Heterogeneity in practices to reduce the risk of transmission of Clostridioides difficile in healthcare settings: a survey of ESCMID Study Group for Clostridioides difficile (ESGCD) members.

Clostridioides difficile is a leading cause of healthcare-associated infections. The main objective was to assess the current landscape of CDI infection prevention and control (IPC) practices. An anonymous survey of IPC practices for CDI was conducted between July 25 and October 31, 2022. Precautions for symptomatic patients were applicable for 75.9% and were discontinued 48 h minimum after the resolution of diarrhea for 40.7% of respondents. Daily cleaning of CDI patients' rooms was reported by 23 (42.6%). There was unexpected heterogeneity in IPC practices regarding the hospital management of CDI.

Ribotypes and New Virulent Strains Across Europe.

Clostridioides (formerly Clostridium) difficile is a major bacterial cause of post-antibiotic diarrhoea. The epidemiology of C. difficile infections (CDIs) has dramatically changed since the early 2000s, with an increasing incidence and severity across Europe. This trend is partly due to the emergence and rapid worldwide spread of the hypervirulent and epidemic PCR ribotype 027. Profiles of patients with CDI have also evolved, with description of community-acquired (CA) infections in patients with no traditional risk factors for CDI. However, epidemiological studies indicated that some European countries have successfully controlled the dissemination of the 027 clone whereas other countries reported the emergence of other virulent or unusual strains. The aims of this review are to summarize the current European CDI epidemiology and to describe the new virulent C. difficile strains circulating in Europe, as well as other potential emerging strains described elsewhere. Standardized typing methods and surveillance programmes are mandatory for a better understanding and monitoring of CDI in Europe.

Clostridioides difficile positivity rate and PCR ribotype distribution on retail potatoes in 12 European countries, January to June 2018.

BackgroundWhile human-to-human transmission of Clostridioides difficile occurs often, other infection sources, including food, animals and environment, are under investigation.AimWe present a large study on C. difficile in a food item in Europe, encompassing 12 European countries (Austria, France, Greece, Ireland, Italy, the Netherlands, Poland, Slovakia, Spain, Sweden, Romania and the United Kingdom).MethodsPotato was selected because of availability, ease of sampling and high C. difficile positivity rates. Identical protocols for sampling and isolation were used, enabling a direct comparison of the C. difficile positivity rate.ResultsFrom C. difficile-positive potato samples (33/147; 22.4%), we obtained 504 isolates, grouped into 38 PCR ribotypes. Positivity rates per country varied (0-100%) and were at least 10% in 9/12 countries. No geographical clustering of samples with high positivity rates or in PCR ribotype distribution was observed. The most frequently detected PCR ribotypes (014/020, 078/126, 010 and 023) are also commonly reported in Europe among human clinically relevant isolates, in animal isolates and in the environment. Whole genome sequencing revealed several genetically related strain pairs (Spain/RT126, France/RT010, Austria and Sweden/RT276) and a cluster of very similar strains in RT078/126.ConclusionOur results suggest, the high potato contamination rates could have public health relevance. They indicate potatoes can serve as a vector for introducing C. difficile spores in the household environment, where the bacterium can then multiply in sensitive hosts with disrupted or unmature microbiota. Potato contamination with PCR ribotypes shared between humans, animals and soil is supportive of this view.

A point-prevalence study on community and inpatient Clostridioides difficile infections (CDI): results from Combatting Bacterial Resistance in Europe CDI (COMBACTE-CDI), July to November 2018.

BackgroundThere is a paucity of data on community-based Clostridioides difficile infection (CDI) and how these compare with inpatient CDI.AimTo compare data on the populations with CDI in hospitals vs the community across 12 European countries.MethodsFor this point-prevalence study (July-November 2018), testing sites sent residual diagnostic material on sampling days to a coordinating laboratory for CDI testing and PCR ribotyping (n = 3,163). Information on whether CDI testing was requested at the original site was used to identify undiagnosed CDI. We used medical records to identify differences between healthcare settings in patient demographics and risk factors for detection of C. difficile with or without free toxin.ResultsThe CDI positivity rate was 4.4% (country range: 0-16.2) in hospital samples, and 1.3% (country range: 0-2.2%) in community samples. The highest prevalence of toxinotype IIIb (027, 181 and 176) was seen in eastern European countries (56%; 43/77), the region with the lowest testing rate (58%; 164/281). Different predisposing risk factors were observed (use of broad-spectrum penicillins in the community (OR: 8.09 (1.9-35.6), p = 0.01); fluoroquinolones/cephalosporins in hospitals (OR: 2.2 (1.2-4.3), p = 0.01; OR: 2.0 (1.1-3.7), p = 0.02)). Half of community CDI cases were undetected because of absence of clinical suspicion, accounting for three times more undiagnosed adults in the community compared with hospitals (ca 111,000 vs 37,000 cases/year in Europe).ConclusionThese findings support recommendations for improving diagnosis in patients presenting with diarrhoea in the community, to guide good practice to limit the spread of CDI.

COVID-19 rapid diagnostics: practice review.

Point-of-care tests for SARS-CoV-2 could enable rapid rule-in and/or rule-out of COVID-19, allowing rapid and accurate patient cohorting and potentially reducing the risk of nosocomial transmission. As COVID-19 begins to circulate with other more common respiratory viruses, there is a need for rapid diagnostics to help clinicians test for multiple potential causative organisms simultaneously.However, the different technologies available have strengths and weaknesses that must be understood to ensure that they are used to the benefit of the patient and healthcare system. Device performance is related to the deployed context, and the diagnostic characteristics may be affected by user experience.This practice review is written by members of the UK's COVID-19 National Diagnostic Research and Evaluation programme. We discuss relative merits and test characteristics of various commercially available technologies. We do not advocate for any given test, and our coverage of commercially supplied tests is not intended to be exhaustive.

Haem is crucial for medium-dependent metronidazole resistance in clinical isolates of Clostridioides difficile.

BACKGROUND: Until recently, metronidazole was the first-line treatment for Clostridioides difficile infection and it is still commonly used. Though resistance has been reported due to the plasmid pCD-METRO, this does not explain all cases. OBJECTIVES: To identify factors that contribute to plasmid-independent metronidazole resistance of C. difficile. METHODS: Here, we investigate resistance to metronidazole in a collection of clinical isolates of C. difficile using a combination of antimicrobial susceptibility testing on different solid agar media and WGS of selected isolates. RESULTS: We find that nearly all isolates demonstrate a haem-dependent increase in the MIC of metronidazole, which in some cases leads to isolates qualifying as resistant (MIC >2 mg/L). Moreover, we find an SNP in the haem-responsive gene hsmA, which defines a metronidazole-resistant lineage of PCR ribotype 010/MLST ST15 isolates that also includes pCD-METRO-containing strains. CONCLUSIONS: Our data demonstrate that haem is crucial for medium-dependent metronidazole resistance in C. difficile.

Is point-of-care testing feasible and safe in care homes in England? An exploratory usability and accuracy evaluation of a point-of-care polymerase chain reaction test for SARS-CoV-2.

INTRODUCTION: Reliable rapid testing for COVID-19 is needed in care homes to reduce the risk of outbreaks and enable timely care. This study aimed to examine the usability and test performance of a point of care polymerase chain reaction (PCR) test for detection of SARS-CoV-2 (POCKITTM Central) in care homes. METHODS: POCKITTM Central was evaluated in a purposeful sample of four UK care homes. Test agreement with laboratory real-time PCR and usability and used errors were assessed. RESULTS: No significant usability-related hazards emerged, and the sources of error identified were found to be amendable with minor changes in training or test workflow. POCKITTM Central has acceptable sensitivity and specificity based on RT-PCR as the reference standard, especially for symptomatic cases.Asymptomatic specimens showed 83.3% (95% confidence interval (CI): 35.9-99.6%) positive agreement and 98.7% negative agreement (95% CI: 96.2-99.7%), with overall prevalence and bias-adjusted kappa (PABAK) of 0.965 (95% CI: 0.932- 0.999). Symptomatic specimens showed 100% (95% CI: 2.5-100%) positive agreement and 100% negative agreement (95% CI: 85.8-100%), with overall PABAK of 1.Recommendations are provided to mitigate the frequency of occurrence of the residual use errors observed. Integration pathways were discussed to identify opportunities and limitations of adopting POCKIT™ Central for screening and diagnostic testing purposes. CONCLUSIONS: Point-of-care PCR testing in care homes can be considered with appropriate preparatory steps and safeguards. Further diagnostic accuracy evaluations and in-service evaluation studies should be conducted, if the test is to be implemented more widely, to build greater certainty on this initial exploratory analysis.

Development and clinical validation of an automated cell cytotoxicity neutralization assay for detecting Clostridioides difficile toxins in clinically relevant stools samples.

OBJECTIVES: To improve the diagnostic accuracy of Clostridioides difficile infection, current U.S. and E.U. guidelines recommend multistep testing that detects the presence of C. difficile and toxin in clinically relevant stool samples to confirm active disease. An accepted gold standard to detect C. difficile toxins is the cell cytotoxicity neutralization assay (CCNA). Although highly sensitive, the traditional CCNA has limitations. One such limitation is the subjective interpretation of an analyst to recognize cytopathic effects in cultured cells exposed to a fecal sample containing toxin. To overcome this limitation, an automated CCNA was developed that replaces most human pipetting steps with robotics and incorporates CellTiterGlo® for a semi-quantitative, non-subjective measure of cell viability instead of microscopy. METHODS: To determine sample positivity and control for non-specific cytopathic effects, two thresholds were defined and validated by evaluating the sample with/without antitoxin antisera (sample-antitoxin/sample + antitoxin): 1) a >70% cell viability threshold was validated with samples containing anti-toxin, and 2) a >1.2-fold difference cut-off where sample results above the cut-off are considered positive. RESULTS: Assay validation demonstrated excellent accuracy, precision, and sample linearity with an LOD of 126.9 pg/mL toxin-B in stool. The positivity cut-offs were clinically validated by comparing 322 diarrheal stool sample results with those run in a predicate, microscopic readout-based CCNA. The automated CCNA demonstrated 96% sensitivity and 100% specificity compared with the predicate CCNA. CONCLUSIONS: Overall, the automated CCNA provides a specific, sensitive, and reproducible tool to support determination of CDI epidemiology or the efficacy of interventions such as vaccines.

Ultrasensitive Clostridioides difficile Toxin Testing for Higher Diagnostic Accuracy.

Currently available diagnostic tests for Clostridioides difficile infection (CDI) lack specificity or sensitivity, which has led to guideline recommendations for multistep testing algorithms. Ultrasensitive assays for detection of C. difficile toxins provide measurements of disease-specific markers at very low concentrations. These assays may show improved accuracy compared to that of current testing methods and offer a potential standalone solution for CDI diagnosis, although large studies of clinical performance and accuracy are lacking.

Factors affecting reported Clostridioides difficile infection rates; the more you look the more you find, but should you believe what you see?

Reported rates of C. difficile infection (CDI) have increased in many settings; however, these can be affected by factors including testing density (test-density) and diagnostic methods. We aimed to describe the impact of multiple factors on CDI rates. Hospitals (n = 182) across five countries (France, Germany, Italy, Spain, and UK) provided data on; size and type of institution, CDI testing methodology, number of tests/month and patient-bed-days (pbds)/month over one year. Incidence rates were compared between countries, different sized institutions, types of institutions and testing method. After univariate analyses, the highest CDI rates were observed in Italy (average 11.8/10,000pbds/hospital/month), acute/primary hospitals (12.3/10,000pbds/hospital/month), small hospitals (16.7/10,000pbds/hospital/month), and hospitals using methods that do not detect toxin (NO-TOXIN) (e.g. GDH/NAAT or standalone NAAT) (10.7/10,000pbds/hospital/month). After adjusting for test-density, highest incidence rates were still in Italy, acute/primary hospitals and those using NO-TOXIN. The relative rate in long-term healthcare facilities (LTHCFs) increased, but size of institution no longer influenced the CDI rate. Test-density appears to have the largest effect on reported CDI rates. NO-TOXIN testing still influences CDI rates, even after adjusting for test-density, which is consistent with tests that 'overcall' true CDI. Low test-density can mask the true burden of CDI, e.g. in LTHCFs, highlighting the importance of good quality surveillance.

The perils of PCR-based diagnosis of Clostridioides difficile infections: Painful lessons from clinical trials.

Diagnostic tests favoured to detect C. difficile infections (CDI) have undergone successive changes. The problem of over-diagnosis with polymerase chain reaction (PCR) testing is recognized in the clinical setting; here we discuss the parallel of the clinical trial setting. We summarize and discuss four examples of the impact of method used to diagnose CDI on clinical trial outcomes. Bezlotoxumab, a human monoclonal antibody neutralizing toxin B, was found to be protective against recurrent CDI (rCDI) in clinical trials. A post hoc analysis showed that the magnitude of the relative reduction in rCDI rates of bezlotoxumab over placebo in patients diagnosed with toxin-based testing was almost double that in patients diagnosed with PCR. SER-109, a microbiome therapeutic developed to prevent rCDI, showed promise in a phase 1b trial, but results were not replicated in a phase 2 trial in which diagnosis was in majority PCR-based. Surotomycin, an oral lipopeptide antibiotic, was found to be non-inferior to vancomycin in phase 2 study, but development was discontinued after unfavourable phase 3 results in which the majority of CDI were diagnosed by PCR. Finally, a C. difficile vaccine program for a toxoid vaccine developed by Sanofi/Pasteur was terminated after interim analysis of a phase 3 trial, in which CDI diagnosis was based solely on PCR. We highlighted the perils of using PCR alone in studies involving different aspects of C. difficile clinical research, including immunotherapies, microbiome-based therapies, treatments, and vaccines. The importance of designing C. difficile clinical trials with careful consideration to the diagnostic testing method cannot be overemphasized.

Two Distinct Patterns of Clostridium difficile Diversity Across Europe Indicating Contrasting Routes of Spread.

Background: Rates of Clostridium difficile infection vary widely across Europe, as do prevalent ribotypes. The extent of Europe-wide diversity within each ribotype, however, is unknown. Methods: Inpatient diarrheal fecal samples submitted on a single day in summer and winter (2012-2013) to laboratories in 482 European hospitals were cultured for C. difficile, and isolates the 10 most prevalent ribotypes were whole-genome sequenced. Within each ribotype, country-based sequence clustering was assessed using the ratio of the median number of single-nucleotide polymorphisms between isolates within versus across different countries, using permutation tests. Time-scaled Bayesian phylogenies were used to reconstruct the historical location of each lineage. Results: Sequenced isolates (n = 624) were from 19 countries. Five ribotypes had within-country clustering: ribotype 356, only in Italy; ribotype 018, predominantly in Italy; ribotype 176, with distinct Czech and German clades; ribotype 001/072, including distinct German, Slovakian, and Spanish clades; and ribotype 027, with multiple predominantly country-specific clades including in Hungary, Italy, Germany, Romania, and Poland. By contrast, we found no within-country clustering for ribotypes 078, 015, 002, 014, and 020, consistent with a Europe-wide distribution. Fluoroquinolone resistance was significantly more common in within-country clustered ribotypes (P = .009). Fluoroquinolone-resistant isolates were also more tightly clustered geographically with a median (interquartile range) of 43 (0-213) miles between each isolate and the most closely genetically related isolate, versus 421 (204-680) miles in nonresistant pairs (P < .001). Conclusions: Two distinct patterns of C. difficile ribotype spread were observed, consistent with either predominantly healthcare-associated acquisition or Europe-wide dissemination via other routes/sources, for example, the food chain.

Patient and Strain Characteristics Associated With Clostridium difficile Transmission and Adverse Outcomes.

Background: No study has used whole-genome sequencing (WGS) to investigate risk factors for Clostridium difficile (CD) transmission between cases, or assessed the impact of recent acquisition on patient outcome. Methods: This 20 month retrospective cohort study included consecutive cytotoxin-positive diarrheal samples, which underwent culture, ribotyping, and WGS (Illumina). Sequenced isolates were compared using single nucleotide variants (SNVs). Independent predictors of acquisition from another case, onward transmission, 120-day recurrence, and 30-day mortality were identified using logistic regression with backwards elimination. Results: Of 660 CD cases, 640 (97%) were sequenced, of which 567 (89%) shared a ribotype with a prior case, but only 227 (35%) were ≤2 SNVs from a prior case, supporting recent acquisition. Plausible (<2 SNVs) recent ward-based acquisition from a symptomatic case was more frequent in certain ribotypes; 64% (67/105) for ribotype-027 cases, compared with 11% (6/57) for ribotype-078. Independent risk factors (adjusted P < .05) for CD acquisition included older age, longer inpatient duration, and ribotype; these factors, and male sex, increased onward transmission. Patients with a plausible donor had a greater risk of recurrence (adjusted P = .001) and trended towards greater 30-day mortality (adjusted P = .06). Ribotype had no additional mortality or recurrence impact after adjusting for acquisition (P > .1). Conclusions: Greater transmission of certain lineages suggests CD may have different reservoirs and modes of transmission. Acquiring CD from a recent case is associated with poorer clinical outcomes. Clinical characteristics associated with increased healthcare-associated CD transmission could be used to target preventative interventions.

Sensitivity of Single-Molecule Array Assays for Detection of Clostridium difficile Toxins in Comparison to Conventional Laboratory Testing Algorithms.

Guidelines recommend the use of an algorithm for the laboratory diagnosis of Clostridium difficile infection (CDI). Enzyme immunoassays (EIAs) detecting C. difficile toxins cannot be used as standalone tests due to suboptimal sensitivity, and molecular tests suffer from nonspecificity by detecting colonization. Sensitive immunoassays have recently been developed to improve and simplify CDI diagnosis. Assays detecting CD toxins have been developed using single-molecule array (SIMOA) technology. SIMOA performance was assessed relative to a laboratory case definition of CDI defined by positive glutamate dehydrogenase (GDH) screen and cell cytotoxicity neutralizing assay (CCNA). Samples were tested with SIMOA assays and a commercial toxin EIA to compare performance, with discrepancy resolution using a commercial nucleic acid-based test and a second cell cytotoxicity assay. The SIMOA toxin A and toxin B assays showed limits of detection of 0.6 and 2.9 pg/ml, respectively, and intra-assay coefficients of variation of less than 10%. The optimal clinical thresholds for the toxin A and toxin B assays were determined to be 22.1 and 18.8 pg/ml, respectively, with resultant sensitivities of 84.8 and 95.5%. In contrast, a high-performing EIA toxin test had a sensitivity of 71.2%. Thus, the SIMOA assays detected toxins in 24% more samples with laboratory-defined CDI than the high performing toxin EIA (95% [63/66] versus 71% [47/66]). This study shows that SIMOA C. difficile toxin assays have a higher sensitivity than currently available toxin EIA and have the potential to improve CDI diagnosis.

Ribotypes and New Virulent Strains Across Europe.

Clostridium difficile is a major bacterial cause of post-antibiotic diarrhoea. The epidemiology of C. difficile infections (CDI) has dramatically changed since the early 2000s, with an increasing incidence and severity across Europe. This trend is partly due to the emergence and rapid worldwide spread of the hypervirulent and epidemic PCR ribotype 027. Profiles of patients with CDI have also evolved, with description of community-acquired (CA) infections in patients with no traditional risk factors for CDI. However, recent epidemiological studies indicated that some European countries have successfully controlled the dissemination of the 027 clone whereas other countries recently reported the emergence of other virulent or unusual strains. The aims of this review are to summarize the current European CDI epidemiology and to describe the new virulent C. difficile strains circulating in Europe, as well as other potential emerging strains described elsewhere. Standardized typing methods and surveillance programmes are mandatory for a better understanding and monitoring of CDI in Europe.

Enhanced surveillance of Clostridium difficile infection occurring outside hospital, England, 2011 to 2013.

There are limited national epidemiological data for community-associated (CA)-Clostridium difficile infections (CDIs). Between March 2011 and March 2013, laboratories in England submitted to the Clostridium difficile Ribotyping Network (CDRN) up to 10 diarrhoeal faecal samples from successive patients with CA-CDI, defined here as C. difficile toxin-positive diarrhoea commencing outside hospital (or less than 48 hours after hospital admission), including those cases associated with community-based residential care, with no discharge from hospital within the previous 12 weeks. Patient demographics and C. difficile PCR ribotypes were compared for CA-CDIs in our study and presumed healthcare-associated (HA) CDIs via CDRN. Ribotype diversity indices, ranking and relative prevalences were very similar in CA- vs HA-CDIs, although ribotypes 002 (p ≤ 0.0001),020 (p = 0.009) and 056 (p < 0.0001) predominated in CA-CDIs; ribotype 027 (p = 0.01) predominated in HA-CDIs. Epidemic ribotypes 027 and 078 predominated in institutional residents with CDI (including care/nursing homes) compared with people with CDI living at home. Ribotype diversity decreased with increasing age in HA-CDIs, but not in CA-CDIs. Ribotype 078 CA-CDIs were significantly more common in elderly people (3.4% (6/174) vs 8.7% (45/519) in those aged < 65 and ≥ 65 years, respectively; p = 0.019). No antibiotics were prescribed in the previous four weeks in about twofold more CA-CDI vs HAs (38.6% (129/334) vs 20.3% (1,226/6,028); p < 0.0001). We found very similar ribotype distributions in CA- and HA-CDIs, although a few ribotypes significantly predominated in one setting. These national data emphasise the close interplay between, and likely common reservoirs for, CDIs, particularly when epidemic strains are not dominant.

Diversity of Clostridium difficile PCR ribotypes in Europe: results from the European, multicentre, prospective, biannual, point-prevalence study of Clostridium difficile infection in hospitalised patients with diarrhoea (EUCLID), 2012 and 2013.

Clostridium difficile infection (CDI) is the major cause of infective diarrhoea in healthcare environments. As part of the European, multicentre, prospective, biannual, point-prevalence study of Clostridium difficile infection in hospitalised patients with diarrhoea (EUCLID), the largest C. difficile epidemiological study of its type, PCR ribotype distribution of C. difficile isolates in Europe was investigated. PCR ribotyping was performed on 1,196 C. difficile isolates from diarrhoeal samples sent to the European coordinating laboratory in 2012-13 and 2013 (from two sampling days) by 482 participating hospitals from 19 European countries. A total of 125 ribotypes were identified, of which ribotypes 027 (19%, n =222), 001/072 (11%, n = 134) and 014/020 (10%, n = 119) were the most prevalent. Distinct regional patterns of ribotype distribution were noted. Of 596 isolates from patients with toxin-positive stools (CDI cases), ribotype 027 accounted for 22% (32/144) of infections in cases aged from 18 to less than 65 years, but the prevalence decreased in those aged ≥ 65 years (14% (59/412)) and further decreased in those aged ≥ 81 years (9% (18/195)). The prevalence of ribotype 027 and 176, but not other epidemic strains, was inversely proportional to overall ribotype diversity (R(2) = 0.717). This study highlights an increased diversity of C. difficile ribotypes across Europe compared with previous studies, with considerable intercountry variation in ribotype distribution. Continuous surveillance programmes are necessary to monitor the changing epidemiology of C. difficile.

Characterization of resistance to Pratylenchus thornei (Nematoda) in wheat (Triticum aestivum): attraction, penetration, motility, and reproduction.

Lines from a cross between two wheat (Triticum aestivum) cultivars with contrasting resistance phenotypes to Pratylenchus thornei (Nematoda) were investigated to determine the stage at which resistance occurs. Host resistance was examined at nematode attraction to and penetration of roots and nematode motility, maturation, and reproduction within roots. There was no significant difference in the rate at which P. thornei was attracted toward or penetrated resistant or susceptible roots. However, suppression of migration, juvenile maturation, and reproduction in and near resistant roots was evident, suggesting that resistance acts post penetration. No preferential root penetration zone was observed in contrast to other studies. The inhibitory compounds from resistant wheat plants appeared to be constitutively expressed and water soluble because nematode migration was suppressed in roots and root exudates of unchallenged seedlings. The effects of these compounds were reversible and affected P. thornei but not P. neglectus. Apart from migration, nematode multiplication was greatly inhibited by resistance because only a few juveniles (10%) developed past stage three in roots of resistant compared with susceptible plants. Earlier in the life cycle, egg deposition and hatch of P. thornei were also significantly reduced in resistant roots and root exudates, suggesting the presence of hatching inhibitors.

Ensuring accuracy in the development and application of nucleic acid amplification tests (NAATs) for infectious disease.

Diagnostic tests were heralded as crucial during the Coronavirus disease (COVID-19) pandemic with most of the key methods using bioanalytical approaches that detected larger molecules (RNA, protein antigens or antibodies) rather than conventional clinical biochemical techniques. Nucleic Acid Amplification Tests (NAATs), like the Polymerase Chain Reaction (PCR), and other molecular methods, like sequencing (that often work in combination with NAATs), were essential to the diagnosis and management during COVID-19. This was exemplified both early in the pandemic but also later on, following the emergence of new genetic SARS-CoV-2 variants. The 100 day mission to respond to future pandemic threats highlights the need for effective diagnostics, therapeutics and vaccines. Of the three, diagnostics represents the first opportunity to manage infectious diseases while also being the most poorly supported in terms of the infrastructure needed to demonstrate effectiveness. Where performance targets exist, they are not well served by consensus on how to demonstrate they are being met; this includes analytical factors such as limit of detection (LOD) false positive results as well as how to approach clinical evaluation. The selection of gold standards or use of epidemiological factors such as predictive value, reference ranges or clinical thresholds are seldom correctly considered. The attention placed on molecular diagnostic tests during COVID-19 illustrates important considerations and assumptions on the use of these methods for infectious disease diagnosis and beyond. In this manuscript, we discuss state-of-the-art approaches to diagnostic evaluation and explore how they may be better tailored to diagnostic techniques like NAATs to maximise the impact of these highly versatile bioanalytical tools, both generally and during future outbreaks.