Multiplex RT-PCR in the diagnosis of human picornaviruses and human respiratory viruses

The family Picornaviridae includes many human pathogens. Human enteroviruses (HEVs) exhibit a variety of clinical manifestations ranging from poliomyelitis and encephalomyelitis to respiratory infections and rashes. Human rhinoviruses (HRVs) are the major causes of the common cold. Human parechoviruses (HPeVs) and Aichi virus (AV) are mostly detected in cases of gastroenteritis, and hepatitis A virus (HAV) causes hepatitis with favourable prognosis. In addition to HEVs and HRVs, a large number of viruses are recognized as respiratory pathogens. The conventional respiratory pathogens include influenza A and B viruses, human respiratory syncytial virus (RSV), adenoviruses (AdVs), parainfluenza viruses (PIVs) and the human coronaviruses (hCoVs) OC43 and 229E. Moreover, several new respiratory pathogens, such as human metapneumovirus (hMPV), severe acute respiratory syndrome coronavirus (SARS-CoV), and the hCoVs HKU1 and NL63 have been found during the 2000s. Human bocavirus (hBoV) is also increasingly being recognized as a true pathogen of humans. Since many clinical illnesses may be caused by several different viruses, multiplex assays for simultaneous detection of several viruses are increasingly being applied. Real-time multiplex polymerase chain reaction (PCR) assays for detection of viral nucleic acids offer remarkable benefits, such as short turnaround time and the non-necessity for handling amplified products. Since multiplexing, utilizing real-time PCR, is limited by reduction in amplification efficiency due to multiple primer and probe sets, separate amplification and hybridization reactions have re-emerged in attempts to develop tests with broad diagnostic range. With this approach microarrays, which have the potential for resolving complex mixtures of amplification products, may be applied. In this study, a multiplex reverse transcription-PCR (RT-PCR) and liquid hybridization assay for sensitive detection of HEV, HRV, HPeV and AV were developed and a single RT-PCR and liquid hybridization assay for detection of HAV was optimized. In analysis of clinical samples, the results obtained by the multiplex assay were consistent with those obtained by routine diagnostic assays. When 68 stool samples were analysed for the presence of HPeV and AV, one sample positive for HPeV was detected. This finding is in line with the current knowledge of neither of these viruses being very common enteric pathogens. More rapid detection of HEV and HRV in respiratory samples was achieved when a real-time duplex RT-PCR assay for detection of these viruses was developed. The same approach was used to develop another assay for more sensitive detection of RSV than with the direct fluorescent assay (DFA) and simultaneous identification of hMPV. Both multiplex real-time RT-PCR assays provided reliable and sensitive detection of their targets, except for detection of HRV, since doubts were raised on the ability of the assay to detect all rhinoviruses. Moreover, two commercial hMPV antibodies were found applicable for detection of the virus in respiratory samples by DFA. Results from analysis of respiratory samples using the duplex real-time RT-PCR assays were compared with those obtained with DFA and the Respiratory Viral Panel (RVP) Fast test, a bead-based suspension microarray test evaluated for routine diagnosis. The RVP Fast assay and PCR showed similar detection rates, except for HEV/HRV, for which a higher detection rate by RVP was observed. All PCR-based assays presented more findings of their target viruses than DFA. The broad detection range of the RVP Fast assay resulted in a nearly threefold overall detection rate, compared with that by DFA. Moreover, analysis of clinical samples resulted in a notable prevalence of hMPV and non-SARS-hCoVs, which emphasizes the role of these viruses as respiratory pathogens. Although the RVP Fast assay demonstrated adequate overall performance, doubts were raised on the ability of the test to detect the H1N1 2009 influenza A virus and all AdV serotypes. Evaluation of the RVP Fast assay demonstrated the remarkable increase in overall viral detection rate that results from adapting a PCR-based multiplex assay to virus diagnostics. The sensitive detection of all the viruses of clinical relevance facilitates efficient infection control measures and appropriate patient management and enables systematic studies on the clinical importance of coinfections. Moreover, collection of data on occurrence of all the viruses of clinical relevance will enable a better understanding of the seasonality, geographical distribution and risk groups of the viral pathogens.Picornavirusten suku käsittää monia ihmiselle patogeeneja viruksia. Ihmisen enterovirukset (HEV) aiheuttavat lukuisia tautitiloja, kuten poliomyeliittiä, enkefalomyeliittiä, hengitystieinfektioita ja ihottumia. Ihmisen rinovirukset (HRV) ovat yleisin flunssan aiheuttaja. Ihmisen parechovirusta (HPeV) ja Aichi virusta (AV) tavataan lähinnä gastroenteriiteissa ja hepatiitti A virus (HAV) aiheuttaa hyväennusteista hepatiittia. Entero- ja rinovirusten lisäksi suuri joukko viruksia aiheuttaa hengitystieinfektioita. Perinteisiä hengitystieviruksia ovat influenssavirukset, respiratory syncytial virus (RSV), adenovirus (AdV), parainfluenssavirukset (PIV) ja ihmisen koronavirukset (hCoV) OC43 ja 229E. Lisäksi 2000-luvulla on löydetty joukko uusia hengitystiepatogeeneja viruksia, kuten ihmisen metapneumovirus (hMPV), severe acute respiratory syndrome -koronavirus (SARS-CoV), ja ihmisen koronavirukset (hCoV) HKU1 ja NL63. Myös ihmisen bokaviruksen (hBoV) rooli todellisena hengitystiepatogeenina on saanut vahvistusta. Koska monet tautitilat voivat olla usean eri viruksen aiheuttamia, on usean viruksen nukleiinihappojen osoittaminen näytteestä samanaikaisesti multiplex testien avulla yleistynyt. Virusten nukleiinihappojen osoittaminen reaaliaikaisilla multiplex polymeraasiketjureaktioon (PCR) perustuvilla testeillä mahdollistaa testitulosten nopean saatavuuden ilman monistustuotteiden käsittelyä. Koska monistustehokkuuden aleneminen käytettäessä useita alukepareja ja koettimia rajoittaa reaaliaikaisen PCR:n käyttöä usean viruksen osoittamiseen, on erillisen monistus- ja hybridisaatioreaktion käyttö uudelleen yleistynyt laajennettaessa testien kattavuutta. Käytettäessä tällaista lähestymistapaa voidaan monistustuotteiden spesifiin osoittamiseen hyödyntää mikrosirutestejä, joilla on suuri kapasiteetti eri monistustuotteiden spesifiin osoittamiseen PCR-reaktioseoksesta. Tässä tutkimuksessa kehitettiin multiplex RT-PCR -monistukseen ja liuoshybridisaatioon perustuva testi HEV, HRV, HPeV ja AV osoittamiseksi. Lisäksi optimoitiin vastaava erillinen testi HAV osoittamiseksi. Ulostenäytteiden analyysissä multiplex RT-PCR testillä löydettiin yksi HPeV-positiivinen näyte. Tulos vastaa hyvin nykykäsitystä, jonka mukaan HPeV ja AV ovat suhteellisen harvinaisia gastroenteriitin aiheuttajia. Tutkimuksessa kehitettiin edelleen reaaliaikainen multiplex RT-PCR -testi, joka mahdollisti entero- ja rinovirusten nopeamman osoittamisen. RSV-osoituksen herkkyyden lisäämiseksi ja hMPV:n samanaikaiseksi osoittamiseksi optimoitiin reaaliaikainen multiplex RT-PCR -testi näiden virusten osoittamiseen. Molemmat reaaliaikaiset RT-PCR -testit olivat luotettavia ja herkkiä kohdevirustensa osoittamisessa, paitsi HRV-osoituksessa, missä heräsi epäilys testin kyvystä osoittaa kaikkia rinoviruksia. Lisäksi kahden kaupallisen hMPV-vasta-aineen todettiin soveltuvan suoraan virusantigeeniosoitukseen hengitystienäytteistä. Hengitystienäytteiden tutkimisesta multiplex RT-PCR testeillä saatuja tuloksia verrattiin virusantigeeniosoituksen tuloksiin ja evaluoitiin RVP Fast liuosmikrosirutesti. Positiivisten löydösten prevalenssi RVP Fast testillä ja reaaliaikaisilla RT-PCR testeillä oli samansuuruinen, paitsi HEV/HRV löydösten määrä, joka oli RVP:lla oli suurempi. Kaikki PCR-perusteiset testit osoittivat virusantigeeniosoitusta enemmän kohdeviruksia. RVP Fast testin suuri kohdevirusten määrä johti lähes kolminkertaiseen detektioprevalenssiin virusantigeeniosoitukseen verrattuna. Potilasnäytteistä merkittävä osa oli positiivisia hMPV:n ja non-SARS-koronavirusten suhteen, mikä korostaa näiden virusten merkitystä hengitystieinfektioiden aiheuttajina. RVP Fast testin toiminta osoittautui yleisesti ottaen luotettavaksi, mutta epäilys heräsi testin kyvystä osoittaa influenssa A viruksen H1N1 (2009) tyyppiä sekä kaikkia adenovirusserotyyppejä. RVP Fast testin evaluaatio osoitti, että PCR-perustaisen multiplex testin käyttöönotto johtaa positiivisten löydösten määrän merkittävään lisääntymiseen virusantigeeniosoitukseen verrattuna. Kaikkien kliinisesti merkittävien virusten osoittaminen mahdollistaa tehokkaan infektiotautien torjunnan ja asianmukaisen hoidon sekä luo puitteet koinfektioiden kliinisen merkityksen systemaattiselle tutkimiselle. Lisäksi kaikkien kliinisesti merkittävien virusten löydösten huomioiminen johtaa parempaan käsitykseen näiden patogeenien kausivaihtelusta, maantieteellisestä esiintyvyydestä ja riskiryhmistä

Analysis of Biologics Molecular Descriptors towards Predictive Modelling for Protein Drug Development Using Time-Gated Raman Spectroscopy

Pharmaceutical proteins, compared to small molecular weight drugs, are relatively fragile molecules, thus necessitating monitoring protein unfolding and aggregation during production and post-marketing. Currently, many analytical techniques take offline measurements, which cannot directly assess protein folding during production and unfolding during processing and storage. In addition, several orthogonal techniques are needed during production and market surveillance. In this study, we introduce the use of time-gated Raman spectroscopy to identify molecular descriptors of protein unfolding. Raman spectroscopy can measure the unfolding of proteins in-line and in real-time without labels. Using K-means clustering and PCA analysis, we could correlate local unfolding events with traditional analytical methods. This is the first step toward predictive modeling of unfolding events of proteins during production and storage

The Finnish New Variant of Chlamydia trachomatis with a Single Nucleotide Polymorphism in the 23S rRNA Target Escapes Detection by the Aptima Combo 2 Test

In 2019, more than 200 cases of Chlamydia trachomatis negative/equivocal by the Aptima Combo 2 assay (AC2, target: 23S rRNA) with slightly elevated relative light units (RLUs), but positive by the Aptima Chlamydia trachomatis assay (ACT, target: 16S rRNA) have been detected in Finland To identify the cause of the AC2 CT false-negative specimens, we sequenced parts of the CT 23S rRNA gene in 40 specimens that were AC2 negative/equivocal but ACT positive. A single nucleotide polymorphism (SNP; C1515T in the C. trachomatis 23S rRNA gene) was revealed in 39 AC2/ACT discordant specimens. No decrease in the number of mandatorily notified C. trachomatis cases was observed nationally in Finland in 2010–2019. When RLUs obtained for AC2 negative specimens were retrospectively evaluated in 2011–2019, a continuous increase in the proportion of samples with RLUs 10–19 was observed since 2014, and a slight increase in the proportion of samples with RLUs 20–84 in 2017–2019, indicating that the Finnish new variant of C. trachomatis might have been spreading nationally for several years. This emphasizes that careful surveillance of epidemiology, positivity rate and test performance are mandatory to detect any changes affecting detection of infections

The Finnish New Variant of Chlamydia trachomatis with a Single Nucleotide Polymorphism in the 23S rRNA Target Escapes Detection by the Aptima Combo 2 Test

In 2019, more than 200 cases of Chlamydia trachomatis negative/equivocal by the Aptima Combo 2 assay (AC2, target: 23S rRNA) with slightly elevated relative light units (RLUs), but positive by the Aptima Chlamydia trachomatis assay (ACT, target: 16S rRNA) have been detected in Finland To identify the cause of the AC2 CT false-negative specimens, we sequenced parts of the CT 23S rRNA gene in 40 specimens that were AC2 negative/equivocal but ACT positive. A single nucleotide polymorphism (SNP; C1515T in the C. trachomatis 23S rRNA gene) was revealed in 39 AC2/ACT discordant specimens. No decrease in the number of mandatorily notified C. trachomatis cases was observed nationally in Finland in 2010–2019. When RLUs obtained for AC2 negative specimens were retrospectively evaluated in 2011–2019, a continuous increase in the proportion of samples with RLUs 10–19 was observed since 2014, and a slight increase in the proportion of samples with RLUs 20–84 in 2017–2019, indicating that the Finnish new variant of C. trachomatis might have been spreading nationally for several years. This emphasizes that careful surveillance of epidemiology, positivity rate and test performance are mandatory to detect any changes affecting detection of infections

Single-Cell Proteomics Reveals the Defined Heterogeneity of Resident Macrophages in White Adipose Tissue

Adipose tissue macrophages (ATMs) regulate homeostasis and contribute to the metabolically harmful chronic inflammation in obese individuals. While evident heterogeneity of resident ATMs has been described previously, their phenotype, developmental origin, and functionality remain inconsistent. We analyzed white adipose tissue (WAT) during homeostasis and diet interventions using comprehensive and unbiased single-cell mass cytometry and genetic lineage tracking models. We now provide a uniform definition of individual subsets of resident ATMs. We show that in lean mice, WAT co-harbors eight kinetically evolving CD206+ macrophage subpopulations (defined by TIM4, CD163, and MHC II) and two CD206- macrophage subpopulations. TIM4-CD163+, TIM4-CD163- and CD206- macrophage populations are largely bone marrow-derived, while the proliferating TIM4+CD163+ subpopulation is of embryonic origin. All macrophage subtypes are active in phagocytosis, endocytosis, and antigen processing in vitro, whereas TIM4+CD163+ cells are superior in scavenging in vivo. A high-fat diet induces massive infiltration of CD206- macrophages and selective down-regulation of MHC II on TIM4+ macrophages. These changes are reversed by dietary intervention. Thus, the developmental origin and environment jointly regulate the functional malleability of resident ATMs.</p

Fetal-derived macrophages persist and sequentially maturate in ovaries after birth in mice

Macrophages, which are highly diverse in different tissues, play a complex and vital role in tissue development, homeostasis, and inflammation. The origin and heterogeneity of tissue-resident monocytes and macrophages in ovaries remains unknown. Here we identify three tissue-resident monocyte populations and five macrophage populations in the adult ovaries using high-dimensional single cell mass cytometry. Ontogenic analyses using cell fate mapping models and cell depletion experiments revealed the infiltration of ovaries by both yolk sac and fetal liver-derived macrophages already during the embryonic development. Moreover, we found that both embryonic and bone marrow-derived macrophages contribute to the distinct ovarian macrophage subpopulations in the adults. These assays also showed that fetal-derived MHC II-negative macrophages differentiate postnatally in the maturing ovary to MHC II-positive cells. Our analyses further unraveled that the developmentally distinct macrophage types share overlapping distribution and scavenging function in the ovaries under homeostatic conditions. In conclusion, we report here the first comprehensive analyses of ovarian monocytes and macrophages. In addition, we show that the mechanisms controlling monocyte immigration, the phenotype of different pools of interstitial macrophages, and the interconversion capacity of fetal-derived macrophages in ovaries are remarkably different from those seen in other tissue niches.</p

Chlamydia trachomatis samples testing falsely negative in the Aptima Combo 2 test in Finland, 2019

Since February 2019, over 160 Chlamydia trachomatis (CT) cases testing negative or equivocal by Aptima Combo 2 (AC2) but positive by Aptima CT test run with Panther instruments occurred in Finland. The AC2 test targets chlamydial 23S rRNA while the CT test targets 16S rRNA. Sequencing of 10 strains revealed a nucleotide substitution in 23S rRNA. The significance of this for the failure of the AC2 test to detect the variant is not yet known.Peer reviewe

The Finnish New Variant of Chlamydia trachomatis with a Single Nucleotide Polymorphism in the 23S rRNA Target Escapes Detection by the Aptima Combo 2 Test

In 2019, more than 200 cases of Chlamydia trachomatis negative/equivocal by the Aptima Combo 2 assay (AC2, target: 23S rRNA) with slightly elevated relative light units (RLUs), but positive by the Aptima Chlamydia trachomatis assay (ACT, target: 16S rRNA) have been detected in Finland To identify the cause of the AC2 CT false-negative specimens, we sequenced parts of the CT 23S rRNA gene in 40 specimens that were AC2 negative/equivocal but ACT positive. A single nucleotide polymorphism (SNP; C1515T in the C. trachomatis 23S rRNA gene) was revealed in 39 AC2/ACT discordant specimens. No decrease in the number of mandatorily notified C. trachomatis cases was observed nationally in Finland in 2010-2019. When RLUs obtained for AC2 negative specimens were retrospectively evaluated in 2011-2019, a continuous increase in the proportion of samples with RLUs 10-19 was observed since 2014, and a slight increase in the proportion of samples with RLUs 20-84 in 2017-2019, indicating that the Finnish new variant of C. trachomatis might have been spreading nationally for several years. This emphasizes that careful surveillance of epidemiology, positivity rate and test performance are mandatory to detect any changes affecting detection of infections

Real-life clinical sensitivity of SARS-CoV-2 RT-PCR test in symptomatic patients

Background Understanding the false negative rates of SARS-CoV-2 RT-PCR testing is pivotal for the management of the COVID-19 pandemic and it has implications for patient management. Our aim was to determine the real-life clinical sensitivity of SARS-CoV-2 RT-PCR. Methods This population-based retrospective study was conducted in March-April 2020 in the Helsinki Capital Region, Finland. Adults who were clinically suspected of SARS-CoV-2 infection and underwent SARS-CoV-2 RT-PCR testing, with sufficient data in their medical records for grading of clinical suspicion were eligible. In addition to examining the first RT-PCR test of repeat-tested individuals, we also used high clinical suspicion for COVID-19 as the reference standard for calculating the sensitivity of SARS-CoV-2 RT-PCR. Results All 1,194 inpatients (mean [SD] age, 63.2 [18.3] years; 45.2% women) admitted to COVID-19 cohort wards during the study period were included. The outpatient cohort of 1,814 individuals (mean [SD] age, 45.4 [17.2] years; 69.1% women) was sampled from epidemiological line lists by systematic quasi-random sampling. The sensitivity (95% CI) for laboratory confirmed cases (repeat-tested patients) was 85.7% (81.5-89.1%) inpatients; 95.5% (92.2-97.5%) outpatients, 89.9% (88.2-92.1%) all. When also patients that were graded as high suspicion but never tested positive were included in the denominator, the sensitivity (95% CI) was: 67.5% (62.9-71.9%) inpatients; 34.9% (31.4-38.5%) outpatients; 47.3% (44.4-50.3%) all. Conclusions The clinical sensitivity of SARS-CoV-2 RT-PCR testing was only moderate at best. The relatively high false negative rates of SARS-CoV-2 RT-PCR testing need to be accounted for in clinical decision making, epidemiological interpretations, and when using RT-PCR as a reference for other tests.Peer reviewe