How to verify and validate a clinical microbiology test before it can be used in routine diagnostics: a practical guide.

BACKGROUND: Before a new test can be routinely used in your laboratory, its reliability must be established in the laboratory where it will be used. International standards demand validation and verification procedures for new tests. The International Organization for Standardization (ISO) 15189 was recently updated, and the European Commission's In Vitro Diagnostic Regulation (IVDR) came into effect. These events will likely increase the need for validation and verification procedures. OBJECTIVES: This paper aims to provide practical guidance in validating or verifying microbiology tests, including antimicrobial susceptibility tests in a clinical microbiology laboratory. SOURCES: It summarizes and interprets certain parts of standards such as ISO 15189:2022, and regulations, such as IVDR 2017/746 regarding validation or verification of a new test in a routine clinical microbiology laboratory. CONTENT: The reasons for choosing a new test and the outline of the validation and verification plan are discussed. Furthermore, the following topics are touched upon: the choice of reference standard, number of samples, testing procedures, how to solve the discrepancies between results from new test and reference standard, and acceptance criteria. Arguments for selecting certain parameters (such as reference standard and sample size) and examples are given. IMPLICATIONS: With the expected increase in validation and verification procedures because of the implementation of IVDR, this paper may aid in planning and executing these procedures.

Evaluating the Clinical Relevance of Routine Sonication for Periprosthetic Hip or Knee Joint Infection Diagnosis.

Periprosthetic joint infection (PJI) is a serious complication after joint arthroplasty. PJI screening and conventional cultures may be inconclusive. Sonication fluid culturing stands out as a valuable adjunct technique for PJI diagnosis. This study aims to determine the clinical relevance of routine sonication for all (a)septic revisions. All patients who underwent (partial) hip or knee revision arthroplasty between 2012 and 2021 were retrospectively reviewed. We formed three groups based on the European Bone and Joint Society PJI criteria: infection confirmed, likely, and unlikely. We analyzed clinical, laboratory, and radiological screening. Sensitivity and specificity were calculated for synovial fluid (preoperative), tissue, and sonication fluid cultures. We determined the clinical relevance of sonication as the percentage of patients for whom sonication confirmed PJI; 429 patients who underwent (partial) revision of hip or knee arthroplasty were included. Sensitivity and specificity were 69% and 99% for synovial fluid cultures, 76% and 92% for tissue cultures, and 80% and 89% for sonication fluid cultures, respectively. Sonication fluid cultures improved tissue culture sensitivity and specificity to 83% and 99%, respectively. In 11% of PJIs, sonication fluid cultures were decisive for diagnosis. This is applicable to acute and chronic infections. Sonication fluid cultures enhanced the sensitivity and specificity of PJI diagnostics. In 11% of PJI cases, causative pathogens were confirmed by sonication fluid culture results. Sonication fluid culture should be performed in all revision arthroplasties.

Clinical outcomes of rifampicin combination therapy in implant-associated infections due to staphylococci and streptococci: A systematic review and meta-analysis.

OBJECTIVES: Adjunctive rifampicin for implant-associated infections is controversial. This study investigated the clinical outcomes of rifampicin combination therapy compared with monotherapy in treating prosthetic joint infection (PJI) or prosthetic valve endocarditis (PVE) due to staphylococci and streptococci. METHODS: A systematic search was performed from inception to 13 June 2022 in Embase, MEDLINE, Cochrane and Web of Science to investigate the clinical outcomes of rifampicin combination therapy compared with monotherapy in treating staphylococcal and streptococcal PJI or PVE. Randomised controlled trials (RCTs) and observational studies were included in the systematic review and meta-analysis. RESULTS: Fourteen studies were included. A moderate quality of evidence was found in favour of rifampicin in patients with staphylococcal PJI who underwent a debridement, antibiotics and implant retention (DAIR) procedure [odds ratio = 2.49, 95% confidence interval (CI) 1.93-3.23]. Including the two RCTs only, adding rifampicin to the antibiotic regimen after DAIR was also in favour of rifampicin, but this was not statistically significant (risk ratio = 1.27, 95% CI 0.79-2.04; n = 126). Pooling data for patients with staphylococcal PJI who underwent a two-stage procedure showed that adding rifampicin was not associated with therapeutic success. Limited evidence was found for the use of rifampicin for PVE caused by staphylococci. CONCLUSIONS: Adding rifampicin in the treatment of staphylococcal PJI treated by DAIR clearly increased the likelihood for therapeutic success. The clinical benefit of adjunctive rifampicin in the treatment of other staphylococci and streptococci implant-associated infections is still unclear.

Less Is More: When to Repeat Antimicrobial Susceptibility Testing.

This study investigated the frequency of change of the antimicrobial susceptibility pattern when the same isolate was found in the same patient in various situations. We used laboratory data collected over a period of 8 years (January 2014 to December 2021) at the clinical microbiology laboratory of a tertiary hospital for Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., Pseudomonas aeruginosa, and Staphylococcus aureus. Antimicrobial susceptibility tests (AST) were performed using Vitek 2 automated system. We determined essential agreement and categorical agreement, and introduced the new terms essential MIC increase and change from nonresistant to resistant to present changes in antimicrobial susceptibility over time. During the study period, 18,501 successive AST were included. The risk for S. aureus to be resistant to any antibiotic upon repeated culture was <10% during a follow-up of 30 days. For Enterobacterales, this risk was approximately 10% during a follow-up of 7 days. For P. aeruginosa, this risk was higher. The longer the follow-up period, the higher the risk that the bacteria would show phenotypic resistance. We also found that some drug-bug combinations were more likely to develop phenotypical resistance (i.e., E. coli/amoxicillin-clavulanic acid and E. coli/cefuroxime). A potential consequence of our finding is that if we regard a risk of resistance below 10% as acceptable, it may be feasible to omit follow-up AST within 7 days for the microorganisms investigated in this study. This approach saves money, time, and will reduce laboratory waste. Further studies are needed to determine whether these savings are in balance with the small possibility of treating patients with inadequate antibiotics.

Epidemiology of Mycoplasma genitalium and Trichomonas vaginalis in the primary health care setting in the Netherlands.

The aim of this paper is to describe the prevalence of Mycoplasma genitalium and Trichomonas vaginalis in patients who visited general practitioners in the Netherlands. Additionally, we describe the prevalence of M. genitalium resistance to azithromycin and moxifloxacin. We used data from 7,411 consecutive female patients who were screened for Chlamydia trachomatis, Neisseria gonorrhoeae, M. genitalium, and T. vaginalis and data from 5,732 consecutive male patients screened for C. trachomatis, N. gonorrhoeae, and M. genitalium. The prevalence of M. genitalium and T. vaginalis in female patients was 6.7% (95% CI: 6.2 to 7.4) and 1.9% (95%CI: 1.6 to 2.2%), respectively. M. genitalium prevalence in male patients was 3.7% (3.3 to 4.3). M. genitalium co-occurred with C. trachomatis in 1.4% (0.3 to 0.6%) of female and in 0.7% (0.5 to 0.9) of male patients. Macrolide resistance gene mutations and fluoroquinolone resistance gene mutations were detected in 73.8% and 9.9%, respectively. We concluded that M.genitalium is relatively infrequently found in a large general practitioner population in the Netherlands. It can co-occur with C. trachomatis, and is often resistant to azithromycin. Therefore, when treating sexually transmitted infections, these prevalence and resistance data should be taken into account.

A narrative review of the intermediate category of the antimicrobial susceptibility test: relation with dosing and possible impact on antimicrobial stewardship.

The interpretation of 'susceptible (S)' or 'resistant (R)' results of antimicrobial susceptibility testing is easily understood, but the interpretation of the 'intermediate (I)' category can be confusing. This review critically discusses how this categorization (clinical breakpoints) comes into being with the emphasis on the use of pharmacokinetics and pharmacodynamic data. It discusses the differences between the 'I' according to the CLSI and the EUCAST. This review also discusses the recent EUCAST change of the 'I' definition, and the impact of this change from laboratory and clinical points of view.

The importance of disease prevalence in clinical decision making: a real practice study on COVID-19 antigen test in Curacao.

The performance of a test can be suboptimal, but in appropriate setting such a test is still useful for clinical decision making. We investigated the role of Antigen Rapid Diagnostic Test (Ag-RDT) for clinical decision making in an Emergency Department (ED) in Curacao during peak of COVID-19 pandemic. Ag-RDT was performed in the naso- and oropharynx-swabs from patients with respiratory insufficiency presented to the ED. Ag-RDT was performed in 153 patients, of which 64 (41.8%) showed positive results. Comparing Ag-RDT results with molecular tests, its sensitivity was 68.8% (95% CI 57.4 to 78.7), and specificity of 94.6% (95% CI 84.9 to 98.9). The positive and negative predictive value were 95.1% (95% CI 86.5 to 98.3) and 66.3 (95% CI 58.6 to 73.3), respectively. All patients with Ag-RDT positive test were admitted to the cohorted COVD-19 department of the hospital. By using Ag-RDT, 35.9% of rapid PCR tests (that are more costly and laborious to perform) could be avoided at cost of 5.8% patients with false positive result. In conclusion, in real practice, disease prevalence is as important as test's performance for clinical decision making. The conclusion may also be applicable for other diagnostic tests than COVID-19 diagnostic.

The State of Microbiology Diagnostic of Prosthetic Joint Infection in Europe: An In-Depth Survey Among Clinical Microbiologists.

Background: This study aims to give an overview on how microbiology diagnosis tests of Prosthetic joint infections (PJI) is performed in Europe, and to explore whether any factor influences the decision on implementing a test. Methods: An extensive online survey of clinical microbiologists from seven European countries (Belgium, Estonia, Germany, Italy, Netherlands, Switzerland, and Spain). Following items were assessed: (i). general information on the laboratory, (ii) preference of the laboratory and clinical microbiologists regarding samples, (iii) transportation and (iv) processing of explanted foreign bodies and tissues and synovial fluid, (v) culture media and culture duration, (vi) reporting (identification and susceptibility testing), and (vii) use of molecular microbiology techniques. Results: Invited were 163 clinical microbiologists. The response rate from each country was above 50% (range 51-78%), except for Germany (36%). Frequent PJI diagnostics were the use of tissue pre-processing (58.1%), culturing synovial fluid in blood culture bottles (45.5%), use of sonication for processing explanted prosthesis (56.8%), reporting the presence of synovial leukocyte counts (67%), use of blood aerobic and anaerobic agar (97.7%), and enrichment media thioglycolate (69.3%). The most common incubation time of the culture media is 7-14 days (34.1-70.5%). The clinicians were called to report the culture results (80.7%), and to give antibiotic recommendation (67%). Conclusion: There are common practices in processing PJI samples and reporting results, which is promising for harmonization of PJI diagnostic in the future. However, variation in diagnostic tests should also be considered in interpreting and comparing clinical microbiology results.

Valid Evidence for Diagnosis and Treatment of Infections in the Intensive Care Unit: Beyond Randomized Control Trial Study Design (Trial Emulation and Machine Learning).

Infection in critically ill patients is an important problem [...].

High Prevalence of ST502 Carrying an OXA-24 Carbapenemase gene in Carbapenem-Nonsusceptible Acinetobacter baumannii-calcoaceticus Isolates in Romania.

Background: Acinetobacter baumannii can cause difficult-to-treat infections because it can acquire extensive antimicrobial resistance mechanisms. We aim to describe the antimicrobial resistance pattern and the genetic basis of carbapenem-nonsusceptible A. baumannii isolates in a University Hospital in Romania, a country where multidrug-resistant A. baumannii is widespread. Methods: We collected 104 consecutive meropenem-nonsusceptible A. baumannii isolates from 104 patients (36% female, mean age [SD] of 63 [16] years) between May 2015 and August 2017 from a large tertiary center in Romania. Whole-genome sequencing of representative isolates from amplified fragment length polymorphism clusters was used to determine clonality and resistance patterns. Results: All isolates were resistant to piperacillin/tazobactam, ceftazidime, and ciprofloxacin; 88.5% to gentamicin; and 90.4% to trimethoprim/sulfamethoxazole. In contrast, 79.8% and 99.0% were susceptible to tobramycin and colistin, respectively. The only isolate resistant to colistin had an minimum inhibitory concentration (MIC) of ≥16 mg/L. The blaOXA-24 gene was detected in 79.1% and blaOXA-23 in 20.9% of the isolates. In one isolate, blaOXA-23 was copresent with blaOXA-24. ST502 (Oxford scheme) was the most prevalent sequence type and was exclusively associated with blaOXA-24. Conclusions: ST502 associated with blaOXA-24 was frequently observed in the region where carbapenem-nonsusceptible A. baumannii was found to be endemic. In these isolates, tobramycin and colistin might be the remaining therapeutic options. Due to differences in gentamicin and tobramycin resistance in these isolates, surveillance data should not group gentamicin, tobramycin, and amikacin together as aminoglycosides.

The unintended contribution of clinical microbiology laboratories to climate change and mitigation strategies: a combination of descriptive study, short survey, literature review and opinion.

OBJECTIVE: Climate change poses a significant threat to humanity and human activity is largely responsible for it. Clinical microbiology laboratories have their unintended shares in carbon dioxide (CO2) emissions. The aim of this study is to estimate CO2 emission of a clinical microbiology laboratory and to propose initiatives to reduce the emissions. METHODS: CO2 emission of instruments was estimated based on their electricity consumption. CO2 emitted in producing consumables was estimated by weighing the consumables needed to perform major tests in a large academic hospital. A systematic literature review was performed to identify studies on the impact of clinical microbiology laboratories on the environment. A short survey was sent to four major manufacturers of agar plates on initiatives to reduce the environmental impact of their products. Opinion was given on activities that can reduce CO2 emission in laboratories. RESULTS: The study shows that the largest amount of CO2 emission in the microbiological laboratories comes from consumables and personnel commuting. For example, the production and transportation of agar plates needed to culture samples for a year in a hospital with 1320 beds result in 16 590 kg CO2 is emitted. All survey participants mentioned that they were committed to reduce environmental impact of their products. The initiatives to reduce CO2 emission can be performed at the laboratory and at policy level, such as reducing the number of tests to only the necessary amount to reduce consumables. DISCUSSION: The calculations contribute to map CO2-related emissions in clinical microbiology laboratory activities, and the proposed initiatives to reduce the CO2 may serve as starting point for further discussions.

Fosfomycin Susceptibility Testing Using Commercial Agar Dilution Test.

The reference standard for fosfomycin antimicrobial susceptibility testing (AST) is agar dilution, but it is laborious and is not routinely used in diagnostic microbiology. In this study, we evaluated the performance of a ready-to-use commercially available agar dilution kit for fosfomycin AST (Liofilchem Diagnostics). We compared this kit with the reference standard agar dilution, performed according to the Clinical & Laboratory Standards Institute (CLSI) in 229 clinical isolates. The isolates were selected to represent both Gram-positive and Gram-negative microorganisms, with various MIC values. It consisted of 43 enterococci (E. faecalis n = 16, E. faecium n = 27), 13 methicillin-resistant S. aureus (MRSA), 118 Enterobacterales (Escherichia coli n = 94, Klebsiella pneumoniae n = 20, and Enterobacter cloacae complex n = 4), 55 Pseudomonas aeruginosa, and three ATCC isolates. Using CLSI breakpoints for enterococci for oral treatment of urinary tract infections, European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints for intravenous dosing for Enterobacterales and Staphylococci, and epidemiological cutoff value for P. aeruginosa, the essential agreement was 87.5%, and 99.6% after discrepancy resolution. There was no very major error, and 1.9% major error before, and 0.9% major error after resolution of discrepancies. The commercial test showed 100% reproducibility. In conclusion, in comparison to the reference standard, the ready-to-use commercially available agar dilution kit for fosfomycin AST showed excellent performance. IMPORTANCE Fosfomycin is increasingly needed to treat infection due to multidrug resistant microorganisms. Yet, the antimicrobial susceptibility test (AST) of fosfomycin is fraught with difficulties or often laborious to perform. An easy-to-use AST method for fosfomycin is thus needed. In this study, we showed that the ready-to-use commercially available agar dilution kit, in comparison to the reference standard, showed excellent performance.

The importance of disease prevalence in clinical decision making: a real practice study on COVID-19 antigen test in Curacao

ABSTRACT The performance of a test can be suboptimal, but in appropriate setting such a test is still useful for clinical decision making. We investigated the role of Antigen Rapid Diagnostic Test (Ag-RDT) for clinical decision making in an Emergency Department (ED) in Curacao during peak of COVID-19 pandemic. Ag-RDT was performed in the naso- and oropharynxswabs from patients with respiratory insufficiency presented to the ED. Ag-RDT was performed in 153 patients, of which 64 (41.8%) showed positive results. Comparing Ag-RDT results with molecular tests, its sensitivity was 68.8% (95% CI 57.4 to 78.7), and specificity of 94.6% (95% CI 84.9 to 98.9). The positive and negative predictive value were 95.1% (95% CI 86.5 to 98.3) and 66.3 (95% CI 58.6 to 73.3), respectively. All patients with Ag-RDT positive test were admitted to the cohorted COVD-19 department of the hospital. By using Ag-RDT, 35.9% of rapid PCR tests (that are more costly and laborious to perform) could be avoided at cost of 5.8% patients with false positive result. In conclusion, in real practice, disease prevalence is as important as test's performance for clinical decision making. The conclusion may also be applicable for other diagnostic tests than COVID-19 diagnostic.

Aspergillus in Critically Ill COVID-19 Patients: A Scoping Review.

Several reports have been published on Aspergillus findings in COVID-19 patients leading to a proposition of new disease entity COVID-19-associated pulmonary aspergillosis. This scoping review is designed at clarifying the concepts on how the findings of Aspergillus spp. in COVID-19 patients were interpreted. We searched Medline to identify the studies on Aspergillus spp. findings in COVID-19 patients. Included were observational studies containing the following information: explicit mention of the total number of the study population, study period, reason for obtaining respiratory samples, case definition, and clinical outcomes. Excluded were case series, case reports and reviews. Identified were 123 publications, and 8 observational studies were included. From the included studies the following issues were identified. The proportion of immunocompromised patients considered as host factors varied from 0 to 17%. Most of the studies did not mention radiographic findings explicitly. Respiratory samples were mostly obtained to investigate clinical deterioration. Aspergillus culture, antigen or PCR testing on bronchoalveolar lavage (BAL) fluid were performed in between 23.3% and 66.3% of the study population. Two studies performed periodic samples of BAL. Galactomannan index (GI) positivity in BAL was between 10% and 28%. GI in blood was found in 0.9% to 6.7% of the available samples. The prevalence of COVID-19-associated pulmonary aspergillosis ranged from 2.7% to 27.7%. Studies compared the mortality between defined cases and non-cases, and all showed increased mortality in cases. No studies showed that antifungal treatment reduced mortality. Concluding, this review showed how studies defined the clinical entity COVID-19-associated pulmonary aspergillosis where positive Aspergillus test in the respiratory sample was the main driver for the diagnosis. There were many differences between studies in terms of test algorithm and Aspergillus test used that largely determined the prevalence. Whether antifungal therapy, either as prophylaxis, pre-emptive or targeted therapy will lead to better outcomes of COVID-19-associated pulmonary aspergillosis patients is still need to be answered.

Pre-processing tissue specimens with a tissue homogenizer: clinical and microbiological evaluation.

BACKGROUND: Tissues are valuable specimens in diagnostic microbiology because they are often obtained by invasive methods, and effort should thus be taken to maximize microbiological yield. The objective of this study was to evaluate the added value of using tissue pre-processing (tissue homogenizer instrument gentleMACS Dissociator) in detecting microorganisms responsible for infections. METHODS: We included 104 randomly collected tissue samples, 41 (39.4 %) bones and 63 (60.6 %) soft tissues, many of those (42/104 (40.4 %)) were of periprosthetic origins. We compared the agreement between pre-processing tissues using tissue homogenizer with routine microbiology diagnostic procedure, and we calculated the performance of these methods when clinical infections were used as reference standard. RESULTS: There was no significant difference between the two methods (McNemar test, p = 0.3). Among the positive culture using both methods (n = 62), 61 (98.4 %) showed at least one similar microorganism. Exactly similar microorganisms were found in 42/62 (67.7 %) of the samples. From the included tissues, 55/ 104 (52.9 %) were deemed as infected. We found that the sensitivity of homogenized tissue procedure was lower (83.6 %) than when tissue was processed using tissue homogenizer (89.1 %). Sub-analysis on periprosthetic tissues and soft or bone tissues showed comparable results. CONCLUSIONS: The added value of GentleMACS Dissociator tissue homogenizer is limited in comparison to routine tissue processing.