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Pathogens
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Molecular Diagnostics for Infectious Diseases
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Molecular Diagnostics for Infectious Diseases

A special issue of Pathogens (ISSN 2076-0817).

Deadline for manuscript submissions: closed (15 July 2022) | Viewed by 36807

Special Issue Editor

Dr. Collette Bromhead

E-Mail Website
Guest Editor
School of Health Sciences, Massey University, Auckland 0632, New Zealand
Interests: molecular diagnostics; infectious disease; sexually transmitted infections; viral haemorrhagic fevers; point of care diagnostics; human papillomavirus; cervical cancer screening; capacity building; antimicrobial stewardship
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

In recent times, we have seen an explosion in the laboratory usage and public profile of molecular microbiology tools in humanity’s fight against the SARS-CoV-2 pandemic. Clinical laboratories have quickly adapted to high-volume RT-PCR testing of nasopharyngeal samples, and sequencing laboratories and bioinformaticians have stepped up with molecular epidemiological data and variant detection that have been essential to good public health advice. Indeed, we applaud all the international scientists and front-line laboratory health workers who have contributed to this monumental work.  

There are numerous challenges in implementing any new molecular diagnostic test. These new techniques are born in the research laboratory and their eventual evolution to routine use in the clinical or veterinary laboratory is dependent on multiple factors, including their complexity and performance, potential clinical impact, and cost. The detection of nucleic acids by molecular diagnostics does not always equate to the detection of a viable organism that is the cause of the patient’s disease. Pathogens detected may represent colonisation, asymptomatic infection, prolonged shedding after resolved infection, or other non-significant reasons. Sequence variants in rapidly mutating viral genomes can cause false negative results. Some sample types are challenging to extract adequate infectious particles from, lowering the sensitivity of molecular tests. Finally, it does not matter how well your test performs if you cannot get your patient to provide the required invasive sample or follow up the test result with the required treatment. 

In tackling some of these challenges in the context of COVID-19, current research has paved the way for innovation in all other areas of molecular diagnostics for infectious disease.

It is in this context of an explosion of change and new literature in molecular diagnostics that this Special Issue is being issued. We feel it is timely to provide this single source to showcase the highlights of this period of change, from all areas of veterinary and clinical molecular diagnostics for infectious disease.

FOCUS

The main focus of this Special Issue is to showcase the rapid development, advantages, and unique contribution of molecular tools in solving infectious disease challenges in the clinical, veterinary, and public health areas.

Both original and review articles are welcomed. Potential topics include, but are not limited to:

  1. New molecular microbiology tools as well as the application of existing techniques in new settings or novel ways.
  2. The application of sequencing and bioinformatics in diagnostics, epidemiologic investigations, strain/variant identification and resistance genotyping of pathogens for antimicrobial stewardship.
  3. Novel use of point of care molecular testing or non-invasive sample types.
  4. Molecular microbiology laboratory process improvements

PURPOSE

We welcome submissions that include molecular diagnostics for either human or veterinary infectious diseases, including bacterial, viral, fungal, parasites, and protists.

Dr. Collette Bromhead
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Pathogens is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • PCR
  • RTPCR
  • sequencing
  • bioinformatics
  • molecular diagnostics
  • zoonoses
  • infectious diseases
  • strain typing
  • metagenomic sequencing
  • resistance genotyping
  • molecular epidemiology
  • point of care
  • variant identification
  • microbiology
  • virology
  • fungi
  • parasites
  • protozoa
  • antimicrobial stewardship

Published Papers (15 papers)

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9 pages, 624 KiB  
Article
Culture-Independent Genotyping Improves Surveillance of Neisseria gonorrhoeae, Especially in Oropharyngeal Samples, the Netherlands, 2017 to 2018
by Michiel H. C. Slaats, Brian M. J. W. van der Veer, Lieke B. van Alphen, Christian J. P. A. Hoebe, Nicole H. T. M. Dukers-Muijrers and Petra F. G. Wolffs
Pathogens 2022, 11(11), 1344; https://0-doi-org.brum.beds.ac.uk/10.3390/pathogens11111344 - 14 Nov 2022
Viewed by 952
Abstract
It is important i to monitor the transmission and antimicrobial resistance of Neisseria gonorrhoeae (NG). Current surveillance relies on culturing, which frequently fails. Previously, a culture-independent genotyping method was developed based on NG multi-antigen sequence typing (NG-MAST). To determine whether crucial sequence types [...] Read more.
It is important i to monitor the transmission and antimicrobial resistance of Neisseria gonorrhoeae (NG). Current surveillance relies on culturing, which frequently fails. Previously, a culture-independent genotyping method was developed based on NG multi-antigen sequence typing (NG-MAST). To determine whether crucial sequence types (STs) are missed during culture-dependent surveillance, NG-positive NAAT samples were genotyped, and the results of the culture-positive and culture-negative samples were compared. In total, 196 NG-positive NAAT samples, from January 2017 until August 2018, which were also routinely cultured, were retrospectively included. Genotyping was successful in 152 NAAT samples (77.0%), 33 NAAT samples failed, and 11 NAAT samples showed possible mixed strain infections. Oropharyngeal samples (n = 16) showed the largest increase in typing rate from 6.3% (1/16) success in culture-dependent genotyping to 81.3% (13/16) in culture-independent genotyping. Nine genogroups (n ≥ 5 samples) were found; all included both culture-positive and culture-negative NG. However, culture-independent surveillance revealed 14 additional STs in the culture-negative samples. Overall, culture-dependent surveillance could detect all genogroups, indicating that major trends could be identified with culture-dependent surveillance. However, culture-independent surveillance provides more STs, mixed infections and more oropharyngeal samples, giving a more detailed view and could result in an earlier detection of outbreaks and transmission. Full article
(This article belongs to the Special Issue Molecular Diagnostics for Infectious Diseases)
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13 pages, 292 KiB  
Article
What Does 16S rRNA Gene-Targeted Next Generation Sequencing Contribute to the Study of Infective Endocarditis in Heart-Valve Tissue?
by Paula Santibáñez, Concepción García-García, Aránzazu Portillo, Sonia Santibáñez, Lara García-Álvarez, María de Toro and José A. Oteo
Pathogens 2022, 11(1), 34; https://0-doi-org.brum.beds.ac.uk/10.3390/pathogens11010034 - 29 Dec 2021
Cited by 7 | Viewed by 1615
Abstract
Infective endocarditis (IE) is a severe and life-threatening disease. Identification of infectious etiology is essential for establishing the appropriate antimicrobial treatment and decreasing mortality. The aim of this study was to explore the potential utility of metataxonomics for improving microbiological diagnosis of IE. [...] Read more.
Infective endocarditis (IE) is a severe and life-threatening disease. Identification of infectious etiology is essential for establishing the appropriate antimicrobial treatment and decreasing mortality. The aim of this study was to explore the potential utility of metataxonomics for improving microbiological diagnosis of IE. Here, next-generation sequencing (NGS) of the V3–V4 region of the 16S rRNA gene was performed in 27 heart valve tissues (18 natives, 5 intravascular devices, and 4 prosthetics) from 27 patients diagnosed with IE (4 of them with negative blood cultures). Metataxonomics matched with conventional diagnostic techniques in 24/27 cases (88.9%). The same bacterial family was assigned to 24 cases; the same genus, to 23 cases; and the same species, to 13 cases. In 22 of them, the etiological agent was represented by percentages > 99% of the reads and in two cases, by ~70%. Staphylococcus aureus was detected in a previously microbiological undiagnosed patient. Thus, microbiological diagnosis with 16S rRNA gene targeted-NGS was possible in one more sample than using traditional techniques. The remaining two patients showed no coincidence between traditional and 16S rRNA gene-targeted NGS microbiological diagnoses. In addition, 16S rRNA gene-targeted NGS allowed us to suggest coinfections that were supported by clinical data in one patient, and minority records also verified mixed infections in three cases. In our series, metataxonomics was valid for the identification of the causative agents, although more studies are needed before implementation of 16S rRNA gene-targeted NGS for the diagnosis of IE. Full article
(This article belongs to the Special Issue Molecular Diagnostics for Infectious Diseases)
21 pages, 2155 KiB  
Article
Qualitative and Quantitative Detection of Potentially Virulent Vibrio parahaemolyticus in Drinking Water and Commonly Consumed Aquatic Products by Loop-Mediated Isothermal Amplification
by Zhengke Shen, Yue Liu and Lanming Chen
Pathogens 2022, 11(1), 10; https://0-doi-org.brum.beds.ac.uk/10.3390/pathogens11010010 - 22 Dec 2021
Cited by 2 | Viewed by 2512
Abstract
Vibrio parahaemolyticus can cause acute gastroenteritis, wound infection, and septicemia in humans. In this study, a simple, specific, and user-friendly diagnostic tool was developed for the first time for the qualitative and quantitative detection of toxins and infection process-associated genes opaR, [...] Read more.
Vibrio parahaemolyticus can cause acute gastroenteritis, wound infection, and septicemia in humans. In this study, a simple, specific, and user-friendly diagnostic tool was developed for the first time for the qualitative and quantitative detection of toxins and infection process-associated genes opaR, vpadF, tlh, and ureC in V. parahaemolyticus using the loop-mediated isothermal amplification (LAMP) technique. Three pairs of specific inner, outer, and loop primers were designed for targeting each of these genes, and the results showed no cross-reaction with the other common Vibrios and non-Vibrios pathogenic bacteria. Positive results in the one-step LAMP reaction (at 65 °C for 45 min) were identified by a change to light green and the emission of bright green fluorescence under visible light and UV light (302 nm), respectively. The lowest limit of detection (LOD) for the target genes ranged from 1.46 × 10−5 to 1.85 × 10−3 ng/reaction (25 µL) for the genomic DNA, and from 1.03 × 10−2 to 1.73 × 100 CFU/reaction (25 µL) for the cell culture of V. parahaemolyticus. The usefulness of the developed method was demonstrated by the fact that the bacterium could be detected in water from various sources and commonly consumed aquatic product samples. The presence of opaR and tlh genes in the Parabramis pekinensis intestine indicated a risk of potentially virulent V. parahaemolyticus in the fish. Full article
(This article belongs to the Special Issue Molecular Diagnostics for Infectious Diseases)
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11 pages, 2392 KiB  
Article
New Reference Genes for qRT-PCR Analysis as a Potential Target for Identification of Trichophyton verrucosum in Different Culture Conditions
by Sebastian Gnat, Dominik Łagowski, Aneta Nowakiewicz, Aleksandra Trościańczyk and Mariusz Dyląg
Pathogens 2021, 10(11), 1361; https://0-doi-org.brum.beds.ac.uk/10.3390/pathogens10111361 - 21 Oct 2021
Cited by 1 | Viewed by 1735
Abstract
Dermatophytes are a group of filamentous fungi infecting skin, hair, and nails that raise great diagnostic difficulties. qRT-PCR is a reliable technique for quantifying gene expression with increasingly frequent use in mycological diagnostics. Knowledge of genes and molecular markers with potential to be [...] Read more.
Dermatophytes are a group of filamentous fungi infecting skin, hair, and nails that raise great diagnostic difficulties. qRT-PCR is a reliable technique for quantifying gene expression with increasingly frequent use in mycological diagnostics. Knowledge of genes and molecular markers with potential to be used in the identification of dermatophytes is of great importance for the development of this branch of diagnostics. In this article, the suitability of six candidate reference genes (TUBB, ACTB, ADPRF, RPL2, SDHA, and EEF1A1) was investigated for gene expression analysis in the dermatophyte Trichophyton verrucosum, which was cultured in various mycological media that are commonly used in a diagnostic laboratory, i.e., Sabouraud, potato dextrose, and keratin-supplemented MM-Cove. The different culture conditions are extremely important factors for the growth and physiology of dermatophytes. Gene expression stability was evaluated using geNorm, NormFinder, BestKeeper, and RefFinder algorithms. Regarding the stability of expression, SDHA was the most stable housekeeping gene; hence, this gene is recommended for future qRT-PCR studies on T. verrucosum strains. These results allow us to conclude that the SDHA gene can be an additional good candidate as an identification target in the qRT-PCR technique. Full article
(This article belongs to the Special Issue Molecular Diagnostics for Infectious Diseases)
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14 pages, 1800 KiB  
Article
Genetic Evidence of the Black Death in the Abbey of San Leonardo (Apulia Region, Italy): Tracing the Cause of Death in Two Individuals Buried with Coins
by Donato Antonio Raele, Ginevra Panzarino, Giuseppe Sarcinelli, Maria Assunta Cafiero, Anna Maria Tunzi and Elena Dellù
Pathogens 2021, 10(11), 1354; https://0-doi-org.brum.beds.ac.uk/10.3390/pathogens10111354 - 20 Oct 2021
Cited by 1 | Viewed by 3171
Abstract
The Abbey of San Leonardo in Siponto (Apulia, Southern Italy) was an important religious and medical center during the Middle Ages. It was a crossroads for pilgrims heading along the Via Francigena to the Sanctuary of Monte Sant’Angelo and for merchants passing through [...] Read more.
The Abbey of San Leonardo in Siponto (Apulia, Southern Italy) was an important religious and medical center during the Middle Ages. It was a crossroads for pilgrims heading along the Via Francigena to the Sanctuary of Monte Sant’Angelo and for merchants passing through the harbor of Manfredonia. A recent excavation of Soprintendenza Archeologica della Puglia investigated a portion of the related cemetery, confirming its chronology to be between the end of the 13th and beginning of the 14th century. Two single graves preserved individuals accompanied by numerous coins dating back to the 14th century, hidden in clothes and in a bag tied to the waist. The human remains of the individuals were analyzed in the Laboratorio di Antropologia Fisica of Soprintendenza ABAP della città metropolitana di Bari. Three teeth from each individual were collected and sent to the Istituto Zooprofilattico Sperimentale di Puglia e Basilicata to study infectious diseases such as malaria, plague, tuberculosis, epidemic typhus and Maltese fever (Brucellosis), potentially related to the lack of inspection of the bodies during burial procedures. DNA extracted from six collected teeth and two additional unrelated human teeth (negative controls) were analyzed using PCR to verify the presence of human DNA (β-globulin) and of pathogens such as Plasmodium spp., Yersinia pestis, Mycobacterium spp., Rickettsia spp. and Brucella spp. The nucleotide sequence of the amplicon was determined to confirm the results. Human DNA was successfully amplified from all eight dental extracts and two different genes of Y. pestis were amplified and sequenced in 4 out of the 6 teeth. Molecular analyses ascertained that the individuals buried in San Leonardo were victims of the Black Death (1347–1353) and the data confirmed the lack of inspection of the corpses despite the presence of numerous coins. This study represents molecular evidence, for the first time, of Southern Italy’s involvement in the second wave of the plague pandemic. Full article
(This article belongs to the Special Issue Molecular Diagnostics for Infectious Diseases)
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12 pages, 305 KiB  
Article
Comparison of Three Real-Time PCR Assays Targeting the SSU rRNA Gene, the COWP Gene and the DnaJ-Like Protein Gene for the Diagnosis of Cryptosporidium spp. in Stool Samples
by Felix Weinreich, Andreas Hahn, Kirsten Alexandra Eberhardt, Torsten Feldt, Fred Stephen Sarfo, Veronica Di Cristanziano, Hagen Frickmann and Ulrike Loderstädt
Pathogens 2021, 10(9), 1131; https://0-doi-org.brum.beds.ac.uk/10.3390/pathogens10091131 - 02 Sep 2021
Cited by 7 | Viewed by 2534
Abstract
As qualified microscopy of enteric parasitoses as defined by high diagnostic accuracy is difficult to maintain in non-endemic areas due to scarce opportunities for practicing with positive sample materials, molecular diagnostic options provide less investigator-dependent alternatives. Here, we compared three molecular targets for [...] Read more.
As qualified microscopy of enteric parasitoses as defined by high diagnostic accuracy is difficult to maintain in non-endemic areas due to scarce opportunities for practicing with positive sample materials, molecular diagnostic options provide less investigator-dependent alternatives. Here, we compared three molecular targets for the real-time PCR-based detection of Cryptosporidium spp. From a population of 1000 individuals comprising both Ghanaian HIV (human immunodeficiency virus) patients and military returnees after deployment in the tropics, stool samples were assessed for Cryptosporidium spp. by real-time PCR targeting the small subunit ribosomal RNA (SSU rRNA) gene, the Cryptosporidium oocyst wall (COWP) gene, and the DnaJ-like protein gene (DnaJ), respectively. In declining order, sensitivity of 100% for the SSU rRNA gene PCR, 90.0% for the COWP PCR and 88.8% for the DnaJ PCR, respectively, as well as specificity of 99.6% for the COWP PCR and 96.9% for both the SSU rRNA gene PCR and the DnaJ PCR, respectively, were recorded. Substantial agreement (kappa value 0.663) between the three assays was observed. Further, an accuracy-adjusted Cryptosporidium spp. prevalence of 6.0% was calculated for the study population. In conclusion, none of the assessed real-time PCR assays were associated with perfect test accuracy. However, a combination of highly sensitive SSU rRNA gene PCR for screening purposes and more specific COWP PCR for confirmatory testing should allow reliable diagnosis of Cryptosporidium spp. in stool samples even in low prevalence settings. Full article
(This article belongs to the Special Issue Molecular Diagnostics for Infectious Diseases)
9 pages, 230 KiB  
Article
Evaluation of a PlexZyme-Based PCR Assay and Assessment of COVID-19 Surge Testing Throughput Compared to Cobas SARS-CoV-2
by Todd M. Pryce, Erin J. Haygarth, Jessica Bordessa and Peter A. Boan
Pathogens 2021, 10(9), 1088; https://0-doi-org.brum.beds.ac.uk/10.3390/pathogens10091088 - 26 Aug 2021
Cited by 1 | Viewed by 2270
Abstract
Reliable high-throughput methods are required for the detection of severe acute respiratory coronavirus 2 (SARS-CoV-2). We evaluated the new research use only (RUO) SpeeDx PlexZyme SARS-CoV-2 components (Plex) compared to the Roche cobas SARS-CoV-2 assay (cobas). A collection of positive (n = [...] Read more.
Reliable high-throughput methods are required for the detection of severe acute respiratory coronavirus 2 (SARS-CoV-2). We evaluated the new research use only (RUO) SpeeDx PlexZyme SARS-CoV-2 components (Plex) compared to the Roche cobas SARS-CoV-2 assay (cobas). A collection of positive (n = 214) and negative samples (n = 201) was tested in parallel comparing Plex with cobas. The overall agreement comparing the qualitative outcomes was 96.9%. Using an in-house quantitative PCR method, correlation comparing Plex ORF1ab to cobas ORF1a was r2 = 0.95. The median Plex ORF1ab change in target copy number compared to cobas ORF1a was +0.48 log10 copies/mL respectively. Inter- and intra-assay reproducibility of each assay was compared, including a limit-of-detection study. Reproducibility was comparable; however cobas was more sensitive than Plex by 1-log dilution. Throughput was evaluated during a COVID-19 testing surge of 4324 samples in a 30-h period. Plex demonstrated less hands-on time per reportable result (19% decrease) and increased throughput (155% increase of 102 results/hour) compared to cobas (40 results/hour). Our study demonstrates good qualitative and quantitative correlation of Plex compared to cobas and that Plex is well-suited for high throughput testing. Full article
(This article belongs to the Special Issue Molecular Diagnostics for Infectious Diseases)
16 pages, 1516 KiB  
Article
Loop-Mediated Isothermal Amplification (LAMP) Assay for Detecting Burkholderia cepacia Complex in Non-Sterile Pharmaceutical Products
by Soumana Daddy Gaoh, Ohgew Kweon, Yong-Jin Lee, John J. LiPuma, David Hussong, Bernard Marasa and Youngbeom Ahn
Pathogens 2021, 10(9), 1071; https://0-doi-org.brum.beds.ac.uk/10.3390/pathogens10091071 - 24 Aug 2021
Cited by 7 | Viewed by 2670
Abstract
Simple and rapid detection of Burkholderia cepacia complex (BCC) bacteria, a common cause of pharmaceutical product recalls, is essential for consumer safety. In this study, we developed and evaluated a ribB-based colorimetric loop-mediated isothermal amplification (LAMP) assay for the detection of BCC [...] Read more.
Simple and rapid detection of Burkholderia cepacia complex (BCC) bacteria, a common cause of pharmaceutical product recalls, is essential for consumer safety. In this study, we developed and evaluated a ribB-based colorimetric loop-mediated isothermal amplification (LAMP) assay for the detection of BCC in (i) nuclease-free water after 361 days, (ii) 10 μg/mL chlorhexidine gluconate (CHX) solutions, and (iii) 50 μg/mL benzalkonium chloride (BZK) solutions after 184 days. The RibB 5 primer specifically detected 20 strains of BCC but not 36 non-BCC strains. The limit of detection of the LAMP assay was 1 pg/μL for Burkholderia cenocepacia strain J2315. Comparison of LAMP with a qPCR assay using 1440 test sets showed higher sensitivity: 60.6% in nuclease-free water and 42.4% in CHX solution with LAMP vs. 51.3% and 31.1%, respectively, with qPCR. These results demonstrate the potential of the ribB-based LAMP assay for the rapid and sensitive detection of BCC in pharmaceutical manufacturing. Full article
(This article belongs to the Special Issue Molecular Diagnostics for Infectious Diseases)
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15 pages, 299 KiB  
Article
Low Sensitivity of Real Time PCRs Targeting Retrotransposon Sequences for the Detection of Schistosoma japonicum Complex DNA in Human Serum
by Hagen Frickmann, Ulrike Loderstädt, Beatrice Nickel, Sven Poppert, Peter Odermatt, Somphou Sayasone, Marjan Van Esbroeck, Isabel Micalessi, Lieselotte Cnops, Poom Adisakwattana, Gérard Leboulle, Olfert Landt, Thorsten Thye and Egbert Tannich
Pathogens 2021, 10(8), 1067; https://0-doi-org.brum.beds.ac.uk/10.3390/pathogens10081067 - 22 Aug 2021
Cited by 4 | Viewed by 1974
Abstract
While hybridization probe-based real-time PCR assays targeting highly repetitive multi-copy genome sequences for the diagnosis of S. mansoni complex or S. haematobium complex from human serum are well established, reports on the evaluation of respective assays for the identification of S. japonicum complex [...] Read more.
While hybridization probe-based real-time PCR assays targeting highly repetitive multi-copy genome sequences for the diagnosis of S. mansoni complex or S. haematobium complex from human serum are well established, reports on the evaluation of respective assays for the identification of S. japonicum complex DNA in human serum are scarce. Here, we assessed the potential use of the retrotransposon sequences SjR2 and SjCHGCS19 from S. japonicum, S. mekongi and S. malayensis for the diagnosis of Asian Schistosoma infections. Based on available S. japonicum sequences and newly provided S. mekongi and S. malayensis sequences, hybridization probe-based real-time PCRs targeting SjR2 and SjCHGCS19 of the S. japonicum complex were designed both as consensus primer assays as well as multi-primer assays for the coverage of multiple variants of the target sequences. The assays were established using plasmids and S. mekongi DNA. While the consensus primer assays failed to detect S. mekongi DNA in human serum samples, the multi-primer assays showed positive or borderline positive results but only in 9.8% (6/61) of serum samples from patients with confirmed S. mekongi infections. Some cross-reactions with samples positive for S. mansoni or S. haematobium were observed but with the SjCHGCS19-PCR only. In spite of the low sensitivity, the presented experience may guide future evaluations of S. japonicum-complex-specific PCRs from human serum. Full article
(This article belongs to the Special Issue Molecular Diagnostics for Infectious Diseases)
9 pages, 294 KiB  
Article
Comparison of Two Real-Time PCR Assays Targeting Ribosomal Sequences for the Identification of Cystoisospora belli in Human Stool Samples
by Martin Blohm, Andreas Hahn, Ralf Matthias Hagen, Kirsten Alexandra Eberhardt, Holger Rohde, Gérard Leboulle, Torsten Feldt, Fred Stephen Sarfo, Veronica Di Cristanziano, Hagen Frickmann and Ulrike Loderstädt
Pathogens 2021, 10(8), 1053; https://0-doi-org.brum.beds.ac.uk/10.3390/pathogens10081053 - 19 Aug 2021
Cited by 5 | Viewed by 2565
Abstract
Cystoisospora (C.) belli is a coccidian parasite associated with acute or chronic gastroenteritis in immunocompromised patients. Dissatisfactory sensitivity of microscopy as the diagnostic standard approach has been described. Here, we comparatively evaluated two real-time PCRs targeting ribosomal RNA gene sequences of C. belli [...] Read more.
Cystoisospora (C.) belli is a coccidian parasite associated with acute or chronic gastroenteritis in immunocompromised patients. Dissatisfactory sensitivity of microscopy as the diagnostic standard approach has been described. Here, we comparatively evaluated two real-time PCRs targeting ribosomal RNA gene sequences of C. belli in stool in a test comparison without a reference standard applying latent class analysis. Therefore, 1000 stool samples from Ghanaian HIV (human immunodeficiency virus) patients (n = 905) as well as military returnees from the tropics (n = 95) were assessed by both assays in parallel. After the exclusion of 33 samples showing PCR inhibition, 29 and 33 positive results were recorded with the 5.8S rRNA gene/ITS-2 sequence PCR and the ITS-2 sequence PCR, respectively, resulting in an accuracy-adjusted prevalence of 3.2%. Nearly perfect agreement between both assays was indicated by Fleiss’ kappa of 0.933 with sensitivity and specificity of 92.8% and 100% as well as 100% and 99.8% for the 5.8S rRNA gene/ITS-2 sequence PCR and the ITS-2 sequence PCR, respectively. Both assays proved to be suitable for the diagnosis of C. belli in human stool samples with slightly better sensitivity of the ITS-2 sequence assay, while the 5.8S rRNA gene/ITS-2 sequence PCR may be considered for confirmatory testing. Full article
(This article belongs to the Special Issue Molecular Diagnostics for Infectious Diseases)
13 pages, 1641 KiB  
Article
Pragmatic Strategy for Fecal Specimen Storage and the Corresponding Test Methods for Clostridioides difficile Diagnosis
by Seong Won Nho, Minjae Kim, Seong-Jae Kim, Steven L. Foley, Rajesh Nayak, Ohgew Kweon and Carl E. Cerniglia
Pathogens 2021, 10(8), 1049; https://0-doi-org.brum.beds.ac.uk/10.3390/pathogens10081049 - 18 Aug 2021
Cited by 1 | Viewed by 2520
Abstract
The quality of fecal specimens is one of the factors responsible for successful Clostridioides difficile infection (CDI) diagnosis. The quality depends largely on the storage conditions, including the temperature and time period. In this study, we organized the outputs of previous studies, filled [...] Read more.
The quality of fecal specimens is one of the factors responsible for successful Clostridioides difficile infection (CDI) diagnosis. The quality depends largely on the storage conditions, including the temperature and time period. In this study, we organized the outputs of previous studies, filled experimental gaps in the knowledge of storage conditions, and introduced a pragmatic strategy for fecal storage for CDI diagnosis. A 5-step pathway was adopted to develop the fecal specimen storage strategy as follows: step 1, bibliomic analysis; step 2, experimental gap-filling; step 3, comparative evaluation; step 4, strategy development; step 5, internal review. Step 1 identified eight articles providing experimental information on the effects of fecal specimen storage conditions on the effectiveness of C. difficile detection methods. Step 2 provided additional quantitative data on C. difficile vegetative and spore cell viability and DNA stability. All previous and current results were compared (step 3). In step 4, fir general and nine special strategies were developed, followed by an internal review of the overall approaches (step 5). It is recommended to separate fecal samples into aliquots before testing and storing them. It is particularly recommended that fecal specimen samples be stored for CDI diagnosis at 4 °C for up to 60 days for all test methods. Full article
(This article belongs to the Special Issue Molecular Diagnostics for Infectious Diseases)
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11 pages, 280 KiB  
Article
Comparison of Three In-House Real PCR Assays Targeting Kinetoplast DNA, the Small Subunit Ribosomal RNA Gene and the Glucose-6-Phosphate Isomerase Gene for the Detection of Leishmania spp. in Human Serum
by Konstantin Tanida, Carsten Balczun, Andreas Hahn, Alexandra Veit, Beatrice Nickel, Sven Poppert, Patrick Leander Scheid, Ralf Matthias Hagen, Hagen Frickmann, Ulrike Loderstädt and Egbert Tannich
Pathogens 2021, 10(7), 826; https://0-doi-org.brum.beds.ac.uk/10.3390/pathogens10070826 - 30 Jun 2021
Cited by 2 | Viewed by 1781
Abstract
To perform PCR from serum for the diagnosis of visceral leishmaniasis is convenient and much less invasive than the examination of deeper compartments such as bone marrow. We compared three Leishmania-specific real-time PCRs with three different molecular targets (kinetoplast DNA, the small [...] Read more.
To perform PCR from serum for the diagnosis of visceral leishmaniasis is convenient and much less invasive than the examination of deeper compartments such as bone marrow. We compared three Leishmania-specific real-time PCRs with three different molecular targets (kinetoplast DNA, the small subunit-ribosomal RNA-(ssrRNA-)gene, the glucose-6-phosphate isomerase-(gpi-)gene) regarding their sensitivity and specificity in human serum. Residual sera from previous diagnostic assessments at the German National Reference Center for Tropical Pathogens Bernhard Nocht Institute for Tropical Medicine Hamburg and the Swiss Tropical and Public Health Institute were used. The sensitivities of kinetoplast DNA-PCR, ssrRNA-gene PCR, and gpi-PCR were 93.3%, 73.3%, and 33.3%, respectively, with 15 initial serum samples from visceral leishmaniasis patients, as well as 9.1%, 9.1%, and 0.0%, respectively, with 11 follow-up serum samples taken at various time points following anti-leishmanial therapy. Specificity was 100.0% in all assays as recorded with 1.137 serum samples from deployed soldiers and migrants without clinical suspicion of visceral leishmaniasis. Kinetoplast-DNA PCR from serum was confirmed as a sensitive and specific approach for the diagnosis of visceral leishmaniasis. The results also indicate the suitability of serum PCR for diagnostic follow-up after therapy, in particular regarding therapeutic failure in case of persisting positive PCR results. Full article
(This article belongs to the Special Issue Molecular Diagnostics for Infectious Diseases)
11 pages, 283 KiB  
Article
Comparative Assessment of In-House Real-Time PCRs Targeting Enteric Disease-Associated Microsporidia in Human Stool Samples
by Konstantin Tanida, Andreas Hahn, Kirsten Alexandra Eberhardt, Egbert Tannich, Olfert Landt, Simone Kann, Torsten Feldt, Fred Stephen Sarfo, Veronica Di Cristanziano, Hagen Frickmann and Ulrike Loderstädt
Pathogens 2021, 10(6), 656; https://0-doi-org.brum.beds.ac.uk/10.3390/pathogens10060656 - 26 May 2021
Cited by 7 | Viewed by 2361 | Correction
Abstract
Microsporidiosis is an infection predominantly occurring in immunosuppressed patients and infrequently also in travelers. This study was performed to comparatively evaluate the diagnostic accuracy of real-time PCR assays targeting microsporidia with etiological relevance in the stool of human patients in a latent class [...] Read more.
Microsporidiosis is an infection predominantly occurring in immunosuppressed patients and infrequently also in travelers. This study was performed to comparatively evaluate the diagnostic accuracy of real-time PCR assays targeting microsporidia with etiological relevance in the stool of human patients in a latent class analysis-based test comparison without a reference standard with perfect accuracy. Thereby, two one-tube real-time PCR assays and two two-tube real-time PCR assays targeting Enterocytozoon bieneusi and Encephalocytozoon spp. were included in the assessment with reference stool material (20), stool samples from Ghanaian HIV-positive patients (903), and from travelers, migrants and Colombian indigenous people (416). Sensitivity of the assays ranged from 60.4% to 97.4% and specificity from 99.1% to 100% with substantial agreement according to Cohen’s kappa of 79.6%. Microsporidia DNA was detected in the reference material and the stool of the HIV patients but not in the stool of the travelers, migrants, and the Colombian indigenous people. Accuracy-adjusted prevalence was 5.8% (n = 78) for the study population as a whole. In conclusion, reliable detection of enteric disease-associated microsporidia in stool samples by real-time PCR could be demonstrated, but sensitivity between the compared microsporidia-specific real-time PCR assays varied. Full article
(This article belongs to the Special Issue Molecular Diagnostics for Infectious Diseases)

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2 pages, 170 KiB  
Correction
Correction: Tanida et al. Comparative Assessment of In-House Real-Time PCRs Targeting Enteric Disease-Associated Microsporidia in Human Stool Samples. Pathogens 2021, 10, 656
by Konstantin Tanida, Andreas Hahn, Kirsten Alexandra Eberhardt, Egbert Tannich, Olfert Landt, Simone Kann, Torsten Feldt, Fred Stephen Sarfo, Veronica Di Cristanziano, Hagen Frickmann and Ulrike Loderstädt
Pathogens 2022, 11(2), 256; https://0-doi-org.brum.beds.ac.uk/10.3390/pathogens11020256 - 17 Feb 2022
Viewed by 989
Abstract
In the original publication [...] Full article
(This article belongs to the Special Issue Molecular Diagnostics for Infectious Diseases)
7 pages, 1905 KiB  
Case Report
Feline Demodicosis Case Report—First Molecular Characterization of Demodex Mites in Romania
by Marius Stelian Ilie, Mirela Imre, Simona Giubega, Iasmina Luca, Tiana Florea and Sorin Morariu
Pathogens 2021, 10(11), 1474; https://0-doi-org.brum.beds.ac.uk/10.3390/pathogens10111474 - 12 Nov 2021
Cited by 2 | Viewed by 4544
Abstract
Cat demodicosis is uncommon to rare, and is caused by Demodex cati, Demodex gatoi and another unnamed species. The investigated patient was a mix-breed, 10-year-old feline with no dermatological history. Alopecia, erythema, minor erosions and ulcerations and crusts, associated with pruritus and [...] Read more.
Cat demodicosis is uncommon to rare, and is caused by Demodex cati, Demodex gatoi and another unnamed species. The investigated patient was a mix-breed, 10-year-old feline with no dermatological history. Alopecia, erythema, minor erosions and ulcerations and crusts, associated with pruritus and self-trauma, were observed on the head. Dark, agglutinated cerumen was also present in the external ear canal. The agent causing the skin condition in the feline patient was identified as being a Demodex genus mite, based on the specific, morphological characteristics noticed upon the microscopic examination of deep skin scrapes. Biological samples were collected from the patient with to perform a PCR assay for clear species-determination and morphological assessment. PCR amplification of DNA extracted from the Demodex mites produced a single band of ~330 bp, indicating the presence of the D. cati species. The acaricidal treatment consisted of topical treatment using a fluralaner and moxidectin-based spot-on. Upon follow-up appointments, scheduled three times at a monthly interval, the patient failed to provide a positive result upon deep skin scrapes. The negative scrapes were also accompanied by the complete resolution of the existing lesions. In conclusion, this is the first molecular study to highlight the presence of Demodex cati within the feline population of Romania, and the fluralaner-moxidectin spot-on therapy has led to a complete recovery of the feline patient affected by feline demodicosis. Full article
(This article belongs to the Special Issue Molecular Diagnostics for Infectious Diseases)
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