Clinical validation of automated and rapid mariPOC SARS-CoV-2 antigen test.

COVID-19 diagnostics was quickly ramped up worldwide early 2020 based on the detection of viral RNA. However, based on the scientific knowledge for pre-existing coronaviruses, it was expected that the SARS-CoV-2 RNA will be detected from symptomatic and at significant rates also from asymptomatic individuals due to persistence of non-infectious RNA. To increase the efficacy of diagnostics, surveillance, screening and pandemic control, rapid methods, such as antigen tests, are needed for decentralized testing and to assess infectiousness. A novel automated mariPOC SARS-CoV-2 test was developed for the detection of conserved structural viral nucleocapsid proteins. The test utilizes sophisticated optical laser technology for two-photon excitation and individual detection of immunoassay solid-phase particles. We validated the new method against qRT-PCR. Sensitivity of the test was 100.0% (13/13) directly from nasopharyngeal swab specimens and 84.4% (38/45) from swab specimens in undefined transport mediums. Specificity of the test was 100.0% (201/201). The test's limit of detection was 2.7 TCID50/test. It showed no cross-reactions. Our study shows that the new test can detect infectious individuals already in 20 min with clinical sensitivity close to qRT-PCR. The mariPOC is a versatile platform for syndromic testing and for high capacity infection control screening of infectious individuals.

Import and transmission of Mycobacterium orygis and Mycobacterium africanum, Norway.

BACKGROUND: The aim of the current study was to improve our understanding of the origins and transmission of Mycobacterium africanum (MAF) in Norway. METHODS: Whole-genome sequences (WGS) were generated for all (n = 29) available clinical isolates received at the Norwegian National Reference Laboratory for Mycobacteria (NRL) and identified as MAF in Norway, in the period 2010-2020. Phylogenetic analyses were performed. RESULTS: The analyses indicated several imports of MAF lineage 6 from both East and West African countries, whereas MAF lineage 5 was restricted to patients with West African connections. We also find evidence for transmission of MAF in Norway. Finally, our analyses revealed that a group of isolates from patients originating in South Asia, identified as MAF by means of a commercial line-probe assay, in fact belonged to Mycobacterium orygis. CONCLUSIONS: Most MAF cases in Norway are the result of import, but transmission is occurring within Norway.

Clinical Validation of Automated and Rapid mariPOC SARS-CoV-2 Antigen Test

Novel SARS coronavirus causing COVID-19 was recognized in late 2019. Diagnostics was quickly ramped up worldwide based on the detection of viral RNA. Based on the scientific knowledge for pre-existing coronaviruses, it was expected that the RNA of this novel coronavirus will be detected from symptomatic and at significant rates also from asymptomatic individuals due to persistence of non-infectious RNA. To increase the efficacy of diagnostics, surveillance, screening and pandemic control, rapid methods, such as antigen tests, are needed for decentralized testing and to assess infectiousness. The objective was to validate the analytical and clinical performance, and usability of a novel automated mariPOC SARS-CoV-2 test, which is based on the detection of structural viral proteins using sophisticated optical laser technology. Clinical performance of the test was evaluated against qRT-PCR with nasopharyngeal swab specimens collected from patients suspected of acute SARS-CoV-2 infection. Sensitivity of the mariPOC test was 100.0% (13/13) directly from swab specimens and 84.4% (38/45) from swab specimens in undefined transport mediums. Specificity of the test was 100.0% (201/201). The tests limit of detection was 2.7 TCID50/test and had no cross-reactions with the tested respiratory microbes. Our study shows that the mariPOC can detect infectious individuals already in 20 minutes with clinical sensitivity close to qRT-PCR. The test targets conserved epitopes of SARS-CoV-2 nucleoprotein, making it robust against strain variations. The new test is a promising and versatile tool for syndromic testing of symptomatic cases and for high capacity infection control screening.

Prospective Evaluation of the mariPOC Test for Detection of Clostridioides difficile Glutamate Dehydrogenase and Toxins A/B.

The objective of this study was to evaluate a novel automated random-access test, mariPOC CDI (ArcDia Ltd., Finland), for the detection of Clostridioides difficile glutamate dehydrogenase (GDH) and toxins A and B directly from fecal specimens. The mariPOC test was compared with both the GenomEra C. difficile PCR assay (Abacus Diagnostica Oy, Finland) and the TechLab C. diff Quik Chek Complete (Alere Inc.; now Abbot) membrane enzyme immunoassay (MEIA). Culture and the Xpert C. difficile assay (Cepheid Inc., USA) were used to resolve discrepant results. In total, 337 specimens were tested with the mariPOC CDI test and GenomEra PCR. Of these specimens, 157 were also tested with the TechLab MEIA. The sensitivity of the mariPOC test for GDH was slightly lower (95.2%) than that obtained with the TechLab assay (100.0%), but no toxin-positive cases were missed. The sensitivity of the mariPOC test for the detection of toxigenic C. difficile by analyzing toxin expression was better (81.6%) than that of the TechLab assay (71.1%). The analytical specificities for the mariPOC and the TechLab tests were 98.3% and 100.0% for GDH and 100.0% and 99.2% for toxin A/B, respectively. The analytical specificity of the GenomEra method was 100.0%. The mariPOC and TechLab GDH tests and GenomEra PCR had high negative predictive values of 99.3%, 98.3%, and 99.7%, respectively, in excluding infection with toxigenic C. difficile The mariPOC toxin A/B test and GenomEra PCR had an identical analytical positive predictive value of 100%, providing highly reliable information about toxin expression and the presence of toxin genes, respectively.

Comparison of phenotypic and genotypic diagnosis of acute human bocavirus 1 infection in children.

BACKGROUND: Diagnosis of human bocavirus 1 (HBoV1) has been based on qualitative PCRs detecting HBoV1 DNA or detection of HBoV1 mRNA. OBJECTIVE: This study aims to assess whether a rapid and automated HBoV1 antigen test is suitable for diagnosis of acute HBoV1 infection. STUDY DESIGN: HBoV1 antigen detection has been compared with quantitative HBoV1 DNA PCR and HBoV1 mRNA RT-PCR. RESULTS AND CONCLUSION: We conclude that HBoV1 antigen detection has higher clinical specificity and positive predictive value than HBoV1 DNA qualitative PCRs, yet a lower sensitivity than HBoV1 mRNA detection. Additionally, HBoV1 antigen detection is beneficial in its rapidity and availability as a point-of-care test.

Detection of human rhinoviruses by reverse transcription strand invasion based amplification method (RT-SIBA).

BACKGROUND: Rhinovirus (RV), a major cause of respiratory infection in humans, imposes an enormous economic burden due to the direct and indirect costs associated with the illness. Accurate and timely diagnosis is crucial for deciding the appropriate clinical approach and minimizing unnecessary prescription of antibiotics. Diagnosis of RV is extremely challenging due to genetic and serological variability among its numerous types and their similarity to enteroviruses. OBJECTIVE: We sought to develop a rapid nucleic acid test that can be used for the detection of Rhinovirus within both laboratory and near patient settings. STUDY DESIGN: We developed and evaluated a novel isothermal nucleic acid amplification method called Reverse Transcription Strand Invasion-Based Amplification (RT-SIBA) to rapidly detect Rhinovirus from clinical specimens. RESULT: The method, RT-SIBA, detected RV in clinical specimens with high analytical sensitivity (96%) and specificity (100%). The time to positive result was significantly shorter for the RV RT-SIBA assay than for a reference RV nucleic acid amplification method (RT-qPCR). CONCLUSION: The rapid detection time of the RV SIBA assay, as well as its compatibility with portable instruments, will facilitate prompt diagnosis of infection and thereby improve patient care.

Genome-based transmission modelling separates imported tuberculosis from recent transmission within an immigrant population.

In many countries the incidence of tuberculosis (TB) is low and is largely shaped by immigrant populations from high-burden countries. This is the case in Norway, where more than 80 % of TB cases are found among immigrants from high-incidence countries. A variable latent period, low rates of evolution and structured social networks make separating import from within-border transmission a major conundrum to TB control efforts in many low-incidence countries. Clinical Mycobacterium tuberculosis isolates belonging to an unusually large genotype cluster associated with people born in the Horn of Africa have been identified in Norway over the last two decades. We modelled transmission based on whole-genome sequence data to estimate infection times for individual patients. By contrasting these estimates with time of arrival in Norway, we estimate on a case-by-case basis whether patients were likely to have been infected before or after arrival. Independent import was responsible for the majority of cases, but we estimate that about one-quarter of the patients had contracted TB in Norway. This study illuminates the transmission dynamics within an immigrant community. Our approach is broadly applicable to many settings where TB control programmes can benefit from understanding when and where patients acquired TB.

Microbial identification from faces and urine in one step by two-photon excitation assay technique.

Two-photon excitation fluorometry (TPX) is a separation-free bioaffinity assay technique which enables accurate diagnostic testing in microvolumes. The technology is currently commercially applied in an automated mariPOC® test system for rapid phenotypic multi-microbe detection of pathogen antigens. The first TPX applications for diagnostics were intended for respiratory infection testing from nasopharyngeal and oropharyngeal samples. Feces and urine are more complex sample matrices and contain substances that may interfere with immunoassay binding or fluorescence detection. Our objective was to study the suitability of these complex matrices in the TPX technique. As expected, feces and urine elevated fluorescence levels but the methodology has the unique property of compensating for matrix effects. Compensation allows reliable separation of specific fluorescence from the fluorescence caused by the matrix. The studied clinical samples did not contain immunoassay inhibitors. The results suggest that the methodology is robust and may provide reliable testing of feces and urine samples with high accuracy.

Tuberculosis Outbreak in an Educational Institution in Norway.

Within 1 week in April 2013, three cases of pulmonary tuberculosis (TB) were reported among students attending training sessions at an educational institution in Oslo, Norway. By the end of October 2013, a total of nine epidemiologically linked cases had been reported. The outbreak encompassed a total of 24 cases from 2009 to 2014, among which all of the 22 Mycobacterium tuberculosis isolates available had identical mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) profiles (MtbC15-9 code 10287-189) belonging to the Beijing lineage. Whole-genome sequencing (WGS) of the M. tuberculosis isolates revealed 20 variable nucleotide positions within the cluster, indicating a clonal outbreak. The most likely index case was identified and diagnosed in Norway in 2009 but was born in Asia. WGS analyses verified that all of the cases were indeed part of a single transmission chain. However, even when combining WGS and intensified contact tracing, we were unable to fully reconstruct the TB transmission events.

Armed conflict and population displacement as drivers of the evolution and dispersal of Mycobacterium tuberculosis.

The "Beijing" Mycobacterium tuberculosis (Mtb) lineage 2 (L2) is spreading globally and has been associated with accelerated disease progression and increased antibiotic resistance. Here we performed a phylodynamic reconstruction of one of the L2 sublineages, the central Asian clade (CAC), which has recently spread to western Europe. We find that recent historical events have contributed to the evolution and dispersal of the CAC. Our timing estimates indicate that the clade was likely introduced to Afghanistan during the 1979-1989 Soviet-Afghan war and spread further after population displacement in the wake of the American invasion in 2001. We also find that drug resistance mutations accumulated on a massive scale in Mtb isolates from former Soviet republics after the fall of the Soviet Union, a pattern that was not observed in CAC isolates from Afghanistan. Our results underscore the detrimental effects of political instability and population displacement on tuberculosis control and demonstrate the power of phylodynamic methods in exploring bacterial evolution in space and time.

Detection of Group A Streptococcus from Pharyngeal Swab Samples by Bacterial Culture Is Challenged by a Novel mariPOC Point-of-Care Test.

mariPOC is a novel point-of-care test system for rapid detection of respiratory tract infections. We compared the performance of the mariPOC test to that of bacterial culture for detecting group A streptococcus (GAS) in 219 pharyngitis patients (ages 1-64 years) and 109 healthy asymptomatic controls (ages 19-69 years). In addition, 42 patient samples were analyzed by quantitative PCR (qPCR). Of the 219 pharyngeal patient samples, 32 were positive in a GAS bacterial culture (prevalence 15%) and 65 (30%) in the mariPOC test. The amount of GAS in samples reported positive by the mariPOC test and negative by culture was, on average, 10-fold less than that of those positive in both methods. This indicated that the negative results in bacterial cultures were due to lower sensitivity. The qPCR results were positive and in line with the mariPOC results in 43% of the discordant samples studied. Two GAS culture-positive samples were negative by the mariPOC test. The prevalences of GAS in the control subjects were 2% and 6% by culture and mariPOC results, respectively. We conclude that the mariPOC antigen detection test is more sensitive than the conventional bacterial culture for the detection of GAS among symptomatic pharyngitis patients. The higher prevalence of GAS by the mariPOC test among symptomatic patients was probably not due to carriership, since among the control patients, the difference in the prevalence of GAS by the mariPOC test and culture was not nearly as high, 15% versus 4%, respectively. Clinical trials are needed to show the clinical importance of our findings.

Elevated concentration of C-reactive protein is associated with pregnancy-related co-morbidities but not with relapse activity in multiple sclerosis.

During pregnancy, alterations take place in mother's immune system with the goal of maintaining a successful pregnancy, and delivering healthy offspring. Immune alterations include activation of the innate immune system and dampening of cell-mediated adaptive immunity. Due to these alterations, cell-mediated autoimmune diseases typically ameliorate during pregnancy. The objectives of this study were to evaluate whether C-reactive protein (CRP) concentration, a sensitive marker of systemic inflammation (1) is increased during MS pregnancy (2) predicts pregnancy-related co-morbidities associated with MS (3) predicts MS disease activity after delivery. CRP concentration was measured using a high sensitivity assay from seven prospectively collected serum samples of 41 MS patients and 19 controls during pregnancy and 6 months after delivery. Annualized relapse rates, EDSS, fatigue scores and obstetric details of the patients were recorded. Delivery-related CRP levels were significantly elevated both among MS patients and in controls. CRP levels were higher during pregnancy than during the postpartum period in both study groups. Delivery-related elevated CRP levels did not correlate with postpartum disease activity. MS patients with eventual gestational diabetes had a significantly higher median CRP in the beginning of pregnancy compared to non-diabetic MS patients (9.28 vs. 2.98 mg/l, p = 0.0025). MS patients reporting fatigue had a significantly higher CRP throughout pregnancy compared to patients without fatigue. Higher CRP values were associated with pregnancy-related co-morbidities but not with MS disease activity.

Evaluation of the automated multianalyte point-of-care mariPOC® test for the detection of influenza A virus and respiratory syncytial virus.

The aim of the study was to evaluate the performance of the new mariPOC(®) method against the direct fluorescent antibody assay (DFA) as the primary reference method for rapid virus detection from nasopharyngeal aspirates and swab samples. The study was an open prospective evaluation during the seasonal winter epidemics in the Mikkeli Central Hospital, Finland. Altogether, 283 samples were analyzed; 124 (43.8%) were from young children (<5 years old). Discrepant samples were resolved by PCR. With nasopharyngeal aspirate samples, the sensitivity and clinical specificity of the mariPOC(®) assay for influenza A virus and respiratory syncytial virus, were 85.7% (CI 69.7-95.2) and 90% (CI 52.0-80.5), and 100% and 99.5%, respectively. The mariPOC(®) performed less well with swab samples having sensitivities at 77.3% (CI 54.6-92.2) and 67.4% (CI 52-80.5), respectively. The specificities were as for nasopharyngeal aspirates. Importantly, similar performance was observed regardless of the cohort age group. In conclusion, the mariPOC(®) test system has a high potential and utility in duty units because it is fast, simple, and multianalyte. The importance of personnel training for proper sample collection should be emphasized.

Androgen-regulated miR-32 targets BTG2 and is overexpressed in castration-resistant prostate cancer.

The androgen receptor (AR) signaling pathway is involved in the emergence of castration-resistant prostate cancer (CRPC). Here, we identified several androgen-regulated microRNAs (miRNAs) that may contribute to the development of CRPC. Seven miRNAs, miR-21, miR-32, miR-99a, miR-99b, miR-148a, miR-221 and miR-590-5p, were found to be differentially expressed in CRPC compared with benign prostate hyperplasia (BPH) according to microarray analyses. Significant growth advantage for LNCaP cells transfected with pre-miR-32 and pre-miR-148a was found. miR-32 was demonstrated to reduce apoptosis, whereas miR-148a enhanced proliferation. Androgen regulation of miR-32 and miR-148a was confirmed by androgen stimulation of the LNCaP cells followed by expression analyses. The AR-binding sites in proximity of these miRNAs were demonstrated with chromatin immunoprecipitation (ChIP). To identify target genes for the miRNAs, mRNA microarray analyses were performed with LNCaP cells transfected with pre-miR-32 and pre-miR-148a. Expression of BTG2 and PIK3IP1 was reduced in the cells transfected with pre-miR-32 and pre-miR-148a, respectively. Also, the protein expression was reduced according to western blot analysis. BTG2 and PIK3IP1 were confirmed to be targets by 3'UTR-luciferase assays. Finally, immunostainings showed a statistically significant (P<0.0001) reduction of BTG2 protein in CRPCs compared with untreated prostate cancer (PC). The lack of BTG2 staining was also associated (P<0.01) with a short progression-free time in patients who underwent prostatectomy. In conclusion, androgen-regulated miR-32 is overexpressed in CRPC, leading to reduced expression of BTG2. Thus, miR-32 is a potential marker for aggressive disease and is a putative drug target in PC.

Overexpression of androgen receptor enhances the binding of the receptor to the chromatin in prostate cancer.

Androgen receptor (AR) is overexpressed in the majority of castration-resistant prostate cancers (CRPCs). Our goal was to study the effect of AR overexpression on the chromatin binding of the receptor and to identify AR target genes that may be important in the emergence of CRPC. We have established two sublines of LNCaP prostate cancer (PC) cell line, one overexpressing AR 2-3-fold and the other 4-5-fold compared with the control cells. We used chromatin immunoprecipitation (ChIP) and deep-sequencing (seq) to identify AR-binding sites (ARBSs). We found that the number of ARBSs and the AR-binding strength were positively associated with the level of AR when cells were stimulated with low concentrations of androgens. In cells overexpressing AR, the chromatin binding of the receptor took place in 100-fold lower concentration of the ligand than in control cells. We confirmed the association of AR level and chromatin binding in two PC xenografts, one containing AR gene amplification with high AR expression, and the other with low expression. By combining the ChIP-seq and expression profiling, we identified AR target genes that are upregulated in PC. Of them, the expression of ZWINT, SKP2 (S-phase kinase-associated protein 2 (p45)) and FEN1 (flap structure-specific endonuclease 1) was demonstrated to be increased in CRPC, while the expression of SNAI2 was decreased in both PC and CRPC. FEN1 protein expression was also associated with poor prognosis in prostatectomy-treated patients. Finally, the knock-down of FEN1 with small interfering RNA inhibited the growth of LNCaP cells. Our data demonstrate that the overexpression of AR sensitizes the receptor binding to chromatin, thus, explaining how AR signaling pathway is reactivated in CRPC cells.

A novel antibody avidity methodology for rapid point-of-care serological diagnosis.

A novel methodology is introduced for rapid serological diagnosis. This methodology combines the antibody bridging assay principle with the measurement of antibody avidity. The combination allows the determination of the infection phase with a single dilution of a single sample of serum. This is a significant improvement on current serological techniques which often require either paired-sample testing (IgG/IgM serology) or testing of the sample in several dilutions (IgG avidity testing). Assay methods were developed on two immunoassay platforms; the heterogeneous time-resolved fluoroimmunoassay and the separation-free two-photon excitation fluorometry. The new methods were compared to conventional class-specific IgG/IgM and IgG avidity techniques. The major findings were that the avidity results of the new methodology were independent of the sample dilution (specific antibody concentration in serum) and consistent between immunoassay platforms. This new methodology is simple, rapid, and quick to perform. It provides the possibility of running serodiagnostic tests at point-of-care with bench-top random-access analyzers.

Ospemifene effectively treats vulvovaginal atrophy in postmenopausal women: results from a pivotal phase 3 study.

OBJECTIVE: The aim of this study was to study the efficacy and safety of ospemifene, a new selective estrogen receptor modulator, in the treatment of vulvovaginal atrophy in postmenopausal women. METHODS: A randomized, double-blind phase 3 study in which 826 postmenopausal women were randomized 1:1:1 to receive treatment with ospemifene 30 or 60 mg/day or placebo orally for 12 weeks was conducted. The primary inclusion criteria were having 5% or less superficial cells on the vaginal smear (maturation index), vaginal pH greater than 5.0, and at least one moderate or severe symptom of vulvovaginal atrophy. The four coprimary endpoints were the change from baseline to 12 weeks in the percentage of superficial and parabasal cells on the vaginal smear, change in vaginal pH, and change in severity of most bothersome symptom (vaginal dryness or dyspareunia) compared with placebo. All participants were given a nonhormonal vaginal lubricant for use as needed. RESULTS: Ospemifene was statistically significantly superior to placebo in each of the coprimary endpoints at the 60-mg dose. Statistically significant results were achieved for all coprimary endpoints with the 30-mg dose except for dyspareunia. Ospemifene was well tolerated at both doses and demonstrated a favorable safety profile. CONCLUSIONS: Ospemifene was shown to be effective and well tolerated for the treatment of the symptoms of vaginal dryness and dyspareunia associated with vulvovaginal atrophy over and above the use of provided lubricants.

Serum glucocorticoids and adiponectin associate with insulin resistance in children born small for gestational age.

OBJECTIVES: Altered glucocorticoid activity is one possible mechanism linking fetal growth restriction with later insulin resistance (IR) and type 2 diabetes. We aimed to investigate whether serum glucocorticoid parameters are related to IR in children born small for gestational age (SGA). DESIGN: A total of 110 children (55 age- and gender-matched pairs born SGA or appropriate for gestational age (AGA) in a case-control setting) were studied at the mean age of 12.2 (s.d. 0.2) years. METHODS: Serum cortisol, corticosteroid-binding globulin (CBG), free cortisol index (FCI=cortisol/CBG), and glucocorticoid bioactivity (GBA, transactivation assay) were analyzed and related to serum adiponectin and insulin-like growth factor-binding protein 1 (IGFBP1) concentrations and homeostasis model assessment for IR (HOMA-IR) and QUICKI indices. RESULTS: In the pooled study population, GBA correlated well with cortisol and FCI (r=0.681 and 0.586 respectively; P<0.001 for both). Serum cortisol, CBG, FCI, GBA, HOMA-IR, or QUICKI did not differ between the SGA and AGA subjects, but the SGA children had lower body mass index (P=0.005) and waist circumference (WC) (P=0.001). The mean GBA in the highest GBA quartile was higher among the SGA subjects than among the AGA subjects (138.6 vs 96.4 nmol/l cortisol equivalents, P<0.001). In the SGA children, GBA correlated positively with HOMA-IR (r=0.522, P<0.001) and inversely with adiponectin (r=-0.278, P=0.042) (WC/height ratio adjustments), and in logistic regression analysis, higher GBA (odds ratio (OR) 1.3; P=0.013), lower adiponectin (OR 1.4; P=0.038), and lower IGFBP1 (OR 1.9; P=0.010) associated independently with higher HOMA-IR. CONCLUSIONS: These findings suggest that increased glucocorticoid activity and low serum adiponectin concentrations associate with IR in SGA children.

Development of a rapid assay methodology for antimicrobial susceptibility testing of Staphylococcus aureus.

Development of a new phenotypic technique for rapid antimicrobial susceptibility testing (AST) of methicillin-resistant Staphylococcus aureus is presented. The new technique combines bacterial culturing and specific immunometric detection in a single separation-free process. The technique uses dry chemistry reagents and the recently developed two-photon excitation detection technology, which allows online detection of bacterium-specific growth. The performance of the new technique was evaluated by monitoring the growth of S. aureus reference strains and determining their susceptibility to oxacillin. In the direct analysis of clinical specimens, method specificity and tolerance to interferences caused by other bacteria present in the sample are pivotal. Other bacteria can compete with the bacteria of interest for nutrients, for example. Specificity and tolerance were studied against Staphylococcus epidermidis reference strains. The results suggest that the new technique could allow rapid AST directly from clinical samples within 6 to 8 h. Such a rapid and simple testing methodology would be a valuable tool in clinical microbiology because it would shorten the turnaround times of microbiologic analyses. Advantages of the new approach in relation to conventional methods are discussed.