16S rDNA-PCR for the identification of a bacterial species is an established method. However, the DNA extraction reagents as well as the PCR reagents may contain residual bacterial DNA, which ...consequently generates false-positive PCR results. Additionally, previously used methods are frequently time-consuming. Here, we describe the results obtained with a new technology that uses DNA-free reagents for automated DNA extraction and subsequent real time PCR using sterile clinical specimens.
In total, we compared 803 clinical specimens using real time PCR and culturing. The clinical specimens were mainly of orthopedic origin received at our diagnostic laboratory. In 595 (74.1%) samples, the results were concordant negative, and in 102 (12.7%) the results were concordant positive. A total of 170 (21.2%) clinical specimens were PCR-positive, of which 62 (36.5% from PCR positive, 7.7% in total) gave an additional benefit to the patient since only the PCR result was positive. Many of these 62 positive specimens were strongly positive based on crossingpoint values (54% < Cp 30), and these 62 positive clinical specimens were diagnosed as medically relevant as well. Thirty-eight (4.2%) clinical specimens were culture-positive (25 of them were only enrichment culture positive) but PCR-negative, mainly for S. epidermidis, S. aureus and C. acnes. The turnaround times for negative specimens were 4 hours (automated DNA extraction and real time PCR) and 1 working day for positive specimens (including Sanger sequencing). Melting-curve analysis of SYBR Green-PCR enables the differentiation of specific and unspecific PCR products. Using Ripseq, even mixed infections of 2 bacterial species could be resolved.
For endocarditis cases, the added benefit of PCR is obvious. The crucial innovations of the technology enable timely reporting of explicit reliable results for adequate treatment of patients. Clinical specimens with truly PCR-positive but culture-negative results represent an additional benefit for patients. Very few results at the detection limit still have to be critically examined.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Steroid analysis in clinical laboratories is dominated by immunoassays (IAs) that have a high sample turnover but are inherently limited in trueness, precision, and sensitivity. Liquid chromatography ...coupled to mass spectrometry (LC-MS/MS) has proved to be a far more capable tool, delivering better sensitivity, specificity, and the possibility of parallel analysis of multiple steroids and metabolites, providing the endocrinologist with more reliable and comprehensive diagnostic information. An LC-MS/MS assay with gradient elution over less than eight minutes and a one-step sample preparation combining protein precipitation with phospholipid removal of off-line solid-phase extraction was developed and validated. It allowed the quantification of 11-deoxycorticosterone (11-DOC), 11-deoxycortisol (11-DF), 17-OH-progesterone (17P), 21-deoxycortisol (21-DF), androstenedione (ANDRO), aldosterone (ALDO), corticosterone (CC), cortisol (CL), cortisone (CN), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), dihydrotestosterone (DHT), estradiol (E2), progesterone (PROG), and testosterone (TES) in human serum. Interday imprecision was generally better than 15%, trueness was proven by recovery experiments with ISO 17034-certified reference materials, proficiency testing (UK NEQAS), and measuring serum reference standards. In-house comparison against IVD-CE-certified immunoassays (IA) for 17P, ANDRO, CL, DHEAS, E2, PROG, and TES was conducted by assessing leftover routine patient samples and purpose-built patient serum pools. None of the compared routine IAs were meeting the standards of the LC-MS/MS. Insufficient overall comparability was found for ANDRO and 17P (mean bias > +65%). Accuracy limitations at lower concentrations were present in IAs for PROG, E2, and TES.
Objectives The sensitivity of molecular and serological methods for COVID-19 testing in an epidemiological setting is not well described. The aim of the study was to determine the frequency of ...negative RT-PCR results at first clinical presentation as well as negative serological results after a follow-up of at least 3 weeks. Methods Among all patients seen for suspected COVID-19 in Liechtenstein (n=1921), we included initially RT-PCR positive index patients (n=85) as well as initially RT-PCR negative (n=66) for follow-up with SARS-CoV-2 antibody testing. Antibodies were detected with seven different commercially available immunoassays. Frequencies of negative RT-PCR and serology results in individuals with COVID-19 were determined and compared to those observed in a validation cohort of Swiss patients (n=211). Results Among COVID-19 patients in Liechtenstein, false-negative RT-PCR at initial presentation was seen in 18% (12/66), whereas negative serology in COVID-19 patients was 4% (3/85). The validation cohort showed similar frequencies: 2/66 (3%) for negative serology, and 16/155 (10%) for false negative RT-PCR. COVID-19 patients with negative follow-up serology tended to have a longer disease duration (p=0.05) and more clinical symptoms than other patients with COVID-19 (p<0.05). The antibody titer from quantitative immunoassays was positively associated with the number of disease symptoms and disease duration (p<0.001). Conclusions RT-PCR at initial presentation in patients with suspected COVID-19 can miss infected patients. Antibody titers of SARS-CoV-2 assays are linked to the number of disease symptoms and the duration of disease. One in 25 patients with RT-PCR-positive COVID-19 does not develop antibodies detectable with frequently employed and commercially available immunoassays.
Premature ventricular contractions (PVCs) are associated with an increased risk of morbidity and mortality. Therefore, it was aimed to assess risk factors for the frequency of PVCs in young and ...healthy adults.
Our population-based study included 2048 healthy adults from the general population aged 25-41 years. PVC frequency was determined by 24-hour Holter ECG. We performed multivariable regression analysis using stepwise backward selection to identify factors independently associated with PVC frequency.
Median age was 37 years, 953 (46.5%) were male. At least one PVC during the 24-hour monitoring period was observed in 69% of participants. Median number of detected PVCs was 2, the 95th percentile was 193. In multivariable regression analyses, we found 17 significant risk factors for PVC frequency. Low educational status (risk ratio (RR) 3.33; 95% CI 1.98 to 5.60), body height>median (1.58, 95% CI 1.11 to 2.24) and increasing levels of waist:hip ratio (2.15, 95% CI 1.77 to 2.61), N-terminal pro brain natriuretic peptide (1.52, 95% CI 1.30 to 1.76) and Sokolow-Lyon Index (1.38, 95% CI 1.15 to 1.66) (all p≤0.01) were associated with a higher PVC frequency. Physical activity (RR fourth vs first quartile 0.51, 95% CI 0.34 to 0.76) and increasing levels of haemoglobin (0.58, 95% CI 0.47 to 0.70) and glucagon-like peptide-1 (0.72, 95% CI 0.64 to 0.82) (all p<0.001) were related to a lower PVC frequency.
PVC occurrence is common even in healthy low-risk individuals, and its frequency is associated with several covariates mainly related to cardiovascular risk factors, markers of cardiac structure and function and socioeconomic status.
Considering sex as a biological variable in modern digital health solutions, we investigated sex-specific differences in the trajectory of four physiological parameters across a COVID-19 infection. A ...wearable medical device measured breathing rate, heart rate, heart rate variability, and wrist skin temperature in 1163 participants (mean age = 44.1 years, standard deviation SD = 5.6; 667 57% females). Participants reported daily symptoms and confounders in a complementary app. A machine learning algorithm retrospectively ingested daily biophysical parameters to detect COVID-19 infections. COVID-19 serology samples were collected from all participants at baseline and follow-up. We analysed potential sex-specific differences in physiology and antibody titres using multilevel modelling and t-tests. Over 1.5 million hours of physiological data were recorded. During the symptomatic period of infection, men demonstrated larger increases in skin temperature, breathing rate, and heart rate as well as larger decreases in heart rate variability than women. The COVID-19 infection detection algorithm performed similarly well for men and women. Our study belongs to the first research to provide evidence for differential physiological responses to COVID-19 between females and males, highlighting the potential of wearable technology to inform future precision medicine approaches.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Certain immunizations including vaccination against tick-borne encephalitis virus (TBEV) have been suggested to confer cross-protection against severe acute respiratory syndrome coronavirus-2 ...(SARS-CoV-2). Within a prospective healthcare worker (HCW) cohort, we assessed the potentially protective role of anti-TBEV antibodies against SARS-CoV-2 infection. Among 3352 HCW, those with ≥ 1 previous TBEV vaccination (n = 2018, 60%) showed a reduced risk of SARS-CoV-2 seroconversion (adjusted odds ratio: 0.8, 95% CI: 0.7-1.0, P = 0.02). However, laboratory testing of a subgroup of 26 baseline and follow-up samples did not demonstrate any neutralizing effect of anti-TBEV antibodies against SARS-CoV-2 in live-virus neutralization assay. However, we observed significantly higher anti-TBEV antibody titers in follow-up samples of participants with previous TBEV vaccination compared to baseline, both TBEV neutralizing (p = 0.001) and total IgG (P < 0.0001), irrespective of SARS-CoV-2 serostatus. Based on these data, we conclude that the observed association of previous TBEV vaccination and reduced risk of SARS-CoV-2 infection is likely due to residual confounding factors. The increase in TBEV follow-up antibody titers can be explained by natural TBEV exposure or potential non-specific immune activation upon exposure to various pathogens, including SARS-CoV-2. We believe that these findings, although negative, contribute to the current knowledge on potential cross-immunity against SARS-CoV-2 from previous immunizations.
Preeclampsia (PE) is associated with severe maternal and fetal morbidity in the acute presentation and there is increasing evidence that it is also an important risk factor for cardiovascular disease ...later in life. Therefore, preventive strategies are of utmost importance. The Fetal Medicine Foundation (FMF) London recently developed a first trimester screening algorithm for placenta-related pregnancy complications, in particular early onset preeclampsia (eoPE) requiring delivery before 34 weeks, and preterm small for gestational age (pSGA), with a birth weight <5th percentile and delivery before 37 weeks of gestation, based on maternal history and characteristics, and biochemical and biophysical parameters. The aim of this study was to test the performance of this algorithm in our setting and to perform an external validation of the screening algorithm.
Between September 2013 and April 2016, all consecutive women with singleton pregnancies who agreed to this screening were included in the study. The proposed cut-offs of ≥1:200 for eoPE, and ≥1:150 for pSGA were applied. Risk calculations were performed with Viewpoint® program (GE, Mountainview, CA, USA) and statistical analysis with GraphPad version 5.0 for Windows.
1372 women agreed to PE screening; the 1129 with complete data and a live birth were included in this study. Nineteen (1.68%) developed PE: 14 (1.24%) at term (tPE) and 5 (0.44%) preterm (pPE, <37 weeks), including 2 (0.18%) with eoPE. Overall, 97/1129 (8.6%) screened positive for eoPE, including both pregnancies that resulted in eoPE and 4/5 (80%) that resulted in pPE. Forty-nine of 1110 (4.41%) pregnancies without PE resulted in SGA, 3 (0.27%) of them in pSGA. A total of 210/1110 (18.9%) non-PE pregnancies screened positive for pSGA, including 2/3 (66.7%) of the pSGA deliveries and 18/46 (39.1%) of term SGA infants.
Our results show that first trimester PE screening in our population performs well and according to expectations, whereas screening for SGA is associated with a high false positive rate.
Obstructive sleep apnea seems to have an important influence on the autonomic nervous system. In this study, we assessed the relations of sleep apnea–related parameters with 24-hour heart rate ...variability (HRV) in a large population of young and healthy adults. Participants aged 25 to 41 years with a body mass index <35 kg/m2 and without known obstructive sleep apnea were included in a prospective population-based cohort study. HRV was assessed using 24-hour electrocardiographic monitoring. The SD of all normal RR intervals (SDNN) was used as the main HRV variable. Apnea-Hypopnea Index (AHI) and oxygen desaturation index (ODI) were obtained from nighttime pulse oximetry with nasal airflow measurements. We defined sleep-related breathing disorders as an AHI ≥5 or an ODI ≥5. Multivariable regression models were constructed to assess the relation of HRV with either AHI or ODI. Median age of the 1,255 participants was 37 years, 47% were men, and 9.6% had an AHI ≥5. Linear inverse associations of SDNN across AHI and ODI groups were found (p for trend = 0.006 and 0.0004, respectively). The β coefficients (95% CI) for the relation between SDNN and elevated AHI were −0.20 (−0.40 to −0.11), p = 0.04 and −0.29 (−0.47 to −0.11), p = 0.002 for elevated ODI. After adjustment for 24-hour heart rate, the same β coefficients (95% CI) were −0.06 (−0.22 to 0.11), p = 0.51 and −0.14 (−0.30 to 0.01), p = 0.07, respectively. In conclusion, even early stages of sleep-related breathing disorders are inversely associated with HRV in young and healthy adults, suggesting that they are tightly linked with autonomic dysfunction. However, HRV and 24-hour heart rate seem to have common information.
antibiotic resistance is increasing worldwide, emphasizing the urgent need for more rapid resistance detection prior to the administration of
eradication regimens. Macrolides and fluoroquinolones are ...widely used to treat
. In this study, we aimed to compare the diagnostic performance of A) 23SrDNA qPCR (with melting curve analysis) and an in-house developed
qPCR followed by Sanger sequencing with a commercial IVD-marked hybridization probe assay (for 23SrDNA and
) using 142 gastric biopsies (skipping culturing) and B) the same two qPCR for 23SrDNA and
(including Sanger sequencing) with whole-genome sequencing (WGS) and phenotypic characterization of clarithromycin and levofloxacin resistance using 76 cultured isolates. The sensitivity of both qPCRs was 100% compared to that of the commercial IVD-marked hybridization probe assay for the detection of
in gastric biopsies (without resistance testing). The specificity of the qPCR
followed by Sanger sequencing was 100%, indicating that the best sequence identity was always
. The results show good agreement between molecular tests, especially between qPCR (inclusive Sanger sequencing) and WGS. Discrepancies (concerning mutated or wild type of positive
gastric biopsies) were observed between Sanger sequencing of the
gene and the corresponding commercial hybridization probe assay, mostly because the high sequence diversity of the
gene even at positions adjacent to the relevant codons of 87 and 91 interfered with obtaining correct results from the hybridization probe assay. Interestingly, we found several mixed sequences, indicating mixed populations in the gastric biopsies (direct detection without culturing). There was a high percentage of both levofloxacin and clarithromycin resistance in gastric biopsies (both between 22% and 29%, direct detection in gastric biopsies). Therefore, we recommend analyzing both targets in parallel. We confirmed that phenotypic resistance is highly correlated with the associated mutations. We concluded that the two qPCR followed by Sanger sequencing of the
gene is a fast, cost-effective and comprehensive method for resistance testing of
directly in gastric biopsies.
Surgical site infections (SSIs) after elective orthopaedic surgery are very stressful for patients due to frequent rehospitalizations with reoperations and poorer functional outcomes. Prevention of ...such events is therefore crucial. Although an evidence-based consensus is still lacking, preoperative decolonization could decrease SSI. Specifically, more information is needed about the effect of a preoperative decolonization procedure on SSI proportions in both Staphylococcus aureus carriers and non-S. aureus carriers after general orthopaedic surgery.
Our study addressed the following questions: (1) Does preoperative decolonization reduce the risk of SSI after general elective orthopaedic surgery in patients colonized with S. aureus? (2) Does preoperative decolonization reduce the risk of SSI among patients who are not colonized with S. aureus?
In this prospective, randomized, single-blinded trial, we recruited patients undergoing general elective orthopaedic surgery in one tertiary care center in Switzerland. Between November 2014 and September 2017, 1318 of 1897 screened patients were enrolled. Patients were allocated into either the S. aureus carrier group (35%, 465 of 1318 patients) or the noncarrier group (65%, 853 of 1318 patients) according to screening culture results. In the S. aureus group, 232 patients were allocated to the intervention arm and 233 were allocated to the control arm. Intervention was 5 days of daily chlorhexidine showers and mupirocin nasal ointment twice a day. Of the 853 noncarriers, 426 were allocated to the intervention arm and 427 were allocated to the control arm. All patients in both groups were analyzed in an intention-to-treat manner. The primary endpoint was SSI occurrence at 90 days postoperative and the secondary endpoint was SSI occurrence at 30 days postoperative.The initial sample size calculation was made for the S. aureus carrier group. Based on the literature review, a 4% proportion of SSI was expected in the control group. Thus, 726 carriers would have been needed to detect a relative risk reduction of 80% with a power of 80% at a two-sided α-error of 0.048 (adjusted for interim analysis). Assuming carrier prevalence of 27%, 2690 patients would have been needed in total. An interim analysis was performed after including half of the targeted S. aureus carriers (363 of 726). Based on the low infection rate in the control group (one of 179), a new sample size of 15,000 patients would have been needed. This was deemed not feasible and the trial was stopped prematurely.
Among carriers, there was no difference in the risk of SSI between the intervention and control arms (decolonized SSI risk: 0.4% one of 232, control SSI risk: 0.4% one of 233, risk difference: 0.0% 95% CI -1.2% to 1.2%, stratified for randomization stratification factors; p > 0.999). For noncarriers, there was no difference in risk between the intervention and control arms (decolonized SSI risk: 0.2% one of 426, control SSI risk: 0.2% one of 247, stratified risk difference: -0.0% 95% CI -0.7 to 0.6; p = 0.973).
We found no difference in the risk of SSI between the decolonization and control groups, both in S. aureus carriers and noncarriers. Because of the low event numbers, no definite conclusion about efficacy of routine preoperative decolonization can be drawn. The results, however, may be helpful in future meta-analyses.
Level II, therapeutic study.
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