Volume 57, Issue 8

The use of saliva samples in clinical studies has increased. However, the diagnostic value of whole saliva is compromised in the presence of blood contamination, owing to the higher levels of analytes in blood compared with those in saliva. The aim of this study was to review the existing methods and their limitations for measuring the levels of blood contamination in saliva. A literature search was performed using Web of Science, SCOPUS, and PubMed databases and 49 articles dealing with salivary diagnostics and measurements of blood contamination were included. Five methods for measuring the degree of blood components in saliva were discussed, including “visual inspection”, use of “strip for urinalysis”, and detection of plasma proteins such as “hemoglobin”, “albumin”, and “transferrin”. Each method has its limitations, and transferrin has been regarded as the most reliable and valid marker for blood contamination in saliva. However, transferrin in whole saliva may not be solely a product of blood, and its level in whole saliva can be influenced by several factors such as age, gonadal hormones, salivary flow rate, chewing performance, and oral microorganisms. In conclusion, when quantitatively analyzing whole saliva samples, the influence of blood contamination should be considered.

Background Central adrenal insufficiency (CAI) is characterized by impaired adrenocorticotropin (ACTH) secretion because of a disease or injury to the hypothalamus or the pituitary, leading to a reduced cortisol production. CAI suspicion arises more frequently in patients with pituitary tumors, cranial irradiation/surgery/injury/infections, as well as after exogenous glucocorticoid withdrawal. Nevertheless, a late diagnosis is not uncommon because CAI may present with nonspecific signs or symptoms, as fatigue or hyponatremia. Content The PubMed database was searched (years 1980–2018), using “central adrenal insufficiency” and “ACTH deficiency” as keywords. Subsequently, reference sections of the retrieved articles were searched. Summary Dynamic tests are needed when morning basal cortisol levels are not sufficient to exclude or to confirm CAI. Short Synacthen Test (SST) is the most used, and Endocrine Society’s guidelines recommend a cortisol peak >500 nmol/L to exclude CAI. Despite thresholds, understanding the pretest probability of ACTH deficiency (the clinical background of the patient) is essential because the diagnostic accuracy of SST in case of a negative result is suboptimal. Glucocorticoid replacement therapy, able to replicate cortisol circadian rhythm, is required in patients with CAI; fludrocortisone treatment is not necessary. Short-acting glucocorticoid drugs (hydrocortisone or cortisone acetate) are the most used; lower doses than previously used are nowadays recommended to reduce cortisol-related comorbidities. Promising results have been obtained with modified-release hydrocortisone, especially regarding glucose metabolism in patients with primary adrenal insufficiency. Outlook An accurate clinical diagnosis and a careful individualized therapy are mandatory in patients with CAI.

Background The process of technology validation and transfer of new molecular diagnostic tests towards the clinic faces challenges and needs to be improved. There is no empirical measure of the chronology and pace of technology transfer of molecular genetic discoveries. Methods We studied these for 29 molecular genetic test discoveries in order to (1) provide estimates of the timeframe between discovery of a clinical application and complete clinical implementation, and (2) compare the trajectories between different new tests to identify common patterns. We identified 11 publicly available “timestamps” for the technology transfer process ranging from discovery of the marker to use in a clinical setting. For each test selected, we searched public databases to identify available timestamps and dates. We plotted and compared trajectories of individual tests, including chronology. Results We show that there is much variability in the chronology of transfer between biomarkers. The median time between discovery of the marker and availability of the clinical test was 9.5 years (minimum 1). There was a median time of 18 years between test discovery and FDA approval (minimum 7 years), and it took a median of 17 years between discovery and the availability of a certified reference material for the 10 assays that have one (minimum 9 years). Conclusions We conclude that new molecular genetic tests take significant time between discovery and clinical implementation, and that further work is needed to pinpoint key factors, including policy and organization factors, that may allow for improving and streamlining this process.

Background The National Institute of Standards and Technology (NIST) Reference Material RM 8366 was developed to improve the quality of gene copy measurements of EGFR (epidermal growth factor receptor) and MET (proto-oncogene, receptor tyrosine kinase), important targets for cancer diagnostics and treatment. The reference material is composed of genomic DNA prepared from six human cancer cell lines with different levels of amplification of the target genes. Methods The reference values for the ratios of the EGFR and MET gene copy numbers to the copy numbers of reference genes were measured using digital PCR. The digital PCR measurements were confirmed by two additional laboratories. The samples were also characterized using Next Generation Sequencing (NGS) methods including whole genome sequencing (WGS) at three levels of coverage (approximately 1 ×, 5 ×  and greater than 30 ×), whole exome sequencing (WES), and two different pan-cancer gene panels. The WES data were analyzed using three different bioinformatic algorithms. Results The certified values (digital PCR) for EGFR and MET were in good agreement (within 20%) with the values obtained from the different NGS methods and algorithms for five of the six components; one component had lower NGS values. Conclusions This study shows that NIST RM 8366 is a valuable reference material to evaluate the performance of assays that assess EGFR and MET gene copy number measurements.

Background Identifying frail elderly subjects is of paramount importance in order to conduct a tailored care. The characterization of frailty status is currently based on the collection of clinical data and on the use of various tools such as Fried’s criteria, which constitutes a difficult and time-consuming process. Up to now, no biological markers have been described as reliable tools for frailty characterization. We tested the hypothesis that a link between frailty and protein molecular aging existed. This study aimed therefore at determining whether post-translational modification derived products (PTMDPs), recognized as biomarkers of protein aging, were associated with frailty status in elderly subjects. Methods Frailty status was determined according to Fried’s criteria in 250 elderly patients (>65 years old) hospitalized in a short-term care unit. Serum concentrations of protein-bound PTMDPs, including carboxymethyllysine (CML), pentosidine, methylglyoxal-hydroimidazolone-1 and homocitrulline (HCit), were determined by liquid chromatography coupled with tandem mass spectrometry, and tissue content of advanced glycation end-products was assessed by skin autofluorescence (SAF) measurement. Associations between PTMDPs and frailty status were analyzed using logistic regression models. Results Frail patients had significantly (p<0.01) higher CML, HCit, and SAF values compared to non-frail and pre-frail subjects. By multivariate analysis, only HCit concentrations and SAF values remained associated with frailty status (p=0.016 and p=0.002, respectively), independently of age, comorbidities, renal function, C-reactive protein and albumin concentrations. Conclusions HCit and SAF are significantly associated with frailty status in elderly subjects. This study suggests that PTMDPs constitute promising biomarkers for identifying frail patients and guiding personalized patient care.

Background Excessive sodium intake is a risk factor for hypertension, cardiovascular disease and the risk for kidney failure in chronic kidney disease (CKD) patients. Methods We tested the diagnostic performance and the feasibility of an inexpensive method based on urine chloride strips for self-monitoring sodium intake in a series of 72 CKD patients. Results Twenty-four hour urinary chloride as measured by the reactive strips and 24 h urinary sodium were interrelated (r=0.59, p<0.001). Forty-nine out of 72 patients (78%) had a 24 h urinary sodium >100 mmol/24 h, i.e. the upper limit recommended by current CKD guidelines. The strip method had 75.5% sensitivity and 82.6% specificity to correctly classify patients with urine sodium >100 mmol/24 h. The positive and the negative predictive values were 90.2% and 61.3%, respectively. The overall accuracy (ROC curve analysis) of urine chloride self-measurement for the >100 mmol/24 h sodium threshold was 87% (95% CI: 77%–97%). The large majority of patients (97%) perceived the test as useful to help compliance with the prescribed dietary sodium and considered the test as simple and of immediate application (58%) or feasible but requiring attention (39%). Conclusions A simple and inexpensive test for urine chloride measurement has a fairly good performance for the diagnosis of excessive sodium intake. The test is feasible and it is perceived by CKD patients as helpful for enhancing compliance to the dietary sodium recommendations. The usefulness of this test for improving hypertension control in CKD patients will be tested in a clinical trial (Clinicaltrials.gov RF-2010-2314890).

Background Urinary conductivity allows a coarse prediction of urinary osmolality in most cases but is insensitive to the osmolal contribution of uncharged particles and the presence of roentgen contrast media. Urinary osmolality can be estimated on the recently introduced Sysmex UF-5000 urine analyzer using conductivity. In this study, we evaluated the analytical performance of this research parameter. Secondly, we aimed to improve the manufacturer’s algorithm for estimating urinary osmolality, based on standard urinalysis parameters (creatinine, glucose, relative density). Methods The analytical performance was determined and a prediction model to estimate urinary osmolality based on urinalysis parameters was developed. We further developed and validated a prediction model using another set of routine urine samples. In addition, the influence of roentgen contrast media on urinary osmolality was studied. Results The within-run and between imprecision for osmolality and conductivity measured on the Sysmex UF-5000 ranged from 1.1% to 4.9% and 0.7% to 4.8%, respectively. Multiple regression analysis revealed urinary creatinine, conductivity and relative density to be the strongest predictors to estimate urinary osmolality. A mean difference of 1.3 mOsm/kg between measured and predicted osmolality demonstrated that the predictive performance of our model was favorable. An excellent correlation between the relative density and % contrast media was demonstrated. Conclusions Urinary osmolality is an important parameter for assessing specimen dilution in urinalysis. Urinary conductivity, along with relative density and urinary creatinine allows a coarse prediction of urinary osmolality and is insensitive to the osmolal contribution of uncharged particles and the presence of roentgen contrast media.

Background The addition of S100B protein to guidelines for the management of mild traumatic brain injury (mTBI) decreases the amount of unnecessary computed tomography (CT) scans with a significant decrease in radiation exposure and an increase in cost savings. Both DiaSorin and Roche Diagnostics have developed automated assays for S100B determination. Recently, bioMérieux developed a prototype immunoassay for serum S100B determination. For the first time, we present the evaluation of the S100B measurement using a bioMérieux Vidas ® 3 analyzer. Methods We evaluated the matrix effects of serum and plasma, and their stability after storage at 2–8 °C, −20 °C and −80 °C. The new measurement prototype (bioMérieux) was compared with an established one (Roche Diagnostics), and a precision study was also conducted. Lastly, clinical diagnostics performance of the bioMérieux and Roche Diagnostics methods were compared for 80 patients referred to the Emergency Department for mTBI. Results Stability after storage at 2–8 °C, −20 °C, and −80 °C and validation of the serum matrix were demonstrated. The bioMérieux analyzer was compared to the Roche Diagnostics system, and the analytical precision was found to be efficient. Clinical diagnosis performance evaluation confirmed the predictive negative value of S100B in the management of mTBI. Conclusions The study’s data are useful for interpreting serum S100B results on a bioMérieux Vidas ® 3 analyzer.

Background The present study was undertaken to evaluate the clinical impact of a thyroglobulin (Tg) minirecovery test (Tg-mRec) in a large series of differentiated thyroid carcinoma (DTC) patients treated and monitored homogeneously in a tertiary referral center. Methods Included were 1120 serum samples from 798 DTC patients. Tg, Tg autoantibodies (TgAb) and Tg-mrec measurements were performed on the automated Kryptor ® platform and results compared to the corresponding clinical status. Results Among included samples 228 (20%) were TgAb-positive (TgAb+) and 892 (80%) TgAb-negative (TgAb−), respectively. Tg cutoff points were settled at 0.31 μg/L and 0.15 μg/L for TgAb− and TgAb+ patients, respectively, by ROC curve analysis. The diagnostic performance of serum Tg was reduced in TgAb+ compared to TgAb− patients, however, 87% of TgAb+ patients with recurrent disease and, particularly, all patients with distant metastases were correctly detected by adopting an optimized Tg cutoff for TgAb+ patients. A disturbed recovery was found in only 1% of TgAb− patients and in these cases no clinically relevant information was added by the Tg-mRec. Among TgAb+ patients with undetectable Tg and undisturbed Tg-mRec, no one had recurrent disease. However, a falsely undetectable Tg was demonstrated in two patients with recurrent disease who next to increased TgAb also had a disturbed Tg-mRec test. Conclusions There is no additional clinical benefit from performing Tg-mRec in most patients. It can however be considered in TgAb+ patients with undetectable Tg levels as it may help differentiate between patients with true negative and false negative Tg levels in the presence of such antibodies.

Background Heterophile antibody (HAb) interferences in immunoassays can cause falsely elevated hCG concentrations leading to incorrect diagnosis and treatments options. When results are not consistent with the clinical findings, hCG HAb interference investigation may be requested by the physician. A retrospective evaluation of the frequency of HAb interference was performed among cases of physician-requested investigations and the effectiveness of commercially available blocking reagents to detect HAb interference in two immunoassay systems was evaluated. Methods One hundred and thirteen physician requests for hCG HAb investigation from 2008 to 2017 were reviewed. The primary method used to measure hCG was the Beckman Coulter Access Total βhCG (2008–2010) and the Roche Elecsys HCG+β (2014–2017). HAb investigation included measurement by two immunoassays before and after treatment of samples with heterophile blocking reagents and serial dilution studies. Results Five cases of HAb and HAb-like interference were identified. The interference frequency was 6.7% for the Beckman assay and 2.9% for the Roche assay. The presence of HAb was detected using heterophile blocking reagents and an alternative method in three cases. The other two cases were detected due to discrepant results with an alternative method and non-linear serial dilutions (HAb-like). Conclusions HAb interference was observed in the Beckman and the Roche assays. The heterophile blocking reagents failed to detect 40% of interference cases. Blocking reagents should not solely be used for these investigations. Multiple strategies including the use of serial dilutions and using an alternative platform are critical when troubleshooting interferences in hCG immunoassays.

Background The importance of the standardisation of immunoassays for autoantibodies has been widely discussed. The appropriate use of certified reference materials (CRM) could contribute to a more accurate diagnosis and follow-up of a series of diseases such as small vessel-associated vasculitis. This is a systemic autoimmune disorder during which two autoantibodies can be present, MPO ANCA IgG and PR3 ANCA IgG. Results from different commercially available immunoassays used for PR3 ANCA IgG measurement can vary significantly. Therefore the potential for improvement using a suitable certified reference material was assessed and led to the development of a CRM. Methods Thirty clinical samples were evaluated using 10 immunoassays. The correlation between results from these assays was assessed in a pairwise manner. Feasibility studies were conducted in order to find a reference material format most suitable for the preparation of a CRM. Results The evaluation of two sets of 30 clinical samples with 10 assays showed that differences between assays can result in different interpretations for individual clinical samples. Most of the samples had the same result classification in all assays. However, six of the samples tested led to inconsistent results. Conclusions The correlation between results from clinical samples was systematically good for combinations of eight of those assays. Therefore, it should be possible to improve the comparability of results using a commutable CRM for calibration. Based on these studies, a final format for the CRM was selected and eventually produced and certified for its PR3 ANCA IgG content.

Background An automated multiplex platform using capillary blood can promote greater throughput and more comprehensive studies in celiac disease (CD). Diagnostic accuracy should be improved using likelihood ratios for the post-test probability of ruling-in disease. Methods The Ig_plex™ Celiac Disease Panel on the sqidlite™ automated platform measured IgA and IgG antibodies to tTG and DGP in n = 224 CD serum or plasma samples. Diagnostic accuracy metrics were applied to the combined multiplex test results for several CD populations and compared to conventional single antibody ELISA tests. Results With multiple positive antibody results, the post-test probability for ruling-in untreated and treated CD increased to over 90%. The number of samples positive for more than one antibody also increased in untreated CD to ≥90%. Measurement of all four CD antibodies generate cut-off dependent accuracy profiles that can monitor response to treatment with the gluten-free diet (GFD). Higher positive tTG and DGP antibodies are seen more frequently in confirmed CD without (81%–94%) than with GFD treatment (44%–64%). In CD lacking biopsy confirmation, overall agreement of plasma to serum was ≥98% for all antibodies, and 100% for venous to capillary plasma. Conclusions The Ig_plex Celiac Disease Panel increases the likelihood of confirming CD based on the post-test probability of disease results for multi-reactive markers. Specific positivity profiles and cut-off intervals can be used to monitor GFD treatment and likely disease progression. Using serum, venous and capillary plasma yield comparable and accurate results.

Background Bile acids (BAs) have been demonstrated to exert a variety of metabolic effects and alterations in BAs have been reported in patients with obesity, insulin resistance (IR) and type 2 diabetes mellitus (T2DM). However, it is unclear which metabolic condition is the main contributor to alterations in BAs. In this study, we investigate the associations between different BA profiles with glycemia, obesity or IR status. Methods Fasting serum concentrations of 15 BA species were determined in a total of 241 individuals (71 drug-naïve patients with T2DM, 95 patients with impaired fasting glucose [IFG], and 75 healthy controls. Results A comparison of the mean values of the BAs revealed no significant differences between normoglycemic controls and patients with IFG or T2DM. However, when the entire cohort was divided according to the presence of IR as determined by a homeostasis model assessment of insulin resistance (HOMA-IR) value >2.5, the levels of total BA and most species of BAs were significantly higher in patients with IR than in patients without. In the correlation analysis, most species of BAs, as well as total BA, were significantly associated with HOMA-IR levels. Furthermore, when the subjects were divided into four groups according to IR and diabetic status, subjects with IR had significantly higher total BAs than participants without IR both in diabetic and non-diabetic groups. Ultimately, multiple linear regression analysis identified HOMA-IR as the only significant contributor to most serum BA species. Conclusions Our findings support the essential role of IR in regulating BA metabolism and that this effect is independent of diabetic status.

Background The most common cause of activated protein C (aPC) resistance is a missense substitution ( Arg506Gln ), known as Factor V Leiden ( FVL ). Due to its low cost, many laboratories use the aPC ratio as a primary test with a unisex cut-off. However, the association between the aPC ratio and FVL including any relation to gender has been sparsely investigated. Methods Results of the aPC ratio and FVL analyses from 1081 patients referred to the Thrombophilia Clinic at Odense University Hospital were compared. Results In 153 FVL positive patients, the mean aPC ratio was 2.1 ± 0.3, which differed from 2.7 ± 0.4 in FVL negative individuals (p < 0.01). The receiver operating characteristics (ROC), with area under the curve (AUC) of 0.93, indicated the optimal aPC cut-off at 2.3–2.4, with sensitivity 89%–94%, specificity 71%–84%, positive predictive value 35%–48% and negative predictive value 98%–99%. In FVL positive females, the mean aPC ratio was 2.0 ± 0.3, which differed from males (2.1 ± 0.3, p < 0.05). In FVL negative females, the mean aPC ratio was 2.6 ± 0.4, also different from males (2.8 ± 0.5, p < 0.01). Of note, 35% of FVL negative females had an aPC ratio ≤2.4 against 18% in males (p < 0.01). Conclusions Our results indicate that the aPC ratio is lower in females than in males. Due to a high negative predictive value the aPC ratio can be used as a first line test for FVL , but those found positive must be confirmed with a DNA test.

Background Internal quality control (QC) rules for laboratory tests can be derived from analytical performance specifications (APS) using the six-sigma method. We tested the applicability of this paradigm to routine haemostasis measurements. Methods Three laboratories using different instruments and reagents calculated sigma scores for their prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen and antithrombin (AT) measurements. Sigma scores were calculated using biological variation (BV) data from the literature in combination with internal and external QC data. Results Wide ranges in sigma scores for the PT (0.1–6.8), APTT (0.0–4.3), fibrinogen (1.5–8.3) and AT (0.1–2.4) were observed when QC data was combined with the minimum, median and maximum value of BV data, due in particular to a large variation in within-subject and between-subjects coefficients of variation. When the median BV values were applied, most sigma scores were below 3.0, for internal QC data; 75% and for external QC data; 92%. Conclusions Our findings demonstrate that: (1) The sigma scores for common haemostasis parameters are relatively low, and (2) The application of the six-sigma method to BV-derived APS is hampered by the large variation in published BV data. As the six-sigma concept is based on requirements for monitoring, and many haemostasis tests are only designed for diagnostic purposes, a fit-for-purpose APS is needed to achieve clinically relevant quality goals.

Background There is only limited information on serum reference ranges of calcitonin (CT) in infants, children and adolescents. This gap hampers valid diagnostics in patients with multiple endocrine neoplasia type 2 (MEN 2) and planned prophylactic thyroidectomy. In addition, age-dependent reference ranges for CT are necessary to define a cure in medullary thyroid carcinoma (MTC). We asked whether the reference ranges for CT levels were age- and gender-dependent in the serum of a pediatric cohort. Methods A total of 6090 serum samples of 2639 subjects of the LIFE-Child cohort aged between 1 month and 17.9 years were analyzed by the CT electrochemiluminescence immunoassay (ECLIA). Reference intervals were estimated using the LMS method. For clinical validation the serum of 28 patients (61 samples) with MEN 2 and 106 patients (136 samples) with thyroid diseases were analyzed. Results CT levels showed a clear age- and gender-dependence with significantly higher values in boys (p<0.01). An accelerated decline of CT levels from newborn to children at the age of 4 and 5 years was observed for both sexes. A cure for MTC was demonstrated in 71% of MEN 2 patients after thyroidectomy, whereas 5 patients remained suspicious for micrometastasis or relapse. Only 1.5% of our patients with thyroid diseases revealed increased CT levels. Conclusions This is the largest study to establish novel pediatric reference ranges from the CT values of healthy subjects. It allows a precise laboratory monitoring of CT in pediatric patients with MEN 2. Thyroid diseases did not have a relevant influence on CT levels in our pediatric cohort.

Background Kallikrein-related peptidases ( KLKs ) are a subgroup of serine proteases located on chromosome 19q13.3. Most KLKs have been extensively studied as potential biomarkers for several carcinomas and other diseases. KLK5 was originally identified from a keratinocyte library, and its enzyme was purified from the stratum corneum of human skin. KLK5 was shown to be differentially expressed in a variety of endocrine tumors, although it is not as yet examined widely in colorectal cancer (CRC). Methods In this study, we quantitatively assessed the mRNA expression status of KLK5 in 197 colorectal tissues from 133 patients (70 cancerous and their paired normal colonic mucosa for 64 of them, as well as 63 colorectal adenomas) by quantitative real-time PCR (qPCR) using TaqMan probes. Statistical analysis evaluated the results. Results It was shown that KLK5 expression is reduced following the histologically non-cancerous-adenoma sequence (p<0.001), whereas it is increased during the sequence adenoma-carcinoma (p<0.001). Furthermore, KLK5 positive expression is associated with positive nodal status (p=0.022), advanced tumor stage (p=0.038) and high histological grade (p=0.033). Cox univariate analysis revealed that KLK5 positive expression is associated with disease-free survival (DFS) (p=0.028) and overall survival (OS) of patients (p=0.048). Kaplan-Meyer survival models showed that patients with positive KLK5 expression have lower DFS (p=0.009) and OS (p=0.019). Receiver operating characteristic (ROC) analysis demonstrated for first time that KLK5 expression had significant discriminatory values between cancer and adenoma tissues (area under the curve [AUC] 0.77; 95% confidence interval [CI]=0.69–0.85, p=0.03). Conclusions KLK5 mRNA expression may be useful for the differentiation of CRC from colorectal adenoma and represents a potential unfavorable prognostic biomarker for CRC.

Background High sensitivity assays for the determination of cardiac troponin I (cTnI) are able to reliably measure cTnI far below the 99th percentile of healthy persons (hs-cTnI) and display sex-specific differences. There is uncertainty regarding the clinical utility of hs-cTnI in asymptomatic hemodialysis (HD) patients and if sex-specific differences also apply in this cohort. Methods In this multicenter study we measured hs-cTnI and sensitive cTnI (s-TnI) concentrations (both on Siemens Centaur) in 215 HD patients from a predialytic sample to determine the prevalence of elevated concentrations above the 99th percentile, the association with baseline characteristics, prognostic accuracy for death, and sex-specific differences. Results Hs-cTnI and s-cTnI concentrations were below the 99th percentile in 93% and 85% of patients with a median concentration of 12 ng/L (interquartile range 7–66) and 19 ng/L (12; 31, p < 0.0001). Hs-cTnI and s-cTnI concentrations were independently associated with age (p < 0.05) and ischemic cardiac disease (p < 0.05), but not with residual renal function. Both hs-cTnI and s-cTnI were predictors of death after median follow-up of 2.6 years with an AUC of 0.733 and 0.744, respectively (both p < 0.0001). Important sex-differences emerged for hs-cTnI, but not for s-cTnI: first, women had significantly lower hs-cTnI concentrations than men (p = 0.03); second, hs-cTnI had significantly higher prognostic accuracy for death in women than for men (AUC 0.824 vs. 0.674, p = 0.04). Conclusions The majority of HD patients have (h)s-cTnI concentrations below the 99th percentile. High normal values are predictive of death. Hs-cTnI allows to elucidate important sex-differences in HD patients with lower concentrations and higher prognostic accuracy in women.

Background The increasing number of multi-drug resistant (MDR) bacteria provides enormous challenges for choosing an appropriate antibiotic therapy in the early phase of sepsis. While bacterial identification has been greatly accelerated by the introduction of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), the antibiotic susceptibility testing (AST) remains time-consuming. Here, we present a rapid susceptibility testing method for testing Gram-negative bacteria, exemplarily validated for Escherichia coli and Klebsiella spp. Methods Gram-negative isolates ( E. coli and Klebsiella spp.) were either taken as single colonies from agar plates (n=136) or directly extracted and identified from positive blood cultures (n=42) using MALDI-TOF MS. Bacteria were incubated in glucose-supplemented Luria broths (LBs) each containing one antibiotic (ceftazidime, piperacillin, imipenem and ciprofloxacin), routinely used to classify Gram-negative bacteria in Germany. To determine susceptibility the dynamics of glucose utilization in bacterial suspensions were quantitatively measured in the presence or absence of antibiotics designated liquid-AST (L-AST). Results The L-AST can be run on clinical-chemistry analyzers and integrated into laboratory routines. It yields critical resistance information within 90–150 min downstream of a MS-based identification. The results showed a high concordance with routine susceptibility testing, with less than 1% very major errors (VME) and 3.51% major errors (ME) for 178 assessed isolates. Analysis of turnaround time (TAT) for 42 clinical samples indicated that L-AST results could be obtained 34 h earlier than the routine results. Conclusions As exemplified for E. coli and Klebsiella spp., L-AST provides substantial acceleration of susceptibility testing following MALDI-TOF MS identification. The assay is a simple and low-cost method that can be integrated into clinical laboratory to allow for 24/7 AST. This approach could improve antibiotic therapy.