Evaluation of DNA Extraction Methods for Reliable Quantification of <i>Acinetobacter baumannii</i>, <i>Klebsiella pneumoniae</i>, and <i>Pseudomonas aeruginosa</i>

Detection and quantification of DNA biomarkers relies heavily on the yield and quality of DNA obtained by extraction from different matrices. Although a large number of studies have compared the yields of different extraction methods, the repeatability and intermediate precision of these methods have been largely overlooked. In the present study, five extraction methods were evaluated, using digital PCR, to determine their efficiency in extracting DNA from three different Gram-negative bacteria in sputum samples. The performance of two automated methods (GXT NA and QuickPick genomic DNA extraction kit, using Arrow and KingFisher Duo automated systems, respectively), two manual kit-based methods (QIAamp DNA mini kit; DNeasy UltraClean microbial kit), and one manual non-kit method (CTAB), was assessed. While GXT NA extraction kit and the CTAB method have the highest DNA yield, they did not meet the strict criteria for repeatability, intermediate precision, and measurement uncertainty for all three studied bacteria. However, due to limited clinical samples, a compromise is necessary, and the GXT NA extraction kit was found to be the method of choice. The study also showed that dPCR allowed for accurate determination of extraction method repeatability, which can help standardize molecular diagnostic approaches. Additionally, the determination of absolute copy numbers facilitated the calculation of measurement uncertainty, which was found to be influenced by the DNA extraction method used

Metrological evaluation of DNA extraction method effects on the bacterial microbiome and resistome in sputum

Targeted high-throughput sequencing (HTS) has revolutionized the way we look at bacterial communities. It can be used for the species-specific detection of bacteria as well as for the determination of the microbiome and resistome and can be applied to samples from almost any environment. However, the results of targeted HTS can be influenced by many factors, which poses a major challenge for its use in clinical diagnostics. In this study, we investigated the impact of the DNA extraction method on the determination of the bacterial microbiome and resistome by targeted HTS using principles from metrology and diagnostics such as repeatability and analytical sensitivity. Sputum samples spiked with Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa at three different concentrations (103–106 cells/mL) were used. DNA was extracted from each sample on 2 separate days in three replicates each using three different extraction methods based on cetrimonium bromide, magnetic beads, and silica membranes. All three spiked bacteria were detected in sputum, and the DNA extraction method had no significant effect on detection. However, the DNA extraction method had significant effects on the composition of the microbiome and the resistome. The sequencing results were repeatable in the majority of cases. The silica membrane-based DNA extraction kit provided the most repeatable results and the highest diversity of the microbiome and resistome. Targeted HTS has been shown to be a reliable tool for determining the microbiome and resistomehowever, the method of DNA extraction should be carefully selected to minimize its impact on the results

Usefulness of rapid antigen testing for SARS-CoV-2 screening of healthcare workers : ǂa ǂpilot study

Background. Identification of infected healthcare workers (HCWs) is an important step in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) transmission control. Rapid antigen tests (RATs) are considered an important addition to molecular tests in diagnosing coronavirus disease 2019 (COVID-19), mainly because of their fast turnaround time, easier analytical procedure and lower price. However, real-life studies on the usefulness of such testing for screening of HCWs are limited. Methods. Physicians, nurses and hospital attendants currently working at the University Clinic of Respiratory and Allergic Diseases Golnik were invited to participate in the pilot study. Nasopharyngeal swabs were obtained three times per week for two consecutive weeks and tested with a point-of-care RAT and reverse transcription polymerase chain reaction (RT-PCR). Serum samples were obtained at the beginning of the study and 2 weeks after the last swab was collected to evaluate the serological status. Results. A total of 191 nasopharyngeal swabs from 36 HCWs were obtained. None of the samples tested was positive for the presence of SARS-CoV-2 antigen, whereas two HCWs tested positive on RT-PCR. Of these, one HCW had a newly identified SARS-CoV-2 infection, whereas RT-PCR probably detected a previous but recent infection in the other HCW. Conclusio.n Based on the results of this pilot study, it is unlikely that RAT will reliably detect novel SARS-CoV-2 infections among asymptomatic HCWs despite serial sampling. Although RT-PCR-based screening of HCWs may not be feasible due to high sample volume, molecular methods may identify SARS-CoV-2-infected HCWs already during the presymptomatic stage

Nephila spider male aggregation: preference for optimal female size and web clustering

Sexual size dimorphism theory predicts biased operational sex ratios (OSRs) and an uneven distribution of males among certain females. We studied this phenomenon through a field census of the giant wood spider Nephila pilipes (family Nephilidae) in Singapore, a species where females are, on average, 6.9 times larger than males. Specifically, we tested two hypotheses concerning male distribution, given their tendency to aggregate in certain female webs. The optimal female size hypothesis predicts that males would predominantly occupy webs of intermediate-sized females. The web clustering hypothesis posits that more males would be found in webs closer together compared to those farther apart. Our snapshot census revealed a female-biased OSR (females: males = 1.85) with an uneven distribution of males in female webs. Most males were found in webs of intermediate-sized females aligning with the optimal female size hypothesis. Proximity among female webs was indicative of male presence, lending support to the web clustering hypothesis. While our study\u27s limited sample size warrants caution, we conclude that in N. pilipes, male occupation of female webs is facilitated by the clustering of webs, and males prefer to cohabit with optimally sized, receptive females

Survey results on nucleic acid tests of infectious diseases

This survey discusses current and emerging isothermal and rapid polymerase chain reaction (PCR) based nucleic acid amplification methods for near-patient diagnostics. To assess the clinical need of rapid diagnostics for infectious diseases based on nucleic acid tests (NATs) we performed and analysed a questionnaire among laboratories participating in corresponding INSTAND ring trials for external quality assurance. The questions concerning new amplification technologies like isothermal nucleic acid amplification, potentially suited to significantly decrease turnaround times, were complemented by questions to evaluate the present status of NATs. Besides end-users, companies were also addressed by sending out a manufacturer specific questionnaire. Analysis of the answers from 48 laboratories in 14 European countries revealed that a much shorter turnaround time is requested for selected pathogens compared to about 2 h or longer when applying temperature cycling amplification, i.e. PCR. In this context, most frequently mentioned were methicillin-resistant Staphylococcus aureus (MRSA), norovirus, influenza A and B viruses, cytomegalovirus (CMV) as well as hepatitis B virus (HBV) and hepatitis C virus (HCV). At present, 8% of the laboratories having participated in this survey apply isothermal amplification of nucleic acids to identify infectious pathogens

Genetska opredelitev za meticilin občutljivih in proti meticilinu odpornih sevov Staphylococcus aureus, izoliranih iz hemokultur v slovenskih bolnišnicah, s tipizacijo SPA

Staphylococcus aureus je drugi najpogostejši bakterijski povzročitelj invazivnih okužb v Sloveniji. S tipizacijo izolatov za meticilin občutljivih S. aureus (MSSA) in proti meticilinu odpornih S. aureus (MRSA) iz hemokultur bolnikov z bakteriemijo iz vse Slovenije v 6- mesečnem obdobju smo poskušali ugotoviti, katerim spa tipom pripadajo invazivni izolati MSSA in MRSA v Sloveniji, kakšna je genetska raznolikost MSSA in MRSA ter preveriti uporabnost tipizacije spa za epidemiološke raziskave S. aureus v Sloveniji. Od 1. septembra 2006 do 28. februarja 2007 smo v 11 slovenskih laboratorijih, ki izvajajo mikrobiološko diagnostiko za 15 bolnišnic, zbirali zaporedno izolirane primoizolate MSSA (največ pet) in zaporedno izolirane primoizolate MRSA (največ pet) iz hemokultur bolnikov. Izolate S. aureus smo tipizirali s tipizacijo spa, to je z določanjem nukleotidnega zaporedja polimorfnega X dela gena spa, ki kodira protein A. Od 58 zbranih izolatov S. aureus iz vse Slovenije smo uspeli tipizirati 54 izolatov. Prevladujoči spa tipi med izolatiMSSA so bili t091, t015 in t005, med izolati MRSA pa t041. Ugotovili smo, da spa tipi, ki prevladujejo med invazivnimi izolati S. aureus pri nas, prevladujejo tudi v sosednjih državah. Tipizacija S. aureus z metodo spa je pokazala veliko genetsko raznolikost invazivnih sevov MSSA in manjšo raznolikost invazivnih sevov MRSA. Tipizacija spa je uporabna tipizacijska metoda za lokalno epidemiologijo MSSA v kratkem in daljšem časovnem obdobju.Staphylococcus aureus is the second leading cause of invasive bacterial infections in Slovenia. The aims of this 6-month study were to identify the most prevalent spa types among invasive isolates of methicillin-susceptible S.aureus (MSSA) and methicillin-resistant S. aureus (MRSA) in Slovene hospitals, to determine genetic diversity of MSSA and MRSA, and to test the applicability of spa typing to epidemiological studies of S. aureus in Slovenia. Over a period of six months(1 September 2006 - 28 February 2007), upto five successive methicillin-susceptible (MSSA) blood culture isolates anda maximum of five successive methicillin- resistant (MRSA) blood culture isolates per hospital were obtained from different patients with bacteremia. The isolates were characterized by spa typing based on sequencing of polymorphic region X of the S. aureus protein A gene. Among the 58 isolates collected 54 were succesfully spa typed. The predominant spa types among MSSA isolates were t091, t015, t005, and among MRSA, t041 . The predominant spa types among invasive S. aureus strains isolated in Slovenia predominated also in the neighbouring countries. The study showed high genetic diversity of invasive MSSA isolates and lower genetic diversity of invasive MRSA isolates in Slovenia. Spa typing is a good typing method for short- and long-term epidemiological studies of MSSA on a local scale

Decontamination strategies and bloodstream infections with antibiotic-resistant microorganisms in ventilated patients : a randomized clinical trial

Importance: The effects of chlorhexidine (CHX) mouthwash, selective oropharyngeal decontamination (SOD), and selective digestive tract decontamination (SDD) on patient outcomes in ICUs with moderate to high levels of antibiotic resistance are unknown. Objective: To determine associations between CHX 2%, SOD, and SDD and the occurrence of ICU-acquired bloodstream infections with multidrug-resistant gram-negative bacteria (MDRGNB) and 28-day mortality in ICUs with moderate to high levels of antibiotic resistance. Design, setting, and participants: Randomized trial conducted from December 1, 2013, to May 31, 2017, in 13 European ICUs where at least 5% of bloodstream infections are caused by extended-spectrum [beta]-lactamase-producing Enterobacteriaceae. Patients with anticipated mechanical ventilation of more than 24 hours were eligible. The final date of follow-up was September 20, 2017. Interventions: Standard care was daily CHX 2% body washings and a hand hygiene improvement program. Following a baseline period from 6 to 14 months, each ICU was assigned in random order to 3 separate 6-month intervention periods with either CHX 2% mouthwash, SOD (mouthpaste with colistin, tobramycin, and nystatin), or SDD (the same mouthpaste and gastrointestinal suspension with the same antibiotics), all applied 4 times daily. Main outcomes and measures: The occurrence of ICU-acquired bloodstream infection with MDRGNB (primary outcome) and 28-day mortality (secondary outcome) during each intervention period compared with the baseline period. Results: A total of 8665 patients (median age, 64.1 years5561 men [64.2%]) were included in the study (2251, 2108, 2224, and 2082 in the baseline, CHX, SOD, and SDD periods, respectively). ICU-acquired bloodstream infection with MDRGNB occurred among 144 patients (154 episodes) in 2.1%, 1.8%, 1.5%, and 1.2% of included patients during the baseline, CHX, SOD, and SDD periods, respectively. Absolute risk reductions were 0.3% (95% CI, -0.6% to 1.1%), 0.6% (95% CI, -0.2% to 1.4%), and 0.8% (95% CI, 0.1% to 1.6%) for CHX, SOD, and SDD, respectively, compared with baseline. Adjusted hazard ratios were 1.13 (95% CI, 0.68-1.88), 0.89 (95% CI, 0.55-1.45), and 0.70 (95% CI, 0.43-1.14) during the CHX, SOD, and SDD periods, respectively, vs baseline. Crude mortality risks on day 28 were 31.9%, 32.9%, 32.4%, and 34.1% during the baseline, CHX, SOD, and SDD periods, respectively. Adjusted odds ratios for 28-day mortality were 1.07 (95% CI, 0.86-1.32), 1.05 (95% CI, 0.85-1.29), and 1.03 (95% CI, 0.80-1.32) for CHX, SOD, and SDD, respectively, vs baseline. Conclusions and relevance: Among patients receiving mechanical ventilation in ICUs with moderate to high antibiotic resistance prevalence, use of CHX mouthwash, SOD, or SDD was not associated with reductions in ICU-acquired bloodstream infections caused by MDRGNB compared with standard care

Robust saliva-based RNA extraction-free one-step nucleic acid amplification test for mass SARS-CoV-2 monitoring

Early diagnosis with rapid detection of the virus plays a key role in preventing the spread of infection and in treating patients effectively. In order to address the need for a straightforward detection of SARS-CoV-2 infection and assessment of viral spread, we developed rapid, sensitive, extraction-free one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) tests for detecting SARS-CoV-2 in saliva. We analyzed over 700 matched pairs of saliva and nasopharyngeal swab (NSB) specimens from asymptomatic and symptomatic individuals. Saliva, as either an oral cavity swab or passive drool, was collected in an RNA stabilization buffer. The stabilized saliva specimens were heat-treated and directly analyzed without RNA extraction. The diagnostic sensitivity of saliva-based RT-qPCR was at least 95% in individuals with subclinical infection and outperformed RT-LAMP, which had at least 70% sensitivity when compared to NSBs analyzed with a clinical RT-qPCR test. The diagnostic sensitivity for passive drool saliva was higher than that of oral cavity swab specimens (95% and 87%, respectively). A rapid, sensitive one-step extraction-free RT-qPCR test for detecting SARS-CoV-2 in passive drool saliva is operationally simple and can be easily implemented using existing testing sites, thus allowing high-throughput, rapid, and repeated testing of large populations. Furthermore, saliva testing is adequate to detect individuals in an asymptomatic screening program and can help improve voluntary screening compliance for those individuals averse to various forms of nasal collections