanni Franzo ◽  
Michele Drigo ◽  
Matteo Legnardi ◽  
Laura Grassi ◽  
Maria Luisa Menandro ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Clinical Signs ◽  
Northern Italy ◽  
Porcine Circovirus ◽  
Swine Industry ◽  
Positive Interaction ◽  
High Prevalence ◽  
Swine Population ◽  
Clinical Conditions

Differently from alpha- and betaherpesviruses affecting swine, interest in the recently discovered Suid gammaherpesvirus 3, Suid gammaherpesvirus 4, and Suid gammaherpesvirus 5, also known as porcine lymphotropic herpesviruses (PLHV-1, PLHV-2, and PLHV-3), has largely focused on their role as potential zoonotic agents in cases of xenotransplantation. However, their role as primary pathogens of swine or as co-factors for other lymphotropic infections has essentially been neglected. The present study aims at filling this gap, evaluating the association between PLHVs infection and different clinical conditions and/or porcine circovirus (PCV) co-infection. One hundred seventy-six samples were obtained from different animals located in a high-density pig area of Northern Italy in the period 2017–2020. The presence of PLHVs and PCVs was tested and quantified by specific real-time PCR: PLHVs were widespread among pigs (PLHV-1, PLHV-2, and PLHV-3 prevalence was 28.97%, 10.79%, and 4.54%, respectively) and detected in all considered tissues and clinical conditions. Frequent co-infections were also observed among PLHVs and with PCVs, although a significant association was not detected with the exception of a positive interaction between PLHV-1 and PLHV-3, and a negative one between PLHV-2 and PCV-2. Significantly, no association between PLHVs, alone or in co-infection, emerged with any of the considered clinical signs, their frequency being comparable between healthy and diseased animals. Based on these pieces of evidence and despite their high prevalence, PLHVs’ relevance for the swine industry appears negligible, either as primary pathogens or as predisposing factors for circovirus-induced diseases.

scholarly journals Ultra-accurate microbial amplicon sequencing with synthetic long reads

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Benjamin J. Callahan ◽  
Dmitry Grinevich ◽  
Siddhartha Thakur ◽  
Michael A. Balamotis ◽  
Tuval Ben Yehezkel
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
Amplicon Sequencing ◽  
Species Level ◽  
Full Length ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Strain Identification ◽  
Long Reads ◽  
Long Read

Abstract Background Out of the many pathogenic bacterial species that are known, only a fraction are readily identifiable directly from a complex microbial community using standard next generation DNA sequencing. Long-read sequencing offers the potential to identify a wider range of species and to differentiate between strains within a species, but attaining sufficient accuracy in complex metagenomes remains a challenge. Methods Here, we describe and analytically validate LoopSeq, a commercially available synthetic long-read (SLR) sequencing technology that generates highly accurate long reads from standard short reads. Results LoopSeq reads are sufficiently long and accurate to identify microbial genes and species directly from complex samples. LoopSeq perfectly recovered the full diversity of 16S rRNA genes from known strains in a synthetic microbial community. Full-length LoopSeq reads had a per-base error rate of 0.005%, which exceeds the accuracy reported for other long-read sequencing technologies. 18S-ITS and genomic sequencing of fungal and bacterial isolates confirmed that LoopSeq sequencing maintains that accuracy for reads up to 6 kb in length. LoopSeq full-length 16S rRNA reads could accurately classify organisms down to the species level in rinsate from retail meat samples, and could differentiate strains within species identified by the CDC as potential foodborne pathogens. Conclusions The order-of-magnitude improvement in length and accuracy over standard Illumina amplicon sequencing achieved with LoopSeq enables accurate species-level and strain identification from complex- to low-biomass microbiome samples. The ability to generate accurate and long microbiome sequencing reads using standard short read sequencers will accelerate the building of quality microbial sequence databases and removes a significant hurdle on the path to precision microbial genomics.

scholarly journals Virus Adaptation Following Experimental Infection of Chickens with a Domestic Duck Low Pathogenic Avian Influenza Isolate from the 2017 USA H7N9 Outbreak Identifies Polymorphic Mutations in Multiple Gene Segments

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1166
Author(s):  
Klaudia Chrzastek ◽  
Karen Segovia ◽  
Mia Torchetti ◽  
Mary Lee Killian ◽  
Mary Pantin-Jackwood ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Experimental Infection ◽  
Clinical Signs ◽  
Interspecies Transmission ◽  
Clinical Samples ◽  
Domestic Duck ◽  
Receptor Binding Site ◽  
Synonymous Mutations ◽  
Field Samples ◽  
Virus Adaptation

In March 2017, highly pathogenic (HP) and low pathogenic (LP) avian influenza virus (AIV) subtype H7N9 were detected from poultry farms and backyard birds in several states in the southeast United States. Because interspecies transmission is a known mechanism for evolution of AIVs, we sought to characterize infection and transmission of a domestic duck-origin H7N9 LPAIV in chickens and genetically compare the viruses replicating in the chickens to the original H7N9 clinical field samples used as inoculum. The results of the experimental infection demonstrated virus replication and transmission in chickens, with overt clinical signs of disease and shedding through both oral and cloacal routes. Unexpectedly, higher levels of virus shedding were observed in some cloacal swabs. Next generation sequencing (NGS) analysis identified numerous non-synonymous mutations at the consensus level in the polymerase genes (i.e., PA, PB1, and PB2) and the hemagglutinin (HA) receptor binding site in viruses recovered from chickens, indicating possible virus adaptation in the new host. For comparison, NGS analysis of clinical samples obtained from duck specimen collected during the outbreak indicated three polymorphic sides in the M1 segment and a minor population of viruses carrying the D139N (21.4%) substitution in the NS1 segment. Interestingly, at consensus level, A/duck/Alabama (H7N9) had isoleucine at position 105 in NP protein, similar to HPAIV (H7N9) but not to LPAIV (H7N9) isolated from the same 2017 influenza outbreak in the US. Taken together, this work demonstrates that the H7N9 viruses could readily jump between avian species, which may have contributed to the evolution of the virus and its spread in the region.

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