APPLICATION OF A NOVEL ASPERGILLUS LATERAL-FLOW DEVICE IN THE DIAGNOSIS OF ASPERGILLOSIS IN CAPTIVE GENTOO PENGUINS (PYGOSCELIS PAPUA PAPUA).

Aspergillosis is the primary fungal disease affecting captive penguins globally. Its diagnosis remains challenging, and currently no tests are both sensitive and specific for the detection of early infection. The present study evaluated a recently developed Aspergillus lateral-flow device (AspLFD) for the detection of Aspergillus spp. antigen in plasma and glottis mucus from captive penguins. In a pilot retrospective study, banked frozen plasma samples from captive penguins were reviewed: samples from 11 gentoo penguins (Pygoscelis papua papua) and 4 king penguins (Aptenodytes patagonicus) fulfilled the inclusion criteria and were used in the analysis. Positive plasma AspLFD test results were found in 80% (four of five) of the aspergillosis-positive cases tested. All of the aspergillosis-negative cases tested negative (10 of 10) on the AspLFD test. In a cohort prospective study, paired plasma and glottis swab samples were opportunistically and nonrandomly collected from captive gentoo penguins. In total, 26 penguins were tested. In the negative control group, AspLFD test was negative on plasma and swab in 100% of birds (14 of 14). In the aspergillosis-positive group, AspLFD test was positive on plasma samples from 33% (4 of 12) of birds, on swab samples from 50% (6 of 12) of birds, and on either plasma or swab samples from 75% (9 of 12) of birds. The AspLFD is currently used for the diagnosis of aspergillosis in humans and also shows promise for use in penguins. Larger prospective studies are recommended.

The Fungal PCR Initiative's evaluation of in-house and commercial Pneumocystis jirovecii qPCR assays: Toward a standard for a diagnostics assay.

Quantitative real-time PCR (qPCR) is increasingly used to detect Pneumocystis jirovecii for the diagnosis of Pneumocystis pneumonia (PCP), but there are differences in the nucleic acids targeted, DNA only versus whole nucleic acid (WNA), and also the target genes for amplification. Through the Fungal PCR Initiative, a working group of the International Society for Human and Animal Mycology, a multicenter and monocenter evaluation of PCP qPCR assays was performed. For the multicenter study, 16 reference laboratories from eight different countries, performing 20 assays analyzed a panel consisting of two negative and three PCP positive samples. Aliquots were prepared by pooling residual material from 20 negative or positive- P. jirovecii bronchoalveolar lavage fluids (BALFs). The positive pool was diluted to obtain three concentrations (pure 1:1; 1:100; and 1:1000 to mimic high, medium, and low fungal loads, respectively). The monocenter study compared five in-house and five commercial qPCR assays testing 19 individual BALFs on the same amplification platform. Across both evaluations and for all fungal loads, targeting WNA and the mitochondrial small sub-unit (mtSSU) provided the earliest Cq values, compared to only targeting DNA and the mitochondrial large subunit, the major surface glycoprotein or the beta-tubulin genes. Thus, reverse transcriptase-qPCR targeting the mtSSU gene could serve as a basis for standardizing the P. jirovecii load, which is essential if qPCR is to be incorporated into clinical care pathways as the reference method, accepting that additional parameters such as amplification platforms still need evaluation.

Evaluation of the new AspID polymerase chain reaction assay for detection of Aspergillus species: A pilot study.

The newly developed AspID PCR assay for detection of Aspergillus spp. was evaluated with an interlaboratory quality control programme panel and human bronchoalveolar lavage fluid (BALF) samples. With the quality control programme, 8 out of 9 panel members were correctly identified. With the clinical study, 36 BALF samples that had been obtained from 18 patients with invasive pulmonary aspergillosis (IPA) and 18 without IPA were investigated. Sensitivity, specificity, positive and negative likelihood ratio for the AspID assay were 94.1% (95% CI 73.3-99.9), 76.5% (95% CI 50.1-93.2), 4 (95% CI 1.7-9.5) and 0.1 (95% CI 0.01-0.5) respectively.

Determining the analytical specificity of PCR-based assays for the diagnosis of IA: What is Aspergillus?

A wide array of PCR tests has been developed to aid the diagnosis of invasive aspergillosis (IA), providing technical diversity but limiting standardisation and acceptance. Methodological recommendations for testing blood samples using PCR exist, based on achieving optimal assay sensitivity to help exclude IA. Conversely, when testing more invasive samples (BAL, biopsy, CSF) emphasis is placed on confirming disease, so analytical specificity is paramount. This multicenter study examined the analytical specificity of PCR methods for detecting IA by blind testing a panel of DNA extracted from a various fungal species to explore the range of Aspergillus species that could be detected, but also potential cross reactivity with other fungal species. Positivity rates were calculated and regression analysis was performed to determine any associations between technical specifications and performance. The accuracy of Aspergillus genus specific assays was 71.8%, significantly greater (P < .0001) than assays specific for individual Aspergillus species (47.2%). For genus specific assays the most often missed species were A. lentulus (25.0%), A. versicolor (24.1%), A. terreus (16.1%), A. flavus (15.2%), A. niger (13.4%), and A. fumigatus (6.2%). There was a significant positive association between accuracy and using an Aspergillus genus PCR assay targeting the rRNA genes (P = .0011). Conversely, there was a significant association between rRNA PCR targets and false positivity (P = .0032). To conclude current Aspergillus PCR assays are better suited for detecting A. fumigatus, with inferior detection of most other Aspergillus species. The use of an Aspergillus genus specific PCR assay targeting the rRNA genes is preferential.

Aspergillus-Specific Lateral-Flow Device and Real-Time PCR Testing of Bronchoalveolar Lavage Fluid: a Combination Biomarker Approach for Clinical Diagnosis of Invasive Pulmonary Aspergillosis.

Clinical experience with the impact of serum biomarkers for invasive fungal disease (IFD) varies markedly in hemato-oncology. Invasive pulmonary aspergillosis (IPA) is the most common manifestation, so we evaluated biomarkers in bronchoalveolar lavage (BAL) fluid. An Aspergillus-specific lateral-flow device (LFD), quantitative real-time PCR (qPCR), and the galactomannan (GM) test were used with 32 BAL fluid samples from 32 patients at risk of IPA. Eight patients had proven IPA, 3 had probable IPA, 6 had possible IPA, and 15 patients had no IPA by European Organization for Research and Treatment of Cancer Invasive Fungal Infections Cooperative Group/Mycoses Study Group of the National Institute of Allergy and Infectious Diseases (EORTC/MSG) criteria. The diagnostic accuracies of the tests were evaluated, and pairwise agreement between biomarkers was calculated. The diagnostic performance of the EORTC/MSG criteria was evaluated against the test(s) identified to be the most useful for IPA diagnosis. Using the EORTC/MSG criteria, the sensitivities of qPCR and LFD were 100% and the sensitivity of the GM test was 87.5% (GM test index cutoff, >0.8), with the tests having specificities of between 66.7 and 86.7%. The agreement between the results of qPCR and LFD was almost perfect (Cohen's kappa coefficient = 0.93, 95% confidence interval, 0.81 to 1.00). LFD and qPCR combined had a sensitivity of 100% and a specificity of 85.7%. Calcofluor staining and culture of all BAL fluid samples were negative for fungal infection. The median time from the start of mold-active antifungal therapy to the time of collection of BAL fluid was 6 days. Reversing roles and using dual testing by LFD and qPCR to classify cases, the EORTC/MSG criteria had a sensitivity of 83.3%. All three tests are useful for the diagnosis of IPA in BAL fluid samples. Despite the significant delays between the start of antifungal therapy and bronchoscopy, unlike microscopy and culture, the biomarkers remained informative. In particular, the combination of LFD and qPCR allows the sensitive and specific detection of IPA.

Significant decline in Galactomannan Signal during storage of clinical serum samples.

Galactomannan (GM) is widely used for detection of invasive aspergillosis in high-risk haemato-oncology patients. Recent publications have reported a lack of repeatability of GM detection. The objective of this retrospective study was to assess the repeatability of GM levels during storage of clinical samples. In a GM screening strategy, positive sera were repeat tested as per manufacturer's recommendations. Short-term (ST) storage of samples was at +4 °C while long-term (LT) storage was at -80 °C. Bronchoalveolar (BAL) fluid was also repeating tested after ST storage and LT storage. Wilcoxon Signed Ranks Test was employed to assess the repeatability of GM levels. In a subset of 14 GM positive sera, repeat testing was performed on both the original serum and ethylenediaminetetraacetic acid (EDTA) pre-treated sample. There was a significant reduction in GM signals on repeat testing following ST storage (median GM index: 0.65 vs. 0.19; p < 0.001) and LT storage (median GM index: 0.56 vs. 0.10; p < 0.001) of serum samples. Of samples that were initially GM positive, an average GM index reduction of 50% was seen, with approximately two-thirds becoming GM negative on repeat testing of the same sample. In contrast, GM signal loss was not seen on repeat testing of BAL fluid following ST or LT storage. When GM positive serum samples were repeat tested using EDTA pre-treated serum from the first step of the testing protocol, all samples remained GM positive. In contrast, when the same samples were repeat tested from the original collected serum, 9 samples (64%) became GM negative. The significant reduction in GM signals during ST and LT storage of serum samples has implications for clinical management. Although the reasons for GM decline are unknown, they occur prior to the EDTA pre-treatment stage, indicating that the time from phlebotomy to testing should be minimized. BAL fluid GM index values remain stable.

Impact of the revised (2008) EORTC/MSG definitions for invasive fungal disease on the rates of diagnosis of invasive aspergillosis.

Diagnosis of invasive aspergillosis (IA) remains a challenge as the clinical manifestations are not specific, and a histological diagnosis is often unfeasible. The 2002 European Organization for Research and Treatment of Cancer (EORTC) and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (MSG) criteria for classification of cases into possible, probable or proven were revised in 2008. Our objective was to analyze the impact of these revisions on the diagnosis of IA. A retrospective analysis of 589 high risk patient-episodes revealed that 125 of 155 'possible' (81%) and 12 of 16 'probable' (75%) cases of IA should be changed to 'non-classifiable' when the new criteria were applied. We concluded, as expected, that the 2008 EORTC/MSG revised definitions reduced the number of cases classified as 'possible' IA, but additionally, there has been a dramatic reduction in 'probable' cases. These changes have significant implications on the interpretation of clinical trial data based on EORTC/MSG classifications.

Mammalian Digit Tip Regeneration: Moving from Phenomenon to Molecular Mechanism.

In this review, we present the current state of knowledge surrounding mammalian digit tip regeneration. We discuss the origin and formation of the blastema, a structure integral to digit tip regeneration, as well as recent insights driven by single-cell RNA sequencing into the molecular markers and cellular composition of the blastema. The digit tip is a composite of many different tissue types and we address what is known about the role of these separate tissues in regeneration of the whole digit tip. Specifically, we discuss the most extensively studied tissues in the digit tip: bone, nail epithelium, and peripheral nerves. We also address how known molecular pathways in limb development can inform research into digit tip regeneration. Overall, the mouse digit tip is an excellent model of complex mammalian regeneration that can provide insight into inducing regeneration in human tissues.

En1 and Lmx1b do not recapitulate embryonic dorsal-ventral limb patterning functions during mouse digit tip regeneration.

The mouse digit tip regenerates following amputation. How the regenerate is patterned is unknown, but a long-standing hypothesis proposes developmental patterning mechanisms are re-used during regeneration. The digit tip bone exhibits dorsal-ventral (DV) polarity, so we focus on En1 and Lmx1b, two factors necessary for DV patterning during limb development. We investigate whether they are re-expressed during regeneration in a developmental-like pattern and whether they direct DV morphology of the regenerate. We find that both En1 and Lmx1b are expressed in the regenerating digit tip epithelium and mesenchyme, respectively, but without DV polarity. Conditional genetics and quantitative analysis of digit tip bone morphology determine that genetic deletion of En1 or Lmx1b in adult digit tip regeneration modestly reduces bone regeneration but does not affect DV patterning. Collectively, our data suggest that, while En1 and Lmx1b are re-expressed during mouse digit tip regeneration, they do not define the DV axis during regeneration.

Digital Interventions Supporting Self-care in People With Type 2 Diabetes Across Greater Manchester (Greater Manchester Diabetes My Way): Protocol for a Mixed Methods Evaluation.

BACKGROUND: Type 2 Diabetes (T2D) is common, with a prevalence of approximately 7% of the population in the United Kingdom. The quality of T2D care is inconsistent across the United Kingdom, and Greater Manchester (GM) does not currently achieve the National Institute for Health and Care Excellence treatment targets. Barriers to delivery of care include low attendance and poor engagement with local T2D interventions, which tend to consist of programs of education delivered in traditional, face-to-face clinical settings. Thus, a flexible approach to T2D management that is accessible to people from different backgrounds and communities is needed. Diabetes My Way (DMW) is a digital platform that offers a comprehensive self-management and educational program that should be accessible to a wide range of people through mobile apps and websites. Building on evidence generated by a Scotland-wide pilot study, DMW is being rolled out and tested across GM. OBJECTIVE: The overarching objectives are to assess whether DMW improves outcomes for patients with T2D in the GM area, to explore the acceptability of the DMW intervention to stakeholders, and to assess the cost-effectiveness of the intervention. METHODS: A mixed methods approach will be used. We will take a census approach to recruitment in that all eligible participants in GM will be invited to participate. The primary outcomes will be intervention-related changes compared with changes observed in a matched group of controls, and the secondary outcomes will be within-person intervention-related changes. The cost-effectiveness analysis will focus on obtaining reliable estimates of how each intervention affects risk factors such as HbA1c and costs across population groups. Qualitative data will be collected via semistructured interviews and focus groups and organized using template analysis. RESULTS: As of May 10, 2021, a total of 316 participants have been recruited for the quantitative study and have successfully enrolled. A total of 278 participants attempted to register but did not have appropriate permissions set by the general practitioners to gain access to their data. In total, 10 participants have been recruited for the qualitative study (7 practitioners and 3 patients). An extension to recruitment has been granted for the quantitative element of the research, and analysis should be complete by December 2022. Recruitment and analysis for the qualitative study should be complete by December 2021. CONCLUSIONS: The findings from this study can be used both to develop the DMW system and improve accessibility and usability in more deprived populations generally, thus improving equity in access to support for T2D self-management. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/26237.

Cellular Heterogeneity and Lineage Restriction during Mouse Digit Tip Regeneration at Single-Cell Resolution.

Innate regeneration following digit tip amputation is one of the few examples of epimorphic regeneration in mammals. Digit tip regeneration is mediated by the blastema, the same structure invoked during limb regeneration in some lower vertebrates. By genetic lineage analyses, the digit tip blastema has been defined as a population of heterogeneous, lineage-restricted progenitor cells. These previous studies, however, do not comprehensively evaluate blastema heterogeneity or address lineage restriction of closely related cell types. In this report, we present single-cell RNA sequencing of over 38,000 cells from mouse digit tip blastemas and unamputated control digit tips and generate an atlas of the cell types participating in digit tip regeneration. We computationally define differentiation trajectories of vascular, monocytic, and fibroblastic lineages over regeneration, and while our data confirm broad lineage restriction of progenitors, our analysis reveals 67 genes enriched in blastema fibroblasts including a novel regeneration-specific gene, Mest.

Cellular Plasticity in Musculoskeletal Development, Regeneration, and Disease.

In this review, we highlight themes from a recent workshop focused on "Plasticity of Cell Fate in Musculoskeletal Tissues" held at the Orthopaedic Research Society's 2019 annual meeting. Experts in the field provided examples of mesenchymal cell plasticity during normal musculoskeletal development, regeneration, and disease. A thorough understanding of the biology underpinning mesenchymal cell plasticity may offer a roadmap for promoting regeneration while attenuating pathologic differentiation. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:708-718, 2020.

Dissecting the sharp response of a canonical developmental enhancer reveals multiple sources of cooperativity.

Developmental enhancers integrate graded concentrations of transcription factors (TFs) to create sharp gene expression boundaries. Here we examine the hunchback P2 (HbP2) enhancer which drives a sharp expression pattern in the Drosophila blastoderm embryo in response to the transcriptional activator Bicoid (Bcd). We systematically interrogate cis and trans factors that influence the shape and position of expression driven by HbP2, and find that the prevailing model, based on pairwise cooperative binding of Bcd to HbP2 is not adequate. We demonstrate that other proteins, such as pioneer factors, Mediator and histone modifiers influence the shape and position of the HbP2 expression pattern. Comparing our results to theory reveals how higher-order cooperativity and energy expenditure impact boundary location and sharpness. Our results emphasize that the bacterial view of transcription regulation, where pairwise interactions between regulatory proteins dominate, must be reexamined in animals, where multiple molecular mechanisms collaborate to shape the gene regulatory function.

Vibrio harveyi sepsis in a child with cancer.

A paediatric oncology patient presented with central line sepsis caused by Vibrio harveyi, a gram negative bioluminescent marine bacterium known to be pathogenic to fish and marine invertebrates, after swimming in the sea.

Evaluation of Galactomannan Testing, the Aspergillus-Specific Lateral-Flow Device Test and Levels of Cytokines in Bronchoalveolar Lavage Fluid for Diagnosis of Chronic Pulmonary Aspergillosis.

Background: Diagnosis of chronic pulmonary aspergillosis (CPA) is challenging. Symptoms are unspecific or missing, radiological findings are variable and proof of mycological evidence is limited by the accuracy of diagnostic tests. The goal of this study was to investigate diagnostic performance of galactomannan (GM), the newly formatted Aspergillus-specific lateral-flow-device test (LFD), and a number of cytokines in bronchoalveolar lavage fluid (BALF) samples obtained from patients with CPA, patients with respiratory disorders without CPA and healthy individuals. Methods: Patients with CPA (n = 27) and controls (n = 27 with underlying respiratory diseases but without CPA, and n = 27 healthy volunteers) were recruited at the Medical University of Graz, Austria and the Research Center Borstel, Germany between 2010 and 2018. GM, LFD and cytokine testing was performed retrospectively at the Research Center Borstel. Results: Sensitivity and specificity of GM testing from BALF with a cut off level of ≥0.5 optical density index (ODI) was 41 and 100% and 30 and 100% with a cut off level of ≥1.0 ODI. ROC curve analysis showed an AUC 0.718 (95% CI 0.581-0.855) for GM for differentiating CPA patients to patients with other respiratory diseases without CPA. The LFD resulted positive in only three patients with CPA (7%) and was highly specific. CPA patients did not differ significantly in the BALF cytokine profile compared to patients with respiratory disorders without CPA, but showed significant higher values for IFN-γ, IL-1b, IL-6, IL-8, and TNF-α compared to healthy individuals. Conclusion: Both GM and LFD showed insufficient performance for diagnosing CPA, with sensitivities of BALF GM below 50%, and sensitivity of the LFD below 10%. The high specificities may, however, result in a high positive predictive value and thereby help to identify semi-invasive or invasive disease.

Diagnosis of invasive aspergillosis in hematological malignancy patients: Performance of cytokines, Asp LFD, and Aspergillus PCR in same day blood and bronchoalveolar lavage samples.

BACKGROUND: Aspergillus spp. induce elevated levels of several cytokines. It remains unknown whether these cytokines hold value for clinical routine and enhance diagnostic performances of established and novel biomarkers/tests for invasive aspergillosis (IA). METHODS: This cohort study included 106 prospectively enrolled (2014-2017) adult cases with underlying hematological malignancies and suspected pulmonary infection undergoing bronchoscopy. Serum samples were collected within 24 hours of bronchoalveolar lavage fluid (BALF) sampling. Both, serum and BALF samples were used to evaluate diagnostic performances of the Aspergillus-specific lateral-flow device test (LFD), Aspergillus PCR, ß-D-glucan, and cytokines that have shown significant associations with IA before. RESULTS: Among 106 cases, 11 had probable IA, and 32 possible IA; 80% received mold-active antifungals at the time of sampling. Diagnostic tests and biomarkers showed better performance in BALF versus blood, with the exception of serum interleukin (IL)-8 which was the most reliable blood biomarker. Combinations of serum IL-8 with either BALF LFD (sensitivity 100%, specificity 94%) or BALF PCR (sensitivity 91%, specificity 97%) showed promise for differentiating probable IA from no IA. CONCLUSIONS: High serum IL-8 levels were highly specific, and when combined with either the BALF Aspergillus-specific LFD, or BALF Aspergillus PCR also highly sensitive for diagnosis of IA.

Evaluation of BacLite Rapid MRSA, a rapid culture based screening test for the detection of ciprofloxacin and methicillin resistant S. aureus (MRSA) from screening swabs.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen worldwide. The need for accurate and rapid screening methods to detect MRSA carriers has been clearly established. The performance of a novel assay, BacLite Rapid MRSA (Acolyte Biomedica, UK) for the rapid detection (5 h) and identification of hospital associated ciprofloxacin resistant strains of MRSA directly from nasal swab specimens was compared to that obtained by culture on Mannitol salt agar containing Oxacillin (MSAO) after 48 h incubation. RESULTS: A total of 1382 nasal screening swabs were tested by multiple operators. The BacLite Rapid MRSA test detected 142 out of the 157 confirmed MRSA that were detected on MSAO giving a diagnostic sensitivity of 90.4, diagnostic specificity of 95.7% and a negative predictive value of 98.7%. Of the 15 false negatives obtained by the BacLite Rapid MRSA test, seven grew small amounts (< 10 colonies of MRSA) on the MSAO culture plate and five isolates were ciprofloxacin sensitive. However there were 13 confirmed BacLite MRSA positive samples, which were negative by the direct culture method, probably due to overgrowth on the MSAO plate. There were 53 false positive results obtained by the BacLite Rapid MRSA test at 5 h and 115 cases where MRSA colonies were tentatively identified on the MSAO plate when read at 48 h, and which subsequently proved not to be MRSA. CONCLUSION: The BacLite MRSA test is easy to use and provides a similar level of sensitivity to conventional culture for the detection of nasal carriage of MRSA with the advantage that the results are obtained much more rapidly.

Comparative study of diagnosis of PD peritonitis by quantitative polymerase chain reaction for bacterial DNA vs culture methods.

INTRODUCTION: Peritonitis remains one of the main complications that afflict peritoneal dialysis patients. We conducted a pilot study to determine the feasibility and potential advantages of quantitative PCR (qPCR) assays for the presence of bacterial DNA in this clinical scenario. METHODS: 14 patients attending with 'cloudy bags' had PD fluid analyzed in accordance with Renal Association Standards. In addition, quantitative bacterial DNA analysis was performed on 50 mL samples of PD fluid. DNA was extracted using a Qiagen kit. Quantitative PCR assays using primers and probes targeted at 16S rDNA were used to measure the levels of bacterial DNA. Samples from 13 patients attending the department for other reasons served as negative controls. Laboratory staff were blinded to clinical details at the time of analysis. RESULTS: We determined a threshold of bacterial DNA whereby 11 of 13 negative controls were 'negative'. Significant bacterial DNA was found in 6 of 9 culture positive' peritonitis cases (p < 0.05 by chi(2). The 3 cases of 'no growth' peritonitis had 'insignificant' bacterial DNA. Serial DNA analysis was performed in 8 patients. Of the 6 patients who were 'cured' with standard antibiotic therapy, only 1 showed a rise in bacterial DNA from Day 1 to 5. But the 2 patients who relapsed after antibiotics had marked rises in bacterial DNA (p < 0.05 by chi(2). DISCUSSION: We showed that results from quantitative bacterial DNA PCR assays correlate with current microbiological tests despite the small size of this study. We also suggest that this technology might be clinically useful as an adjunct for cases of 'no growth' peritonitis and to identify those patients likely to relapse despite apparent clinical improvement with standard antibiotic therapy.

Homogeneous and digital proximity ligation assays for the detection of Clostridium difficile toxins A and B.

BACKGROUND: The proximity ligation assay (PLA) detects proteins via their interaction with pairs of proximity probes, which are antibodies coupled to noncomplementary DNA oligonucleotides. The binding of both proximity probes to their epitopes on the target protein brings the oligonucleotides together, allowing them to be bridged by a third oligonucleotide with complementarity to the other two. This enables their ligation and the detection of the resulting amplicon by real-time quantitative PCR (qPCR), which acts as a surrogate marker for the protein of interest. Hence PLA has potential as a clinically relevant diagnostic tool for the detection of pathogens where nucleic acid based tests are inconclusive proof of infection. METHODS: We prepared monoclonal and polyclonal proximity probes targeting Clostridium difficile toxins A (TcdA) and B (TcdB) and used hydrolysis probe-based qPCR and digital PCR (dPCR) assays to detect antibody/antigen interactions. RESULTS: The performance of the PLA assays was antibody-dependent but both TcdA and TcdB assays were more sensitive than comparable ELISAs in either single- or dualplex formats. Both PLAs could be performed using single monoclonal antibodies coupled to different oligonucleotides. Finally, we used dPCR to demonstrate its potential for accurate and reliable quantification of TcdA. CONCLUSIONS: PLA with either qPCR or dPCR readout have potential as new diagnostic applications for the detection of pathogens where nucleic acid based tests do not indicate viability or expression of toxins. Importantly, since it is not always necessary to use two different antibodies, the pool of potential antibodies useful for PLA diagnostic assays is usefully enhanced.