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Advances in the Diagnosis of Infectious Diseases and Microorganisms: 2nd Edition

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A special issue of Diagnostics (ISSN 2075-4418). This special issue belongs to the section "Diagnostic Microbiology and Infectious Disease".

Deadline for manuscript submissions: closed (31 December 2025) | Viewed by 16327

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Prof. Dr. Philippe Colson

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IRD, AP-HM, MEPHI, Aix Marseille University, 27 Boulevard Jean Moulin, 13005 Marseille, France
Interests: mimivirus; metagenomics; virophages; megavirales; mobilome; emerging viruses; hiv; viral hepatitis
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Dear Colleagues,

There have recently been interesting advances in virological and microbiological diagnostic laboratories. These have notably consisted of the extended use of diagnostics through a syndromic approach, particularly using multiplex PCR, and also the introduction and increasing use of diagnosis through next-generation sequencing. These technologies can help improve the completeness and informativeness of infection diagnoses. They help, for example, to detect numerous viral, microbial or mixed co-infections, the epidemiological and clinical significance of which deserves careful analysis. Here, we describe examples of the implementation and optimization of virological diagnoses involving these approaches. Such diagnostic strategies can be combined with real-time monitoring of the numbers of clinical specimens sampled to search for infectious etiologies and of the numbers of positive diagnoses of infectious agents, in order to detect abnormal events which may correspond to emerging phenomena and epidemics.

Prof. Dr. Philippe Colson
Guest Editor

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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 250 words) can be sent to the Editorial Office for assessment.

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. Diagnostics is an international peer-reviewed open access semimonthly 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 2600 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

clinical microbiology
antimicrobial resistance
diagnostic microbiology
molecular microbiology
HIV
infectious diseases

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Published Papers (8 papers)

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Research

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11 pages, 250 KB

Open AccessArticle

Improved Microbiological Diagnosis of Bone and Joint Infections Using Mechanical Bead-Milling Extraction of Bone Specimens with the Ultra-Turrax^® System

by Maxime Brunaud, Adeline Boutet-Dubois, Alix Pantel, Florian Salipante, Rémy Coulomb, Albert Sotto, Jean-Philippe Lavigne and Nicolas Cellier

Diagnostics 2026, 16(2), 309; https://doi.org/10.3390/diagnostics16020309 - 18 Jan 2026

Viewed by 395

Abstract

Background: Accurate microbiological diagnosis of bone and joint infections (BJIs) is frequently hampered by low bacterial load, biofilm formation, and suboptimal tissue processing. This study evaluated the diagnostic performance of mechanical bead-milling using the Ultra-Turrax^® Tube Drive system compared with standard vortex homogenization. Methods: In a prospective cohort of 116 patients undergoing surgery for suspected BJIs, 540 intraoperative samples were processed using both methods. Culture and 16S rRNA PCR results were analyzed using classical and Bayesian statistical approaches. Diagnostic performance was assessed globally and across specimen types and anatomical sites. Results: Ultra-Turrax^® significantly improved sensitivity across all sample types (87.1% vs. 75.2%, p < 0.0001), while maintaining comparable specificity (>99%). Culture positivity increased by 17%, with the greatest gains observed in bone samples and hip prosthesis infections. Quantitative cultures demonstrated a 1.5–2 log₁₀ CFU/mL increase in bacterial yield. In culture-negative specimens, 16S rRNA PCR detection doubled with Ultra-Turrax^® processing (26% vs. 13%, p = 0.04). No increase in contamination was observed. Time to positivity was similar between methods, although Ultra-Turrax^® provided earlier results in 17% of cases. Bayesian modeling confirmed superior sensitivity (posterior probability > 0.995). Conclusions: Ultra-Turrax^® bead-milling markedly enhances microbiological detection in BJIs, particularly in low-biomass and bone-derived specimens. Its simplicity, reproducibility, and compatibility with routine workflows support its integration into diagnostic pathways. This pre-analytical optimization may improve etiological identification and guide more targeted antimicrobial therapy. Full article

(This article belongs to the Special Issue Advances in the Diagnosis of Infectious Diseases and Microorganisms: 2nd Edition)

13 pages, 554 KB

Open AccessArticle

Validation of Digital Slide Scanning and a Convolutional Neural Network for the Detection of Intestinal Parasites in Human Stool Samples

by Céline Büschlen, Daniel Rotzer, Nadine Sidler, Ha Thu Trang Nguyen and Alexander Oberli

Diagnostics 2025, 15(23), 2974; https://doi.org/10.3390/diagnostics15232974 - 24 Nov 2025

Viewed by 1248

Abstract

Background: Recent studies have shown that digital microscopy (DM) combined with a convolutional neural network (CNN) model is a valid approach for detecting intestinal protozoa and helminth ova or larvae in both trichrome-stained and wet-mount stool preparations. This study evaluated the diagnostic performance of a DM/CNN workflow for routine detection of intestinal parasites in a clinical microbiology laboratory. Methods: A clinical validation was conducted using the Grundium Ocus 40 scanner combined with the Techcyte Human Fecal Wet Mount (HFW) algorithm. The system was evaluated on (a) 135 reference samples and (b) 208 routine clinical samples submitted for intestinal parasite testing. Analytical sensitivity, precision, and limit of detection (LOD) were assessed. Results: For reference samples, the DM/CNN workflow achieved a positive slide-level agreement of 97.6% (95% CI: 94.4–100%), following a confidence threshold adjustment for Schistosoma mansoni, and a negative agreement of 96.0% (95% CI: 86.6–98.9%) compared with light microscopy (LM). Dilution series with reference samples revealed slightly lower analytical sensitivity of the DM/CNN at higher dilutions. Both intra- and inter-run precision studies demonstrated high reproducibility and stability. In prospective testing on 208 routine samples, overall agreement between DM/CNN and LM was 98.1% (95% CI: 95.2–99.2%) with a Cohen’s Kappa coefficient of κ = 0.915. Minor discrepancies involved Blastocystis spp., with DM/CNN showing slightly higher sensitivity. Conclusions: For the first time, we show that the combination of the Grundium Ocus 40 scanner and the Techcyte HFW algorithm provides a reliable, low-throughput screening solution that can effectively assist diagnostic technicians by pre-classifying putative parasitic structures for targeted expert review. Despite its lower throughput, the system substantially reduces the manual review process and simplifies the parasitological workflow. Implementation in a clinical microbiology laboratory requires extensive site-specific validation to account for differences in sample processing and imaging conditions. Moreover, optimization of confidence thresholds for specific classifiers is essential to ensure consistent analytical performance across different laboratory settings. Full article

(This article belongs to the Special Issue Advances in the Diagnosis of Infectious Diseases and Microorganisms: 2nd Edition)

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11 pages, 581 KB

Open AccessArticle

The Evaluation of a Rapid Syndromic Multiplex Meningitis/Encephalitis RT-qPCR MX-17 Panel

by Naim Mahroum, Meltem Yashar, Feyza Nihal Ugur, Nefise Zulal Oz, Gozde Ulfer, Ayse Istanbullu Tosun and Mesut Yilmaz

Diagnostics 2025, 15(20), 2629; https://doi.org/10.3390/diagnostics15202629 - 17 Oct 2025

Viewed by 1501

Abstract

Background/Objectives: Meningoencephalitis (ME) is a life-threatening infectious disease; therefore, prompt and accurate diagnosis is lifesaving. Traditional diagnostic methods, such as culture, have several limitations related to sensitivity and specificity. Emerging multiplex ME-PCR panels are a comprehensive and rapid tool in a single test. The Bio-Speedy Meningitis/Encephalitis RT-qPCR MX-17 panel (Bioeksen R&D Technologies Inc., Turkey) enables testing for 17 targets. To evaluate the performance of the panel compared to clinical and CSF parameters. Methods: A total of 403 patients with a preliminary diagnosis of ME were reviewed between January 2019 and September 2023. Following revision, 72 patients with clinical, CSF, and laboratory findings were included. The tested panel was used to detect targeted pathogens in CSF samples. The 30-day survival rate and prolonged stay were analyzed. Results: The median CSF protein value was 59.5 mg/dL (14.2–1471 mg/dL) and glucose was 61.95 mg/dL (0.083–165 mg/dL). Forty-one (56.9%) ME panel results were positive, among which 38.9% (28) were viral and 19.4% (14) were bacterial. HHV-6 ranked first with a rate of 15.3%. The Bio-Speedy panel test results outperformed the CSF culture (p < 0.001). The correlation of the Bio-Speedy panel with impaired consciousness was statistically significant (p = 0.004). Six (8.3%) patients from the study group died within 30 days. Conclusions: Compared to traditional methods, Bio-Speedy panel was effective in identifying the causative agents of ME. The Bio-Speedy ME RT-qPCR MX-17 panel offers accurate detection of ME-causing pathogens. The implementation of the panel in clinical practice can impact patient management and improve outcomes. Full article

(This article belongs to the Special Issue Advances in the Diagnosis of Infectious Diseases and Microorganisms: 2nd Edition)

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16 pages, 1942 KB

Open AccessArticle

Fecal Metabolomics for the Diagnosis of Clostridioides difficile Infection

by Carlos Bea-Serrano, Andreu Belmonte-Domingo, Carolina Pinto-Pla, Ana Ferrer-Ribera, Sara Vela-Bernal, Ana Isabel de Gracia-León, Andrea de Castro-Oliver, Lucas Serna-Navarro, Celia Prades-Sirvent, David Ruiz-Raga, María José Galindo, María José Forner-Giner and María Rosa Oltra-Sempere

Diagnostics 2025, 15(18), 2331; https://doi.org/10.3390/diagnostics15182331 - 15 Sep 2025

Cited by 1 | Viewed by 1230

Abstract

Background: Clostridioides difficile infection (CDI) is the leading cause of nosocomial diarrhea. Current diagnostic tools have difficulty distinguishing between colonization and active infection. This study evaluated the utility of fecal metabolomics in diagnosing CDI in hospitalized patients with acute diarrhea. Methods: We conducted a prospective observational study involving hospitalized adults with new-onset diarrhea during admission. Participants were stratified into groups based on clinical and microbiological findings: controls, C. difficile colonized and C. difficile infected. Fecal samples were analyzed using UPLC-MS/MS and GC-MS to quantify selected short-chain fatty acids, amino acids, and bile acids. Multivariate and univariate statistical analyses included PLS-DA, sPLSDA, and tests with FDR correction. Results: Infected patients exhibited significantly higher concentrations of SCFAs and notable alterations in bile acid profiles. Key discriminative metabolites included isovalerate, propionate, isobutyrate and alpha-aminobutyric acid. ROC curve analyses showed strong diagnostic performance for these markers, with AUC values exceeding 0.85. Conclusions: Fecal metabolomic profiling could effectively differentiate between colonization and infection in CDI among hospitalized patients with diarrhea. These results highlight the potential of metabolomic signatures to enhance the diagnostic precision for CDI. Full article

(This article belongs to the Special Issue Advances in the Diagnosis of Infectious Diseases and Microorganisms: 2nd Edition)

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18 pages, 760 KB

Open AccessArticle

Antineutrophil Cytoplasmic Autoantibodies Specific to Bactericidal/Permeability-Increasing Protein: A Cross-Road Between Prolonged Gram-Negative Bacterial Infections and Ulcerative Colitis/Primary Sclerosing Cholangitis

by Dragana Jovanovic, Rada Miskovic, Aleksandra Plavsic, Sara Radovic, Ljudmila Nagorni-Obradovic, Dragan Popovic, Milos M. Nikolic and Branka Bonaci-Nikolic

Diagnostics 2025, 15(18), 2309; https://doi.org/10.3390/diagnostics15182309 - 11 Sep 2025

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Abstract

Background/Objectives: Binding of bactericidal/permeability-increasing (BPI) protein to Gram-negative (GN) bacteria plays a major role in bacterial elimination. The relationship between BPI-antineutrophil cytoplasmic autoantibodies (ANCA), persistent infections and immunoinflammatory diseases has not been elucidated. Methods: In total, 193 ANCA-positive patients detected by IIF with ANCA-associated vasculitides (AAV, n-40), connective tissue diseases (CTD, n-28), drug-induced vasculitides (DIV, n-17), ulcerative colitis (UC, n-24), UC with primary sclerosing cholangitis (UC/PSC, n-14), Crohn’s disease (CD, n-10), autoimmune hepatitis (AIH, n-19) and chronic infections (n-41) were tested using the BPI-ANCA quantitative and semiquantitative ELISA (ANCA-profile: BPI, proteinase 3, myeloperoxidase, elastase, cathepsin G, lactoferrin). BPI-ANCA were analyzed in 52 healthy persons. Results: A total of 46/193 (23.8%) patients had BPI-ANCA positivity. BPI-ANCA were more frequently present in patients with prolonged GN bacterial infections and inflammatory bowel diseases than in AAV, DIV, AIH, CTD and healthy controls (p < 0.001). UC/PSC patients more frequently had BPI-ANCA than UC and CD patients (p < 0.001). GN bacterial infections more frequently had BPI-ANCA than Gram-positive bacterial infections (p < 0.001). Infections caused by Pseudomonas aeruginosa and Mycobacterium tuberculosis had monospecific BPI-ANCA (sensitivity 79% and 71%, respectively). UC/PSC and chronic GN bacterial infections caused by Klebsiella pneumoniae, Proteus mirabilis, or Escherichia coli had multispecific BPI-ANCA (sensitivity 64% and 100%, respectively). Odds ratio analysis showed that patients with IBD who were positive for multispecific BPI-ANCA had a 13.5-fold increased risk of UC/PSC (95% CI 2.98–61.18). Conclusions: Monospecific BPI-ANCA may be a valuable biomarker for persistent Pseudomonas aeruginosa and Mycobacterium tuberculosis infections. In contrast, multispecific BPI-ANCA are associated with UC/PSC and persistent infections caused by intestinal Gram-negative bacteria. Suppression of antimicrobial function by multispecific BPI-ANCA could impair the elimination of Gram-negative bacteria, sustaining the immunoinflammation. Dysregulated antimicrobial response might be the target of immunomodulatory therapy in the initial phase of BPI-ANCA-positive UC/PSC. Full article

(This article belongs to the Special Issue Advances in the Diagnosis of Infectious Diseases and Microorganisms: 2nd Edition)

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20 pages, 3217 KB

Open AccessArticle

Microbiome of the Proximal Small Intestine in Patients with Acute Pancreatitis

by Vladimir V. Kiselev, Stanislav I. Koshechkin, Alexey V. Kurenkov, Vera E. Odintsova, Maria S. Zhigalova, Alekxandr V. Tyakht, Sergey S. Petrikov, Petr A. Yartsev and Ilya V. Dmitriev

Diagnostics 2025, 15(15), 1911; https://doi.org/10.3390/diagnostics15151911 - 30 Jul 2025

Viewed by 1353

Abstract

Currently, due to the complexity of obtaining samples, specific features of laboratory processing and analysis of the results, there is a lack of data on the microbial signature of the small intestine in healthy and diseased states of the upper gastrointestinal tract. Objective: To investigate the characteristics of the small intestinal microbiome in acute pancreatitis of varying severity and to identify correlations with clinical factors. Methods: This study included 30 patients with acute pancreatitis of varying severity treated between 1 January 2019 and 31 December 2021. The composition of the microbiota was analyzed by metagenomic sequencing of the 16S rRNA gene from jejunal samples. Results: The mortality rate in the study group was 23.3%. The small intestinal microbiome was dominated by Streptococcus (median relative abundance 19.2%, interquartile range 6.4–35.1%), Veillonella (3.4%; 0.6–7%), Granulicatella (2.7%; 0.6–5%), Fusobacterium (2.2%; 0.3–5.9%), Prevotella (1.5%; 0.3–8%), Haemophilus (0.9%; 0.2–10%), Gemella (0.8%; 0.2–4.3%), and Lactobacillus (0.2%; 0.1–0.9%). More severe disease was associated with decreased abundance of Neisseria mucosa, Parvimonas micra, and Megasphaera micronuciformis. In contrast, the relative abundance of the genera Streptococcus (species S. rubneri/parasanguinis/australis), Actinomyces, and several genera within the family Enterobacteriaceae was higher in these patients. Conclusions: The state of the microbiota has important prognostic value and correlates with the duration from the onset of the pain syndrome to the time of receiving qualified care in the hospital. Full article

(This article belongs to the Special Issue Advances in the Diagnosis of Infectious Diseases and Microorganisms: 2nd Edition)

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11 pages, 455 KB

Open AccessArticle

Do the Results of Bile Cultures Affect the Outcomes of Patients with Mild-to-Moderate Ascending Cholangitis? A Single Center Prospective Study

by Yoav Krupik, Eran Ariam, Daniel L. Cohen, Anton Bermont, Sergei Vosko, Haim Shirin and Shay Matalon

Diagnostics 2025, 15(6), 695; https://doi.org/10.3390/diagnostics15060695 - 11 Mar 2025

Cited by 1 | Viewed by 2911

Abstract

Background/Objectives: Bile cultures are recommended in patients undergoing endoscopic retrograde cholangiopancreatography (ERCP). We sought to evaluate if bile cultures affect the outcomes of patients with mild-to-moderate ascending cholangitis. Methods: Bile cultures were prospectively obtained from patients undergoing ERCP between 2021 and 2023 at a single gastroenterology unit. The primary outcome was the prognosis of patients with mild-to-moderate ascending cholangitis who received appropriate antibiotic coverage with empiric antibiotics versus those with bacteria resistant to the empiric antibiotics. Additionally, outcomes between those with positive and negative biliary cultures were assessed. Results: One hundred sixty ERCPs were conducted, including 65 (40%) for ascending cholangitis with a naive papilla. Of these, 43 (66.2%) had a positive bile culture. Fourteen (32.6%) described mixed bacteria. Enterococcus spp. was the most common bacteria (22, 51.2%), followed by E. coli (17, 39.6%). Patients that were treated with appropriate antibiotics had similar outcomes compared to those who received inappropriate antibiotics per bile culture susceptibilities in terms of length of hospitalization (7.8 days vs. 7.9 days), in-hospital mortality, 30-day readmissions, and 30-day mortality (p ≥ 0.21, ns). There were also no differences in those outcomes between patients with positive and negative bile cultures (p ≥ 0.09, ns). Conclusions: These results question the need for obtaining bile cultures in every ERCP performed, including those with cholangitis. They imply that decompression of the biliary tree during ERCP is the more significant aspect of treatment, rather than the selection of an appropriate antibiotic regimen. Additional studies are needed to assess the benefits of acquiring bile cultures in all cases of ascending cholangitis. Full article

(This article belongs to the Special Issue Advances in the Diagnosis of Infectious Diseases and Microorganisms: 2nd Edition)

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Review

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24 pages, 1219 KB

Open AccessReview

Advances in Point-of-Care Infectious Disease Diagnostics: Integration of Technologies, Validation, Artificial Intelligence, and Regulatory Oversight

by Moustafa Kardjadj

Diagnostics 2025, 15(22), 2845; https://doi.org/10.3390/diagnostics15222845 - 10 Nov 2025

Cited by 14 | Viewed by 5983

Abstract

Point-of-care (POC) infectious disease diagnostics are reshaping global health by delivering rapid, decentralized, and clinically actionable results that link bedside testing to population-level surveillance. Valued at approximately USD 53 billion in 2024 and projected to nearly double by 2033, the global POC diagnostics market is driven by infectious disease assays and accelerated by innovations in molecular amplification, biosensors, microfluidics, and artificial intelligence (AI). This review integrates current evidence across technological, clinical, regulatory, and public health domains. Immunoassays remain the backbone of volume deployment, while molecular nucleic acid amplification tests (NAATs) and emerging CRISPR-based platforms achieve laboratory-grade sensitivity at the point of care. AI has transitioned from an experimental tool to an embedded analytical layer that enhances image interpretation, multiplex signal deconvolution, and automated quality control. Rigorous validation, including analytical accuracy, clinical performance in intended-use settings, and usability testing under CLIA guidance, remains central to ensuring reliability in decentralized environments. Regulatory frameworks are adapting in parallel: FDA’s lifecycle oversight of AI-enabled devices, the European IVDR’s expanded evidence requirements, and the WHO Prequalification all emphasize continuous post-market surveillance. From a public health perspective, POC diagnostics have improved early case detection, treatment initiation, and outbreak containment for HIV, tuberculosis, malaria, influenza, RSV, and COVID-19. Yet persistent challenges (including limited harmonization of standards, uneven reimbursement, and scarce real-world data from low- and middle-income countries) continue to constrain equitable adoption. POC infectious disease diagnostics are thus entering a pivotal phase of digitization and regulatory maturity. Addressing remaining gaps in validation, lifecycle monitoring, and implementation equity will determine whether these technologies achieve their full promise as clinical accelerators and as cornerstones of global infectious disease preparedness. Full article

(This article belongs to the Special Issue Advances in the Diagnosis of Infectious Diseases and Microorganisms: 2nd Edition)

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