Routine Diagnostics Research Articles

Abstract 3411: Detection and monitoring of tumor-derived mutations in ctDNA using the UltraSEEK Lung Panel on the MassARRAY System in metastatic NSCLC patients

Abstract Background: The analysis of circulating tumor DNA (ctDNA) is a potential minimally invasive molecular tool to guide treatment decision making and disease monitoring. A suitable diagnostic-grade platform is required for detection of tumor-specific mutations with high sensitivity in circulating cell-free DNA (ccfDNA) of cancer patients. Methods: In an international multicenter study conducted by three laboratories participating in the CANCER-ID consortium, a cohort containing 177 cell-free plasma samples at baseline, follow-up or disease progression derived from 72 patients treated for advanced-stage non-small cell lung cancer (NSCLC), relevant mutations were tested using the UltraSEEK® Lung Panel on the MassARRAY® System covering 73 hotspot mutations in EGFR, KRAS, BRAF, ERBB2 and PIK3CA, and compared against mutation-specific droplet digital PCR (ddPCR) and tumor tissue next-generation sequencing (NGS) performed in routine diagnostics. Variant detection accuracy at primary diagnosis and during disease progression, and ctDNA dynamics as a marker of treatment efficacy were analyzed. Results: The UltraSEEK® Lung Panel revealed an overall detection rate of 87.2% irrespective of ccfDNA input, and a specificity of >99.5% using the reference material. When comparing the 131 variants identified in the patient-derived cell-free plasma using UltraSEEK® with mutation-specific ddPCR analyses, a concordance of 90% was found. The 77 diagnostically or clinically relevant variants identified in 66 pretreatment tumor tissue showed an overall concordance with UltraSEEK® of 73% at baseline with a false-negative rate of 3%. When restricting to therapeutically targetable mutations, the concordance elevated to 85%. A decrease in ctDNA levels at 4-6 weeks after start of treatment detected with UltraSEEK® correlated with a prolonged median PFS (46 vs 11 weeks; P<0.05) and OS (145 vs 39 weeks; P<0.001). In four out of thirteen NSCLC patients treated with targeted therapy, resistance mutations were identified using UltraSEEK® at disease progression, implicating the requirement of a therapy switch. Conclusion: Using plasma-derived ccfDNA, the UltraSEEK® Lung Panel with a mid-density set of the most common predictive markers for NSCLC is an alternative tool to detect both mutations at diagnosis and disease progression, and to monitor treatment response. Citation Format: Paul van der Leest, Melanie Janning, Naomi Rifaela, Maria L. Aguirre Azpurua, Jolanthe Kropidlowski, Sonja Loges, Nicholas Lozano, Alexander Sartori, Darryl Irwin, Pierre-Jean Lamy, T. Jeroen N. Hiltermann, Harry J. Groen, Klaus Pantel, Léon C. van Kempen, Harriet Wikman, Ed Schuuring. Detection and monitoring of tumor-derived mutations in ctDNA using the UltraSEEK Lung Panel on the MassARRAY System in metastatic NSCLC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3411.

Improvement of Diagnostic Yield by an Additional Amplicon Module to Hybridization-Based Next-Generation Sequencing Panels

Many approaches aimed at improving next-generation sequencing output for clinical purposes exist. However, sequencing gaps or misalignments for regions that are difficult to cover because of their low complexity or high homology still exist. Our aim was to improve the yield of sequencing data. A hybridization-based next-generation sequencing library was pooled with custom add-on amplicon-based libraries processed by the same commercial test and run in parallel and sequenced simultaneously. Formulas and steps for proper amplicon pooling (250 to 7000 bp) and final library merging are presented. The novel strategy was tested on selected archetypal situations: diagnostics of a gene with many pseudogenes, a genomic region surrounded by Alu repeats, simple one-time addition of an extra gene, and mosaicism detection. The sequence of all supplemented genomic regions was traced with reasonable coverage at the background of a hybridization captured library. The flexible add-on module expands the possibilities of routine diagnostics. The technical solution makes it possible to mix amplicons that differ significantly in size and process them in one tube simultaneously with samples of the hybridization-based panel. The proposed approach reduces turnaround time and increases diagnostic yield.

Conventional ultrasound diagnostics in dermatology

The imaging of changes to the skin, the subcutis and especially the regional lymph nodes by high-resolution ultrasound is an integral part of routine dermatological diagnostics. This is mainly done with electronic scanners operating at frequencies between 7.5 and 20 MHz (conventional ultrasound diagnostics). In addition, there are very high-frequency ultrasound systems (frequencies up to 100 MHz) that are used for special scientific questions. Ultrasound diagnostics has anumber of advantages over other cross-sectional imaging techniques but is more dependent than these on the individual experience of the examiner. Structured training and continuing education are therefore essential for ultrasound diagnostics, also in dermatology. The following overview describes the most important indications for conventional sonography in dermatology in addition to the physical, technical and administrative principles.

Genetic Architecture of Azoospermia—Time to Advance the Standard of Care

BackgroundCrypto- and azoospermia (very few/no sperm in the semen) are main contributors to male factor infertility. Genetic causes for spermatogenic failure (SPGF) include Klinefelter syndrome and Y-chromosomal azoospermia factor microdeletions, and CFTR mutations for obstructive azoospermia (OA). However, the majority of cases remain unexplained because monogenic causes are not analysed. ObjectiveTo elucidate the monogenic contribution to azoospermia by prospective exome sequencing and strict application of recent clinical guidelines. Design, setting, and participantsSince January 2017, we studied crypto- and azoospermic men without chromosomal aberrations and Y-chromosomal microdeletions attending the Centre of Reproductive Medicine and Andrology, Münster. Outcome measurements and statistical analysisWe performed exome sequencing in 647 men, analysed 60 genes having at least previous limited clinical validity, and strictly assessed variants according to clinical guidelines. Results and limitationsOverall, 55 patients (8.5%) with diagnostic genetic variants were identified. Of these patients, 20 (3.1%) carried mutations in CFTR or ADGRG2, and were diagnosed with OA. In 35 patients (5.4%) with SPGF, mutations in 20 different genes were identified. According to ClinGen criteria, 19 of the SPGF genes now reach at least moderate clinical validity. As limitations, only one transcript per gene was considered, and the list of genes is increasing rapidly so cannot be exhaustive. ConclusionsThe number of diagnostic genes in crypto-/azoospermia was almost doubled to 21 using exome-based analyses and clinical guidelines. Application of this procedure in routine diagnostics will significantly improve the diagnostic yield and clinical workup as the results indicate the success rate of testicular sperm extraction. Patient summaryWhen no sperm are found in the semen, a man cannot conceive naturally. The causes are often unknown, but genetics play a major role. We searched for genetic variants in a large group of patients and found causal mutations for one in 12 men; these predict the chances for fatherhood.

Grading Systems for Canine Urothelial Carcinoma of the Bladder: A Comparative Overview.

Simple SummaryTumor histological grading systems are a tool widely used by human pathologists in oncology to support the assessment of tumor behavior and patient prognosis by clinical oncologists. In veterinary medicine, several tumor types already have a histological grading system used for these purposes, but some of these schemes lack reproducibility or correlation with clinical parameters, such as the correlation of the grade with survival time. This is the case for the grading systems proposed for canine bladder urothelial carcinoma. Over the years, some grading systems have been described for this type of tumor in dogs but without any routine use by pathologists and, consequently, without any application in clinical practice either. Based on this fact, the present study aimed to review the histological grading systems that exist in both human and veterinary medicine for bladder urothelial carcinoma, carrying out a critical analysis of their differences and thereby encouraging their real practical use and application in a relevant number of cases, prospectively. In this way, a histological grading system could be chosen or built from the existing ones and the knowledge about the behavior of this neoplasm in canine species could be improved, helping clinicians to establish a prognosis and personalized treatment for each patient with bladder urothelial carcinoma and also consider the predictive markers associated with treatment outcomes.The relationship between tumor morphology and clinical behavior is a key point in oncology. In this scenario, pathologists and clinicians play a pivotal role in the identification and testing of reliable grading systems based on standardized parameters to predict patient prognosis. Dogs with bladder urothelial carcinoma (BUC) were recently proposed as a “large animal” model for the study of human BUCs due to the similar morphology and metastasis locations. BUC grading systems are consolidated in human medicine, while in veterinary medicine, the BUC grading systems that have been proposed for canine tumors are not yet applied in routine diagnostics. These latter systems have been proposed, decade by decade, over the last thirty years, and the reason for their scarce application is mainly related to a lack of specific cutoff values and studies assessing their prognostic relevance. However, for any prognostic study, reliable grading is necessary. The aim of the present article was to give an overview of the BUC grading systems available in both human and veterinary pathology and provide an extensive description and a critical evaluation to support veterinary researchers in the choice of possible grading systems to apply in future studies on canine BUCs.

Anti-double stranded DNA antibodies: A rational diagnostic approach in limited-resource settings

ContextAnti-double-stranded deoxyribonucleic acid antibodies (dsDNA Abs) are highly specific markers of systemic lupus erythematosus (SLE). Multiple methods are employed for their detection in routine diagnostics. ObjectivesThe aim of this study was to evaluate a diagnostic approach for anti-dsDNA Abs using DNA-ELISA and Crithidia luciliae fluorescence test (CLIFT), in combination with antinuclear antibody (ANA) screening. MethodsWe enrolled 113 patients—53 with SLE, 50 with other systemic autoimmune rheumatic diseases (OSARD), and 10 with non-autoimmune clinical conditions (NAICC).Patients’ samples were tested for anti-dsDNA Abs using an enzyme-linked immunosorbent assay (ELISA) and CLIFT, combined to ANA screening by indirect immunofluorescence assay (ANA-IIFA). ResultsThe mean age of patients was 39.94 ± 15 years (ranges: 11–85 years). Overall, specimens from 77.3%, 11.7%, and 20% of patients with SLE, OSARD and NAICC respectively were ELISA-positive; and those from 54.7% to 4% of patients with SLE and OSARD, respectively, were CLIFT-positive. CLIFT positivity was significantly associated with high ELISA titers (p = 0.002) and homogeneous ANA-IIF pattern (p = 0.0002). ConclusionFor better clinical relevance of anti-dsDNA antibodies, we suggest a combined detection strategy based on ELISA, CLIFT and ANA-IIFA, considering the clinical criteria of SLE.

PATH-08. DNA methylation profiling improves routine diagnostics of paediatric CNS tumours: a prospective population-based study

Abstract AIMS: Paediatric brain tumours are rare and establishing a precise diagnosis can be challenging. Analysis of DNA methylation profiles has been shown to be a reliable method to classify central nervous system (CNS) tumours with high accuracy. We aimed to prospectively analyse CNS tumours diagnosed in Sweden, to assess the clinical impact of adding DNA methylation-based classification to standard paediatric brain tumour diagnostics in an unselected cohort. Methods: All CNS tumours diagnosed in children (0-18 years) during 2017-2020 were eligible for inclusion provided sufficient tumour material was available. Tumours were analysed using genome-wide DNA methylation profiling and classified by the MNP brain tumour classifier. The initial histopathological diagnosis was compared to the DNA methylation-based classification. For incongruent results, a blinded re-evaluation was performed by an experienced neuropathologist. Results: 240 tumours with a histopathology-based diagnosis were profiled. A high-confidence methylation score of 0.84 or more was reached in 78% of the cases. In 69%, the histopathological diagnosis was confirmed and for some of these also refined, 6% were incongruent and the re-evaluation favoured the methylation-based classification. In the remaining 3% of cases, the methylation class was non-contributory or could not be predicted. The change in diagnosis would have had a direct impact on the clinical management in 5% of all patients.Conclusions: Integrating DNA methylation-based tumour classification into routine clinical analysis improves diagnostics and molecular information important for treatment decisions. The results from methylation profiling should be interpreted in the context of clinical and histopathological information.

SAMBA: A Multicolor Digital Melting PCR Platform for Rapid Microbiome Profiling.

During the past decade, breakthroughs in sequencing technology have provided the basis for studies of the myriad ways in which microbial communities in and on the human body influence human health and disease. In almost every medical specialty, there is now a growing interest in accurate and quantitative profiling of the microbiota for use in diagnostic and therapeutic applications. However, the current next-generation sequencing approach for microbiome profiling is costly, requires laborious library preparation, and is challenging to scale up for routine diagnostics. Split, Amplify, and Melt analysis of BActeria-community (SAMBA), a novel multicolor digital melting polymerase chain reaction platform with unprecedented multiplexing capability is presented, and the capability to distinguish and quantify 16 bacteria species in mixtures is demonstrated. Subsequently, SAMBA is applied to measure the compositions of bacteria in the gut microbiome to identify microbial dysbiosis related to colorectal cancer. This rapid, low cost, and high-throughput approach will enable the implementation of microbiome diagnostics in clinical laboratories and routine medical practice.

Defining a prognostic risk score in ovarian cancer patients by integrated analysis of serum mesothelin, HE4, CA125 and surgical outcome.

e17611 Background: In ovarian cancer, serum CA125 is currently the only biomarker used in routine clinical practice and has several limitations, e.g. it is not entirely specific for ovarian malignancy. Alternative serum biomarkers have been proposed for ovarian cancer, such as mesothelin or human epididymis protein 4 (HE4). Although it is known that single marker analysis is often insufficient for diagnostic purposes or relapse prediction, no comprehensive studies on the combination of these markers have been conducted, yet. Using a multi-marker prediction model, we herein investigated the clinical usefulness of mesothelin and HE4, detected in a sequence of pre- and post-operative serum samples, in complementing the standard marker CA125. Methods: Serum mesothelin, HE4 and CA125 were quantified by Lumipulse G chemiluminescent enzyme immunoassay (Fujirebio) in a total of 432 serum samples from 90 healthy controls and 192 ovarian cancer patients (thereof 81.8% with FIGO III/IV), including 151 paired pre- and post-operative samples. Results: Pre-operative serum mesothelin and HE4 were significantly elevated compared to healthy controls with a median ratio (IQR) to controls of 2.6 and 9.6, respectively (p < 0.0001). High pre-operative levels of mesothelin or HE4 alone were significantly correlated with advanced FIGO-stage (p < 0.0001) and predicted surgical outcome (p < 0.0001). We retrospectively compiled mesothelin and HE4 levels with CA125, BRCA mutational status and surgical outcome, in order to define a multi-marker setting. Both, mesothelin and HE4 levels were independent from BRCA mutational status, however they positively correlated with CA125. Detection of pre-operative HE4 > 498pM (OR = 4.1, p < 0.0001) combined with pre-operative CA125 > 516U/mL (OR = 2.2, p = 0.001) was the best combination to predict surgical outcome (AUC = 0.815). Finally, we defined a prognostic signature, including pre-operative CA125, post-operative mesothelin, HE4 and surgical outcome, which was superior to the individual prognostic capacity of all included parameters. Our model allowed the calculation of a weighted risk score, stratifying patient at “lower risk” (score 0-4) or at “higher risk” (score 5-10). Patients with higher risk had significantly shorter PFS (19.0 months (95%CI = 16.1-21.8) vs 64.1 months (95%CI = 56.8-71.3) p < 0.0001) and OS (34.0 months (95%CI = 28.6-39.3) vs 71.2 months (95%CI = 64.6-77.7) p < 0.0001). Conclusions: This is the first study, proposing an integrated prognostic risk score for ovarian cancer patients, which could easily be implemented into routine diagnostics and may guide personalized treatment decisions. Patients at “higher risk”, as identified by this model, could potentially benefit from an intensified therapy regime, whereas patients at “lower risk” might be suitable for treatment de-escalation.

Justifying integration of liquid biopsy into routine molecular diagnostics to complement tissue genomic profiling: Experience at Fox Chase Cancer Center.

e15029 Background: Plasma-based liquid biopsy has rapidly evolved from a novel research tool toward translation into clinical use for many diseases including cancer. While there are still some gaps regarding standardization and clinical utility, as tissue next-gen sequencing had previously faced and overcome, there is a growing body of evidence supporting integration of liquid biopsy into standard of care as a diagnostic tool for precision oncology. Selected from a larger cohort, several cases are presented here illustrating the use of the PGDx elio plasma resolve assay to profile circulating tumor DNA (ctDNA) from patients whose tumor and matched normal DNA had also been characterized by a next-generation sequencing panel. Methods: The PGDx elio plasma resolve assay and bioinformatics detects single and multi-nucleotide variants in 33 genes, select amplifications and translocations, as well as microsatellite instability by sequencing on the Illumina NextSeq 500/550 platform. DNA from matched tumor and normal FFPE samples were assessed using the 275 gene Molecular Diagnostics Laboratory’s Comprehensive Cancer Panel (CCP), also on the NextSeq platform. Selected cases represent the range of diagnostic utility that might be expected from ctDNA. Results: Across all cases, there were varying levels of concordance between tissue and plasma as well as novel variants in the plasma that were not present in the tissue DNA demonstrating the benefit of incorporating ctDNA profiling to capture critical genomic information. In one rectal adenocarcinoma case, DNA variants from the primary tumor were detected in ctDNA eight months later during evaluation of a recurrent tumor, suggesting that plasma profiling might have provided early biomarker detection of recurrence. An aggressive and recurrent melanoma, however, showed no concordance between ctDNA and tumor DNA variants, indicating that some tumors may not shed sufficient DNA into the bloodstream to make ctDNA clinically relevant. Another case of melanoma had concordant detection of the driver BRAF V600E mutation in tumor and ctDNA, but ctDNA also contained a pathogenic ATM truncation not observed in the tumor sample. Current clinical trials are investigating the combination of PARP- and ATR-inhibiting drugs, which causes excessive DNA damage and cell death in the context of ATM-deficient cancers. Conclusions: These cases show that combined tissue and ctDNA profiling at the time of initial diagnosis can reveal tumor heterogeneity or distinct metastases not observed in a selected tissue sample, and that profiling during and after therapy may provide early detection of recurrence, appearance of resistance mutations, or monitoring of measurable residual disease.

Retrospective evaluation of viral respiratory tract infections in a university hospital in Ankara, Turkey (2016-2019).

Viruses are responsible for two-thirds of all acute respiratory tract infections. This study aims to retrospectively detect respiratory tract viruses in patients from all age groups who visited the hospital. A total of 1592 samples from 1416 patients with respiratory tract symptoms were sent from several clinics to the Molecular Microbiology Laboratory at Gazi University Hospital from February 2016 to January 2019. Nucleic acid extraction from nasopharyngeal swabs, throat swabs or bronchoalveolar lavage (BAL) samples sent to our laboratory was done using a commercial automated system. Extracted nucleic acids were amplified by a commercial multiplex-real time Polymerase Chain Reaction (PCR) method, which can detect 18 viral respiratory pathogens. Among 1592 samples, 914 (57.4%) were positive for respiratory viruses. The most prevalent were rhinovirus (25.2%) and influenza A virus (12.1%), the least prevalent was the bocavirus (2.6%). Rhinovirus was the most detected as a single agent (21.2%, 194/914) among all positive cases, followed by coronavirus (9.3%, 85/914). The detection rates of coronavirus, human adenovirus, respiratory syncytial virus A/B, human parainfluenza viruses, human metapneumovirus-A/B, human parechovirus, enterovirus and influenza B virus were 9.9%, 8%, 7.7%, 5%, 3.4%, 3.1%, 3%, and 2.8%, respectively. The most detected viral agents in our study were influenza A virus and rhinovirus. Laboratory diagnosis of respiratory viruses is helpful to prevent unnecessary antibiotic use and is essential in routine diagnostics for antiviral treatment. Multiplex Real-time PCR method is fast and useful for the diagnosis of viral respiratory infections.

Evaluation of a Quantitative Taqman Real-Time PCR Assay to Measure Proviral load from Human Immunodeficiency Virus Type 1 individuals

Human Immunodeficiency Virus (HIV) is a virus belonging to the family Retroviridae. HIV – 1 is found to be predominant in India and many parts of Africa. The intention of this study was to quantify the HIV Proviral Deoxyribonucleic Acid (DNA) from newly infected HIV-1 individuals. Fifty patients who were tested positive for HIV were included in this study. Proviral Ribo Nucleic Acid (RNA) was extracted by QIAmp® RNA Mini Kit (QIAGEN, Germany) method. Complementary Deoxyribo Nucleic Acid (cDNA) was synthesized by using Invitrogen Superscript III cDNA synthesis Kit (USA). This cDNA was subjected to Polymerase Chain Reaction (PCR) and Gene cloning by transformation method. The quantification of Real time PCR was done by Applied Bio-System (ABI)-Prism 7700. A linear standard curve was obtained 10 copies to 106 copies per reaction. The assay had good analytic sensitivity and linear dynamic range greater than 6 logs. From the results obtained in this study, It was concluded that Taqman Real-Time PCR Assay plays a major role in monitoring the HIV infected patients in routine diagnostics and clinical practice.

Comparative performance of CDC-modified SARS-CoV-2 real-time PCR assay with four different commercial assays: laboratory-based study.

The coronavirus infectious disease (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses. The pandemic has emerged as a global public health crisis, and the threat of fast-spreading of the latest variants of the coronavirus (such as omicron, delta) is rampant. Therefore, a fast and reliable diagnostic assay is needed to make the clinical decision for further treatment. The study aims to develop a Centers for Disease and Prevention (CDC)–modified qualitative real-time reverse transcriptase PCR (RT-qPCR) assay and parallel assessment of commercially available RT-qPCR assay (Altona, Seegene, BD, and GBC) to detect SARS-CoV-2. Two hundred nine samples were chosen randomly out of around two hundred thousand samples. The panel consisted of SARS-CoV-2-positive (n = 156) and SARS-CoV-2-negative (n = 52) nasopharyngeal swab specimens for a primary clinical evaluation. Furthermore, 29 positive samples were sequenced using Oxford Nanopore Minion technology. Two hundred nine patient sample data of the cycle threshold (Ct) readings for target genes of five assays are 100% sensitive for Ct values. Mean Ct values for N1, N2, RdRp, S, and E of the positive controls in CDC assay, RealStar®, Allplex, GBC, and SD Biosensor were 17.5 ± 0.49, 16.9 ± 0.51, 20 ± 0.49, 21.7 ± 0.38, and 23.1 ± 0.43, respectively. F test value shows ≥ 1, which was statistically significant. All assays showed an efficiency of < 120% and R squares were < 0.99, which is well above the required threshold value. Thus, when taking the CDC-modified assay as a gold standard, the other four assays demonstrated a p value of 0.0000, concordance at 100%, and a Kappa at 1.000. A maximum-likelihood (ML) tree was constructed and compared based on full-length SARS-CoV-2 with Wuhan isolate. These isolates are closely related to the B.1.617 lineage and reference sequences. Therefore, we conclude that all RT-PCR kits assessed in this study shall be used for routine diagnostics of COVID-19 in patients. Supplementary informationThe online version contains supplementary material available at 10.1007/s00580-022-03356-y.

Quality of MALDI-TOF mass spectra in routine diagnostics: results from an international external quality assessment including 36 laboratories from 12 countries using 47 challenging bacterial strains

ObjectivesMatrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is a widely used method for bacterial species identification. Incomplete databases and mass spectral quality (MSQ) still represent major challenges. Important proxies for MSQ are the number of detected marker masses, reproducibility, and measurement precision. We aimed to assess MSQs across diagnostic laboratories and the potential of simple workflow adaptations to improve it. MethodsFor baseline MSQ assessment, 47 diverse bacterial strains, which are challenging to identify by MALDI-TOF MS, were routinely measured in 36 laboratories from 12 countries, and well-defined MSQ features were used. After an intervention consisting of detailed reported feedback and instructions on how to acquire MALDI-TOF mass spectra, measurements were repeated and MSQs were compared. ResultsAt baseline, we observed heterogeneous MSQ between the devices, considering the median number of marker masses detected (range = [2–25]), reproducibility between technical replicates (range = [55%–86%]), and measurement error (range = [147 parts per million (ppm)–588 ppm]). As a general trend, the spectral quality was improved after the intervention for devices, which yielded low MSQs in the baseline assessment as follows: for four out of five devices with a high measurement error, the measurement precision was improved (p-values <0.001, paired Wilcoxon test); for six out of ten devices, which detected a low number of marker masses, the number of detected marker masses increased (p-values <0.001, paired Wilcoxon test). DiscussionWe have identified simple workflow adaptations, which, to some extent, improve MSQ of poorly performing devices and should be considered by laboratories yielding a low MSQ. Improving MALDI-TOF MSQ in routine diagnostics is essential for increasing the resolution of bacterial identification by MALDI-TOF MS, which is dependent on the reproducible detection of marker masses. The heterogeneity identified in this external quality assessment (EQA) requires further study.

Diagnostic potential of nanoparticle aided assays for MUC16 and MUC1 glycovariants in ovarian cancer.

Our study reports the discovery and evaluation of nanoparticle aided sensitive assays for glycovariants of MUC16 and MUC1 in a unique collection of paired ovarian cyst fluids and serum samples obtained at or prior to surgery for ovarian carcinoma suspicion. Selected glycovariants and the immunoassays for CA125, CA15‐3 and HE4 were compared and validated in 347 cyst fluid and serum samples. Whereas CA125 and CA15‐3 performed poorly in cyst fluid to separate carcinoma and controls, four glycovariants including MUC16MGL, MUC16STn, MUC1STn and MUC1Tn provided highly improved separations. In serum, the two STn glycovariants outperformed conventional CA125, CA15‐3 and HE4 assays in all subcategories analyzed with main benefits obtained at high specificities and at postmenopausal and early‐stage disease. Serum MUC16STn performed best at high specificity (90%‐99%), but sensitivity was also improved by the other glycovariants and CA15‐3. The highly improved specificity, excellent analytical sensitivity and robustness of the nanoparticle assisted glycovariant assays carry great promise for improved identification and early detection of ovarian carcinoma in routine differential diagnostics.

A novel Borrelia-specific real-time PCR assay is not suitable for diagnosing Lyme neuroborreliosis

BackgroundDiagnosing Lyme neuroborreliosis (LNB) is complicated by a lack of adequate test systems and by the complex culturing conditions required to grow the causative pathogens in the Borrelia sensu lato complex. Improved testing methods are urgently needed. Here, we evaluate the applicability of a novel commercially available Borrelia-specific real-time PCR assay to diagnose LNB. Materials and methodsThe specificity and sensitivity of the novel alphaCube Borrelia real-time PCR assay (Mikrogen) and the well-tested Micro-Dx™ real-time PCR assay (Molzym) were evaluated in cerebrospinal fluid (CSF) spiked with known amounts of Borrelia garinii and CSF from 19 patients with definite or possible LNB. CSF from patients diagnosed with neurosyphilis or enterovirus meningitis served as controls. ResultsThe alphaCube assay specifically identified Borrelia down to 93 B garinii cells/mL in spiked CSF samples. The Micro-Dx™ real-time PCR assay was able to identify the presence of bacteria down to 9300 cells/mL in spiked samples. In CSF from patients diagnosed with LNB the sensitivity of the alphaCube assay was 0.00 and 0.00 for the Micro-DX. ConclusionAlthough the alphaCube Borrelia assay was able to identify down to 93 cells/mL in spiked CSF samples, the inability to identify Borrelia in CSF samples from patients with LNB suggests that this type of infection carries a bacterial load in CSF below this detection level. Based on these results, neither the alphaCube Borrelia real-time PCR assay nor the Micro-Dx™ real-time PCR assay can be recommended for routine diagnostics of LNB using CSF samples.

A novel RFLP method for identification of morphologically similar avian Sarcocystis species.

Protozoans of genus Sarcocystis are widespread parasites infecting mammals, birds, and reptiles. Morphology of their sarcocysts is an important criterion for species identification. However, as more and more morphologically similar Sarcocystis species are being found and described, additional methods for their routine diagnostics are needed. We investigated restriction fragment length polymorphism (RFLP) as potential alternative to sequencing data analysis for the identification of Sarcocystis species using birds as an intermediate host. The internal transcribed spacer 1 (ITS1) region sequences of seventeen Sarcocystis species (S. albifronsi, S. anasi, S. calchasi, S. columbae, S. cornixi, S. corvusi, S. cristata, S. halieti, S. falcatula, S. fulicae, S. kutkienae, S. lari, S. lindsayi, S. rileyi, S. turdusi, S.wenzeli, and S. wobeseri) of interest were analysed and five best-fitting endonucleases generating most informative restriction fragments were selected for routine testing. In general, RFLP analyses are always inconclusive as they target very short DNA sequences. However, it can be an irreplaceable technique when fast and cheap identification and discrimination of known species are required, which was our main goal and preliminary results indicate that RFLP could be successfully used when identifying closely related avian Sarcocystis species with just two nucleases.

Using High-Resolution Differential Cell Counts (HRDCCs) in Bovine Milk and Blood to Monitor the Immune Status over the Entire Lactation Period

Simple SummarySince leukocytes enter the mammary gland as part of the immune defense, they can be analyzed in raw milk samples and used for diagnostics. In our study, we differentiated and measured several subtypes of immune cells in blood and milk samples of eight dairy cows over their whole lactation to monitor their immune status. In addition to these so-called high-resolution differential cell counts (HRDCCs), we determined routine laboratory biomarkers. We found considerable differences between the documented cell count progressions in the blood and milk. Some cell populations remained mostly stable, while others showed a certain dynamic during the course of lactation. Moreover, two types of lymphocytes were also noticeable due to changes in the case of inflammation. These findings suggest that HRDCCs can be a promising tool for more efficient milk diagnostics, ensuring milk quality, and supporting cattle health and wellbeing.Differential cell counts in milk offer a deeper insight into the immunology of the mammary gland and might even provide information about the systemic health status of a dairy cow. Consequently, their potential as a diagnostic method to identify biomarkers has been a subject of research for quite some time. The objective of our study was to closely monitor the immune status of eight healthy dairy cows throughout their whole lactation. For this, high-resolution differential cell counts in milk and blood were determined by means of flow cytometry, which included 10 subpopulations of the 3 main populations of immune cells and their viability. Milk and blood samples were taken twice a week in the first 100 days after calving and once a week during the remaining lactation period: in total, 55 (52–57) blood and 55 (52–57) milk samples per animal. In addition, six well-established routine laboratory biomarkers, i.e., haptoglobin, calcium, and different metabolic parameters (non-esterified fatty acids, β-hydroxybutyric acid, bilirubin, and glutamate dehydrogenase), were analyzed in all blood samples. Furthermore, a standard differential blood cell count was performed on all blood samples. We found substantial differences between cell count progressions in the blood and milk. The distribution of cell populations in the blood remained mostly stable throughout the lactation, albeit at different individual levels. Several cell populations in the milk showed a noticeable dynamic over time, which caused a separation of different lactation stages in clustering analyses. Gamma delta T cells and CD4+ T cells in the milk stood out as they showed characteristic fluctuations during the course of lactation as well as minor changes in the case of inflammation. The determination of a differential cell count has the potential to be a sensitive diagnostic and prognostic tool in bovine milk. Further studies need to show to what extent the method is suitable for routine diagnostics and how to deal with animal-specific differences.

Microdeletions in 1q21 and 8q12.1 depict two additional molecular subgroups of Silver-Russell syndrome like phenotypes

BackgroundSilver-Russell syndrome (SRS) is a genetic disorder characterized by intrauterine and postnatal growth restriction, relative macrocephaly at birth, body asymmetry and typical facial features. Clinical and molecular heterogeneity is described in SRS. Common causes are loss of methylation of the imprinting center 1 in 11p15 and maternal uniparental disomy of chromosome 7. Other genetic alterations include disturbances of imprinted regions in 14q32, 7q32 and 11p15 as well as submicroscopic deletions and duplications. Single nucleotide variants in genes like IGF2, HMGA2, PLAG1, CDKN1C have also been identified in patients with SRS phenotypes. However, routine molecular diagnostics usually focus on 11p15 and chromosome 7, while less frequent causes are not systematically addressed.ResultsHere we report two patients with SRS features in which molecular karyotyping revealed microdeletions in 1q21 and 8q12.1 respectively. In a 3.5-year-old girl with postnatal growth restriction, feeding difficulties, relative macrocephaly and distinct SRS features a 2 Mb deletion in 1q21.1q21.2 was identified. Our second case is a 1.5-year-old boy with intrauterine and postnatal growth restriction, feeding difficulties and distinct facial features with a 77 kb deletion in 8q12.1 affecting PLAG1 as the only protein-encoding gene with known function.ConclusionsThe 1q21 region has not yet been assigned as an SRS region, although six patients with the same deletion and SRS features including relative macrocephaly have been described before. This new case adds to the evidence that distal 1q21 should be annotated as an SRS candidate region. The PLAGL1 alteration is the smallest deletion in 8q12.1 ever reported in a patient with SRS phenotype and it finally confirms that PLAG1 is the SRS causing gene in 8q12.1. To increase the diagnostic yield in patients with suspected SRS, we recommend both molecular karyotyping and next generation sequencing-based approaches.

Dealing with inconclusive SARS-CoV-2 PCR samples-Our experience.

Early confirmation of SARS-CoV-2 is a key point in the timely management of infected patients and contact persons. Routine diagnostics of COVID-19 cases relies on RT-PCR detection of two or three unique sequences of the virus. A serious problem for the laboratories is how to interpret inconclusive samples which are positive for only one of the SARS-CoV-2 specific genes. A total of 16364 naso-oropharyngeal swabs were collected and tested with SARS-CoV-2 Real-TM kit (Sacace Biotechnologies, Italy) between May and September 2020. We retrospectively analyzed their amplification plots to determine the number of inconclusive samples. We also reviewed the medical records to summarize the patient's COVID-19 testing history and basic demographic characteristics. We obtained 136 (0.8%) inconclusive samples with amplification signal only for the N-gene. Thirty-nine of the samples were excluded from further analysis as no additional data were available for them. Of the rest of the samples, the majority- 48% (95% CI 38-59%) had a previous history of SARS-CoV-2 positivity, 14% (95% CI 8-23%)-a subsequent history of positivity and 37% (95% CI 28-48%) were considered as false positive. A substantial proportion of the inconclusive results should be considered as positive samples at the beginning or the end of the infection. However, the number of false-positive results is also significant and each patient's result should be analyzed separately following the clinical symptoms and epidemiological data.