Nanopore Sequencing in a Clinical Routine Laboratory: Challenges and Opportunities.

BACKGROUND: About forty-five years ago the advent of Sanger sequencing (Sanger and Coulson 1975) was revolutionary as it allowed deciphering of complete genome sequences. A second revolution came when next-generation sequencing (NGS) technologies accelerated and cheapened genome sequencing. Recently, third generation/longread sequencing methods have appeared, which can directly detect epigenetic modifications on native DNA and allow whole-transcript sequencing without the need for assembly. Nanopore sequencing is one of these third-generation approaches, enabling a single molecule of DNA or RNA to be sequenced in real-time without the need for PCR amplification or chemical labelling of the sample. It works by monitoring changes to an electrical current as nucleic acids are passed through protein or synthetic nanopores. METHODS: A literature search was performed in order to collect and summarize current information about the methodological aspects of nanopore sequencing as well as some application examples. RESULTS: The review describes concisely and comprehensibly the technical aspects of nanopore sequencing and stresses the advantages and disadvantages of this technique thereby also giving examples of their potential applications in the clinical routine laboratory as are rapid identification of viral pathogens, monitoring Ebola, environmental and food safety monitoring, human and plant genome sequencing, monitoring of antibiotic resistance, and other applications. CONCLUSIONS: It is a useful incitation for such ones being permanently in search of upgrading their laboratory.

Quantification and identification of aerobic bacteria in holy water samples from a German environment.

Despite its frequent use in many religious institutions, the microbiological quality of holy water is clearly underinvestigated. We analyzed the microbial load of 54 holy water samples, repeatedly taken in five Roman Catholic churches in the greater area of Villingen-Schwenningen, Germany, by means of aerobic colony counting and Matrix-Assisted Laser Desorption/Ionization (MALDI) Biotyping of representative isolates. Over all samples, colony counting indicated an average aerobic microbial load of 5.85 ± 3.98 × 103 colony forming units (CFU) ml-1 (average ± standard error of the mean (SEM)). Urban churches showed significantly higher contaminations than rural churches, probably owing to a greater number of visitors. Out of 145 bacterial isolates, 63 (43%) were identified to genus level and 39 (27%) to species level. The majority of the identified bacteria were typical human skin commensals, mainly affiliated with the genus Staphylococcus. Ten out of 20 (50%) of the identified species were classified as potential pathogens. Appropriate hygiene measures should be taken to control microbial contamination of holy water, e.g., regular water exchange, particularly in highly frequented churches.

Genotyping of familial Mediterranean fever gene (MEFV)-Single nucleotide polymorphism-Comparison of Nanopore with conventional Sanger sequencing.

BACKGROUND: Through continuous innovation and improvement, Nanopore sequencing has become a powerful technology. Because of its fast processing time, low cost, and ability to generate long reads, this sequencing technique would be particularly suitable for clinical diagnostics. However, its raw data accuracy is inferior in contrast to other sequencing technologies. This constraint still results in limited use of Nanopore sequencing in the field of clinical diagnostics and requires further validation and IVD certification. METHODS: We evaluated the performance of latest Nanopore sequencing in combination with a dedicated data-analysis pipeline for single nucleotide polymorphism (SNP) genotyping of the familial Mediterranean fever gene (MEFV) by amplicon sequencing of 47 clinical samples. Mutations in MEFV are associated with Mediterranean fever, a hereditary periodic fever syndrome. Conventional Sanger sequencing, which is commonly applied in clinical genetic diagnostics, was used as a reference method. RESULTS: Nanopore sequencing enabled the sequencing of 10 target regions within MEFV with high read depth (median read depth 7565x) in all samples and identified a total of 435 SNPs in the whole sample collective, of which 29 were unique. Comparison of both sequencing workflows showed a near perfect agreement with no false negative calls. Precision, Recall, and F1-Score of the Nanopore sequencing workflow were > 0.99, respectively. CONCLUSIONS: These results demonstrated the great potential of current Nanopore sequencing for application in clinical diagnostics, at least for SNP genotyping by amplicon sequencing. Other more complex applications, especially structural variant identification, require further in-depth clinical validation.

[SARS-CoV-2 vaccines and reaction of the immune system. Can the epidemic spread of the virus be prevented by vaccination?] / SARS-CoV-2-Impfstoffe und Reaktion des Immunsystems. Kann die epidemische Ausbreitung des Virus durch Impfung verhindert werden?

Since the end of 2019 a new coronavirus, SARS-CoV-2, first identified in Wuhan, China, is spreading around the world partially associated with a high death toll. Besides hygienic measurements to reduce the spread of the virus vaccines have been confected, partially based on the experiences with Ebola virus vaccine, based on recombinant human or chimpanzee adenovirus carrying the spike protein and its ACE2 receptor binding domain (RBD). Further vaccines are constructed by spike protein coding mRNA incorporated in lipid nano vesicles that after entry in human cells produce spike protein. Both vaccine types induce a strong immune response that lasts for months possibly for T-cell immunity a few years. Due to mutations in the coronavirus genome in several parts of the world variants selected, that were partially more pathogenic and partially easier transmissible - variants of concern (VOC). Until now vaccinees are protected against the VOC, even when protection might be reduced compared to the Wuhan wild virus.An open field is still how long the vaccine induced immunity will be sufficient to prevent infection and/or disease; and how long the time period will last until revaccination will be required for life saving protection, whether a third vaccination is needed, and whether revaccination with an adenovirus-based vaccine will be tolerated.

A semi-automated, isolation-free, high-throughput SARS-CoV-2 reverse transcriptase (RT) loop-mediated isothermal amplification (LAMP) test.

Shortages of reverse transcriptase (RT)-polymerase chain reaction (PCR) reagents and related equipment during the COVID-19 pandemic have demonstrated the need for alternative, high-throughput methods for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-mass screening in clinical diagnostic laboratories. A robust, SARS-CoV-2 RT-loop-mediated isothermal amplification (RT-LAMP) assay with high-throughput and short turnaround times in a clinical laboratory setting was established and compared to two conventional RT-PCR protocols using 323 samples of individuals with suspected SARS-CoV-2 infection. Limit of detection (LoD) and reproducibility of the isolation-free SARS-CoV-2 RT-LAMP test were determined. An almost perfect agreement (Cohen's kappa > 0.8) between the novel test and two classical RT-PCR protocols with no systematic difference (McNemar's test, P > 0.05) was observed. Sensitivity and specificity were in the range of 89.5 to 100% and 96.2 to 100% dependent on the reaction condition and the RT-PCR method used as reference. The isolation-free RT-LAMP assay showed high reproducibility (Tt intra-run coefficient of variation [CV] = 0.4%, Tt inter-run CV = 2.1%) with a LoD of 95 SARS-CoV-2 genome copies per reaction. The established SARS-CoV-2 RT-LAMP assay is a flexible and efficient alternative to conventional RT-PCR protocols, suitable for SARS-CoV-2 mass screening using existing laboratory infrastructure in clinical diagnostic laboratories.

Serological and viral genetic features of patients with COVID-19 in a selected German patient cohort-correlation with disease characteristics.

To study host-virus interactions after SARS coronavirus-2 (SARS-CoV-2) infection, genetic virus characteristics and the ensued humoral immune response were investigated for the first time. Fifty-five SARS-CoV-2-infected patients from the early pandemic phase were followed up including serological testing and whole genome sequencing. Anti-spike and nucleocapsid protein (S/N) IgG and IgM levels were determined by screening ELISA and IgG was further characterized by reactivity to S-subunit 1 (anti-S1), S-subunit 2 (anti-S2) and anti-N. In 55 patients, 90 genetic SARS-CoV-2 changes including 48 non-synonymous single nucleotide variants were identified. Phylogenetic analysis of the sequencing data showed a cluster representing a local outbreak and various family clusters. Anti-S/N and anti-N IgG were detected in 49 patients at an average of 83 days after blood collection. Anti-S/N IgM occurred significantly less frequently than IgG whereas anti-S2 was the least prevalent IgG reactivity (P < 0.05, respectively). Age and overweight were significantly associated with higher anti-S/N and anti-S1 IgG levels while age only with anti-N IgG (multiple regression, P < 0.05, respectively). Anti-S/N IgG/IgM levels, blood group A + , cardiovascular and tumour disease, NSP12 Q444H and ORF3a S177I were independent predictors of clinical characteristics with anti-S/N IgM being associated with the need for hospitalization (multivariate regression, P < 0.05, respectively). Anti-SARS-CoV-2 antibody generation was mainly affected by higher age and overweight in the present cohort. COVID-19 traits were associated with genetic SARS-CoV-2 variants, anti-S/N IgG/IgM levels, blood group A + and concomitant disease. Anti-S/N IgM was the only antibody associated with the need for hospitalization.

A view to a kill? - Ambient bacterial load of frames and lenses of spectacles and evaluation of different cleaning methods.

Surfaces with regular contact with the human body are typically contaminated with microorganisms and might be considered as fomites. Despite spectacles being widespread across populations, little is known about their microbial contamination. Therefore, we swab-sampled 11 worn spectacles within a university setting as well as 10 worn spectacles in a nursing home setting. The microbial load was determined by aerobic cultivation. All spectacles were found to be contaminated with bacteria, with nose pads and ear clips having the highest density, i.e. at sites with direct skin contact. Summed over all sites, the median microbial load of the university spectacles (1.4 ± 10.7 x 10(3) CFU cm-2) did not differ significantly from the spectacles tested in the nursing home (20.8 ± 39.9 x 10(3) CFU cm-2). 215 dominant bacterial morphotypes were analyzed by MALDI biotyping. 182 isolates could be assigned to 10 genera, with Staphylococcus being the most common. On genus-level, bacterial diversity was greater on nursing home spectacles (10 genera) compared to the university environment (2 genera). Four cleaning methods were investigated using lenses artificially contaminated with Escherichia coli, Micrococcus luteus, a 1:2 mixture of E. coli and M. luteus, and Staphylococcus epidermidis (the dominant isolate in our study), respectively. Best cleaning results (99% -100% median germ reduction) were obtained using impregnated wipes; dry cleaning was less effective (85% -90% median germ reduction). Finally, 10 additional worn university spectacles were cleaned with wipes impregnated with an alcohol-free cleaning solution before sampling. The average bacterial load was significantly lower (0.09 ± 0.49 x 10(3) CFU cm-2) compared to the uncleaned university spectacles previously investigated. Spectacles are significantly contaminated with bacteria of mostly human skin origin-including significant amounts of potentially pathogenic ones and may contribute to eye infections as well as fomites in clinical environments.

Heparin-mediated extracorporeal low density lipoprotein precipitation as a possible therapeutic approach in preeclampsia.

Preeclampsia is a pregnancy-related hypertensive disease resulting in substantial maternal and neonatal morbidity and mortality. Until today there is no satisfactory treatment to stop disease progression except immediate delivery of the fetus. Heparin-mediated extracorporeal low density lipoprotein (LDL) precipitation (H.E.L.P.) apheresis removes simultaneously circulating LDL, lipoprotein(a) [Lp(a)], fibrinogen, C-reactive protein (CRP) and various proinflammatory and procoagulatory factors. This study was to test the feasibility of H.E.L.P. apheresis in preeclamptic patients and its potential effects on blood and placental markers of preeclampsia. We applied H.E.L.P. apheresis to nine preeclamptic patients and it was well tolerated. Their gestational ages could be continued by 17.7 (3-49) more days. Eight of the nine neonates did well during their neonatal stage. One infant died of late-onset sepsis. H.E.L.P. apheresis reduced significantly circulating levels of triglycerides, total and LDL-cholesterol, Lp(a), fibrinogen, hs-CRP, TNFalpha, sVCAM-1, E-selectin, lipopolysaccharide binding protein (LBP), homocysteine and plasma viscosity. We conclude that H.E.L.P. apheresis reduced maternal circulating levels of proinflammatory and coagulatory markers and plasma viscosity without overt maternal or neonatal clinical side effects.

Using the mean platelet volume MPV for the quality assessment of apheresis procedures.

INTRODUCTION: Referring to current standards the quality of an apheresis procedure is estimated by the quantity of collected cells. Nowadays a new kind of quality measurement could be found in the detection of cell volumina. Recent diagnostics have shown that stem cells and platelets - when separated - are likely to appear in a higher volume inside the cell product. Therefore, in this study the question should be discussed wether platelets of higher volume are more likely to be separated than platelets showing a lesser volume. METHODS: Blood samples of three different apheresis procedures could be observed: allogenic platelet donations (n = 7) (Trima, Terumo), autologous (n = 5) and allogenic stem cell donations (n = 5) (Cobe Spectra, Terumo). To examine the blood samples the Sysmex hematology analyser (XT-2000) has been used. RESULTS: During stem cell apheresis, the volume of the separated platelets was 1.2 fold increased compared to the platelet volume in the peripheral blood before separation. Before apheresis the mean platelet volume in the peripheral blood was found to be 6,21 fl, after apheresis 6,09 fl and inside the platelet concentrate 7,42 fl. The platelet number in the peripheral blood was also significantly decreased (before separation 180.1/nl and after separation 133.5/nl). In the blood products the concentration of platelets was nearly 8 fold higher than in the peripheral blood before separation. CONCLUSION: Overall, the observed apheresis procedures are more likely to separate platelets showing a higher voulme than common in the peripheral blood. This might indicate that not only the amount of separated cells reflects the quality of the apheresis procedure but also that the volume of the separated cells can be used as a parameter for quality assessment.

Postoperative Heparin-Mediated Extracorporeal Low-Density Lipoprotein Fibrinogen Precipitation Aphaeresis Prevents Early Graft Occlusion after Coronary Artery Bypass Grafting.

Background Early graft occlusion due to thromboembolic events is a well-known complication after coronary artery bypass grafting (CABG). Fibrinogen, the coagulation factor I, is a glycoprotein that is transformed by thrombin into fibrin. It plays a major role in thrombus formation and is highly elevated after CABG. Our aim was to determine if postoperative lowering of fibrinogen levels by H.E.L.P. (heparin-mediated extracorporeal low-density lipoprotein [LDL] fibrinogen precipitation) aphaeresis could reduce the rate of early graft occlusion in patients with hypercholesterolemia undergoing CABG. Methods Between December 2004 and September 2009, 36 male patients with hypercholesterolemia (mean LDL cholesterol 128 ± 12 mg/dL), mean age 58 ± 9 years, underwent CABG. Mean preoperative fibrinogen level was 387 ± 17 mg/dL. H.E.L.P. aphaeresis was postoperatively performed when fibrinogen levels exceeded 350 mg/dL on day 1 and 250 mg/dL every consecutive day up to day 8. Pre- and postaphaeresis blood samples were obtained and plasma fibrinogen level reduction was calculated. Early graft occlusion was evaluated by means of coronary angiography or multislice computed tomography before discharge. Results A total of 128 distal anastomoses were performed in 36 patients (mean 3.6/patient). Postoperatively, 191 H.E.L.P. aphaeresis sessions were performed (mean 5.3/patient). Fibrinogen levels were lowered from 391 ± 10 mg/dL (preaphaeresis) to 171 ± 5 mg/dL (postaphaeresis; p < 0.001). Coronary angiography (multislice computed tomography in 7 patients) revealed graft patency in 125 of 128 grafts (98% patency) with three occluded venous grafts to target vessels of 1.5 mm. H.E.L.P. aphaeresis-related complications were limited to hypotensive episodes in two patients and bacteremia in one patient. Conclusions H.E.L.P. apheresis offers an easy, save, and efficient method to decrease fibrinogen postoperatively in patients having CABG. Showing excellent graft patency rates in comparison to the literature, this method is a promising tool to reduce early graft occlusion after CABG.

Heparin-mediated extracorporeal LDL precipitation treating a peripheral arterial disease patient suffering from repeated postoperative bypass occlusion.

Acute occlusion of a peripheral artery is a serious complication in peripheral arterial disease (PAD). Traditionally open surgical intervention in combination with antithrombotic therapy is the choice for treatment but the beneficial effects of both strategies are limited often by the patient's situation and therapeutic side effects. Heparin-mediated extracorporeal low-density lipoprotein precipitation (H.E.L.P.) apheresis efficiently removes circulating atherogenic lipoproteins, fibrinogen and C-reactive proteins as well as various proinflammatory and procoagulatory factors. We first report H.E.L.P. apheresis treating a PAD patient suffering from repeated postoperative femoropopliteal bypass graft occlusion, first, intensively, followed by weekly intervals. Limb amputation was avoided and the patient is doing well now. Angiography revealed bypass graft remained patent half a year after operation. This case report might help to design the regime for preventing postoperative bypass occlusion in patients with hyperlipidemia or hyperfibrinogenemia.

Bacteria on smartphone touchscreens in a German university setting and evaluation of two popular cleaning methods using commercially available cleaning products.

Smartphone touchscreens are known as pathogen carriers in clinical environments. However, despite a rapidly growing number of smartphone users worldwide, little is known about bacterial contamination of smartphone touchscreens in non-clinical settings. Such data are needed to better understand the hygienic relevance of these increasingly popular items. Here, 60 touchscreens of smartphones provided by randomly chosen students of a German university were sampled by directly touching them with contact agar plates. The average bacterial load of uncleaned touchscreens was 1.37 ± 0.33 CFU/cm(2). Touchscreens wiped with commercially available microfiber cloths or alcohol-impregnated lens wipes contained significantly less bacteria than uncleaned touchscreens, i.e., 0.22 ± 0.10 CFU/cm(2) and 0.06 ± 0.02 CFU/cm(2), respectively. Bacteria isolated from cleaned and uncleaned touchscreens were identified by means of MALDI Biotyping. Out of 111 bacterial isolates, 56 isolates (50 %) were identified to genus level and 27 (24 %) to species level. The vast majority of the identified bacteria were typical human skin, mouth, lung, and intestinal commensals, mostly affiliated with the genera Staphylococcus and Micrococcus. Five out of 10 identified species were opportunistic pathogens. In conclusion, the touchscreens investigated here showed low bacterial loads and a species spectrum that is typical for frequently touched surfaces in domestic and public environments, the general health risk of which is still under debate.

Effects of heparin-mediated extracorporeal low-density lipoprotein precipitation beyond lowering proatherogenic lipoproteins--reduction of circulating proinflammatory and procoagulatory markers.

In addition to hypercholesterolemia, proinflammatory and prothrombotic markers have been suggested to play an important role in atherogenesis. We examined whether heparin-mediated extracorporeal low-density lipoprotein precipitation (HELP) therapy modulates the circulating levels of proinflammatory and prothrombotic markers. Twenty-two coronary heart disease (CHD) patients undergoing regular HELP-apheresis (18 males, 4 females, mean age 57.3 +/- 10.9 years) were enrolled in this study. A single HELP therapy treatment significantly decreased the circulating levels of high sensitivity C-reactive protein (hs-CRP), soluble vascular adhesion molecule-1 (sVCAM-1), soluble E-selectin, lipopolysaccharide binding protein (LBP), endothelin-1 (ET-1), and monocyte chemoattractant protein-1 (MCP-1) on average by 67, 37, 24, 27, 24, and 15%, respectively. Prothrombotic factors including fibrinogen, tissue factor (TF), soluble CD40 ligand (sCD40L), and homocysteine were decreased by 66, 27, 16, and 22%, respectively. In accordance with previous studies HELP therapy reduced total cholesterol, low density lipoprotein (LDL) cholesterol, and Lp(a) mass by 50, 61, and 62%, respectively. Our data suggest that simultaneous reduction of proinflammatory and prothrombotic factors together with atherogenic lipoproteins by HELP-apheresis may contribute to improvement of endothelial dysfunction and thereby inhibit progression of atherosclerotic lesions and stabilize the existing plaque.

Heparin-mediated extracorporeal low-density lipoprotein precipitation: rationale for a specific adjuvant therapy in cardiovascular disease.

Various radical measures for the treatment of severe hypercholesterolemia such as partial ileal bypass, portocaval shunt, liver transplantation and plasma exchange have been tested in patients in whom drug and diet failed or were insufficient. Although effective, most of these treatments have severe side effects and are not routinely used. For hypercholesterolemic patients LDL-apheresis has proved to be the most promising and safe way as an adjuvant therapy. Several LDL-apheresis procedures with a varying degree of selectivity and efficiency have subsequently been developed. One of them is the H.E.L.P. system which was introduced in 1984 and has now been widely used. Besides the marked reduction of LDL particles by all techniques it has become apparent that only the H.E.L.P. system results in an equally significant change in hemostaseology, hemorheology and vasomotion because of its simultaneously removal of LDL, Lp(a), fibrinogen and CRP. This contribution reviews the application of the H.E.L.P. system as a valuable therapeutic tool for the treatment of various atherothrombotic and microcirculatory disorders such as prevention of early graft occlusion after coronary artery bypass grafting, treatment of peripheral vascular disease, stroke and preeclampsia.

The efficacy and safety of the new heparin-induced extracorporeal low-density lipoprotein precipitation system (Plasmat Futura) in comparison with the currently used system (Plasmat Secura).

The aim of the present study was to examine whether the recently introduced heparin-mediated extra-corporeal low-density lipoprotein precipitation (HELP) apheresis system Plasmat Futura (since 2001) was comparable to Plasmat Secura system, used to date, in its efficiency to remove atherogenic components, its ease of handling and operating as well as clinical safety and patient compliance. Coronary heart disease (CHD) patients (N = 21) were first treated with Plasmat Secura system and 13 of them were then randomly switched over to the upgraded Plasmat Futura system. Eight patients remained on Secura system. All together, 40 Futura treatments and 40 Secura treatments were performed. Blood samples were collected immediately before and after each apheresis therapy. Our data showed no significant differences in the reduction of plasma low-density lipoprotein, lipoprotein (a) and fibrinogen by Plasmat Futura and Secura system (P > 0.05). However, the major advantages of Plasmat Futura system are the ready-to-use sterile dialysis solutions instead of reverse osmosis device in Plasmat Secura, which ensures flexibility and lower risk of cross infections. Long-term tolerance and safety parameters showed no significant difference (P > 0.05). On the basis of our studies. Plasmat Futura system is easy to use, shows no adverse events and is comparable to Plasmat Secura in its capacity to remove proatherogenic plasma factors.

[Atherogenesis: interplay between cholesterol, inflammation and coagulation]. / Atherogenese: Wechselspiel zwischen Cholesterin, Inflammation und Koagulation.

It is now generally accepted that, in addition to hypercholesterolemia, pro-inflammatory and procoagulatory factors play a major role in atherogenesis. Risk factors such as smoking, hypertension, diabetes and renal diseases alter lipoprotein profile and composition thus rendering them susceptible to modification. Modified lipoproteins induce local inflammation possibly due to activation of nuclear factor (NF-)kappaB and subsequent expression of adhesion molecules, release of pro-inflammatory cytokines, growth factors and mitogens, which are mediators for cell growth, proliferation and lipid deposition. Furthermore, activation of collagenases and proteases in combination with prothrombotic processes attenuate clot formation, plaque rupture and occlusion of vessels. Clinical as well as experimental studies suggest that elevated levels of pro-inflammatory markers may have a diagnostic potential. Thus, a therapeutic approach which modulates circulating cholesterol levels and improves pro-inflammatory and procoagulatory situation may prove beneficial as adjuvant therapy in atherosclerotic disease.