Additional Positive Samples Research Articles

Multi-granularity detector for enhanced small object detection under sample imbalance

This paper delves into the realm of object detection models, pinpointing challenges posed by inadequate performance in discerning small objects, as well as examining the inherent imbalance between positive and negative samples. To address these issues, we introduce the Multi-Granularity Detector (MgD), which represents a sophisticated fusion of multi-granularity feature extraction (MFE) and sequential three-way selection (S3WS). Within the MFE framework, three multi-granularity customizable deformable convolutions span three layers of feature maps that are meticulously tailored for nuanced object analysis across spectra of diverse sizes. Notably, this innovative approach enhances the accuracy of small object detection, thereby improving the overall object detection efficacy. The S3WS mechanism was introduced to rectify the imbalance between positive and negative samples. Within this framework, regional proposals undergo scrutiny, with additional positive samples judiciously selected from the positive and boundary regions. This selection process relies on multiple evaluation functions and two dynamic thresholds that are strategically applied layer by layer. The results of exhaustive experiments using the COCO benchmark unequivocally establish the excellent performance of MgD at the system level. When enhanced with the MFE and S3WS (AP 63.1→64.0, AP/APs 1.97→1.42), SwinV2-G surpassed prevailing state-of-the-art results, whereas MgD1 (AP= 53.9, AP/APs= 1.35) ignificantly enhanced the detection of small objects. In addition, the MFE and S3WS can be seamlessly integrated into ConvNet and transformer-based detectors, yielding significant improvements.

Diagnosing viral gastro-enteritis using the fully automated sample-in, result-out STARlet All in one system (AIOS)

The STARlet All-In-One system is a modular platform that integrates the complete molecular diagnostic workflow from nucleic acid extraction of clinical samples to PCR set-up and amplification. The platform was evaluated in comparison with laboratory developed tests (LDT) on fecal samples from patients with suspected viral gastro-enteritis. In a retrospective study, 72 positive samples were analysed, including all pathogens detected by the Seegene Allplex™ GI-virus assay, adenovirus, astrovirus, norovirus GI and GII, sapovirus, and rotavirus. Concordant results were obtained for 69 samples (96 %). Three discordant results were observed, one norovirus GII positive that gave an invalid result in the AIOS and two samples that were negative in the AIOS. One adenovirus positive that was subtyped as a genotype 2 virus, which is not associated with gastro-enteritis, and a sapovirus. In the prospective part of the study, 661 fecal samples were included. A total of 61 positive samples were detected, of which 60 were also detected by the AIOS. One norovirus GII positive sample (CT 35.2) was tested negative in the AIOS. Two additional sapovirus positive samples, CT 37 and 38, were detected by the AIOS but not by the LDT. The STARlet All-In-One platforms results in an automated molecular workflow with reduced hands-on time and enables running assays during out of office hours. Application of the Seegene Allplex™ GI-virus assay showed excellent concordance to the current diagnostic LDT. In a prospective comparison, only three discordant results were observed, all with CT values over 35 and therefore unlikely of clinical relevance.

Risk-based detection as a cost-effective strategy to reduce foodborne illness due to salmonella

BackgroundSalmonella, a widespread pathogen, poses a significant threat to global food safety, leading to foodborne diseases and substantial economic losses. The timely and accurate detection of foodborne pathogens is pivotal for averting food contamination and outbreaks across the food production chain. This study assesses the cost-effectiveness of traditional culture-based methods versus risk-based approaches, incorporating polymerase chain reaction (PCR) for Salmonella detection. MethodsWe employed a stochastic scenario tree model to simulate scenarios based on the sampling inspection plan for raw aquatic products conducted by the Guangzhou Center for Disease Control and Prevention from 2018 to 2020. Various detection methods (culture or PCR) were applied to these aquatic products based on their categorized risk level. Sensitivity values were derived from published data, and incremental cost-effectiveness ratios were used to compare the different scenarios against the traditional culture method. ResultsA total of 360 samples were collected for analysis. The cost of culture-based detection alone amounted to 125,423.20 Chinese Yuan (CNY) and yielded nine instances of positive Salmonella detections. The risk-based detection strategy, which combined the more sensitive PCR method with high-risk sample characteristics, while employing the culture method for the remaining combinations, imposed a total cost of 128,775.83 CNY and yielded ten positive detections. This approach cost approximately 3391.74 CNY per additional positive sample detected compared to the culture method alone. Meanwhile, PCR-only detection imposed a total cost of 62,960.03 CNY. ConclusionsIn comparison to traditional culture-based methods, both the risk-based detection strategy and the PCR-only approach demonstrated superior capabilities with respect to detecting contaminated aquatic products. Implementing risk-based detection strategies can enhance cost-effectiveness, not only ensuring food safety but also reducing the incidence and economic burden of foodborne diseases.

First report of Fig virus B in Ficus carica in New Zealand.

Fig (Ficus carica) has been cultivated since ancient times, and is now grown worldwide, both for its fruit and as an ornamental plant. Several viruses and viroids are associated with Fig mosaic disease (FMD), a disease complex occurring worldwide (Preising et al. 2021). Fig mosaic virus (FMV), fig leaf mottle-associated virus 1 (FLMaV-1), fig mild mottle-associated virus (FMMaV), and fig badnavirus 1 (FBV-1) are known to infect fig in New Zealand (Minafra et al. 2012; Veerakone et al. 2015). In December 2020, leaf samples from a fig tree growing on the roadside at St Heliers, Auckland, showing dieback with foliar chlorotic mosaic symptoms, was received for virus testing. Total nucleic acid was extracted from the symptomatic leaves using a KingFisher™ mL Purification System (Thermofisher Scientific, Waltham, MA) with an InviMag Plant DNA Mini Kit (Invitek Molecular GmbH, Germany) and subjected to high-throughput sequencing on an Oxford Nanopore Technologies MinION device using the method described in Liefting et al. 2021. All sequence analysis was performed using Geneious Prime 2021.1.1 (https://www.geneious.com). A total of 355,858 reads that passed quality check were subjected to BLASTn search against the NCBI nt database as described in Liefting et al. 2021. The following viruses produced hits: FMV, FBV-1, FMMaV and a fig closterovirus. The presence of FMV, FBV-1 and FMMaV were confirmed by species specific RT-PCRs. To identify the closterovirus, reads were mapped to closteroviruses reported in fig including the recently identified tentative species fig virus A (FiVA; GenBank accession no MN817232) and fig virus B (FiVB; GenBank accession no. MN817233). Five viral contigs ranging from 939 to 2,340 nucleotides (nt) were obtained from mapping to FiVB. Subsequently, a 6.4 kb sequence (GenBank accession no. OQ968551) from the 3' region of the NZ isolate was amplified by overlapping RT-PCR using primers designed from the contig sequences. The sequence shared 79.5% nucleotide (nt) identity with FiVB The original sample and a further 25 symptomatic and 10 asymptomatic fig samples, collected from the Auckland area between 2016 and 2021, were tested using FiVB specific RT-PCR and Sanger sequencing using primers FiVB-F1 (5'-GAGGGAGAGATGTAGATGC-3') and FiVB-R2 (5'-TGTCGTCGATATCGTTGTGT-3'), designed to amplify a 725 nt fragment in the 70 kDa heat shock protein (HSP70) ORF. Products of the expected size were amplified from the original sample and three symptomatic samples and their sequences found to be identical. BLAST searches showed that the sequence (GenBank accession no. ON553403) shared 82.7% nt and 87.3% amino acid (aa) identity to an isolate of FiVB (GenBank accession no MN817233). These additional positive samples were collected from a small home nursery where the plants were propagated from cuttings and have been distributed locally, suggesting the virus is very likely to have a limited spread throughout the Auckland area. All three FiVB infected samples were also positive for FMV. However, the association of FiVB with FMD symptoms is unknown. FiVB was first identified from a latex sample exuded from a fig tree collected from Japan (Park et al. 2021) and is the only report of FiVB in the world to date. Although an identical sequence from Argentina, named fig closterovirus 1, was submitted to GenBank, the origin of this isolate is not known. To our knowledge, this is the first report of FiVB in New Zealand.

First report of Tomato spotted wilt orthotospovirus infecting agapanthus (Agapanthus praecox) in South Africa.

Agapanthus praecox Willd. is an ornamental flowering plant that is indigenous to southern Africa and was reported to be a host of tomato spotted wilt orthotospovirus (TSWV) in Australia in 2000 (Wilson et al. 2000). Tomato spotted wilt orthotospovirus (TSWV) belonging to the genus Orthotospovirus of the family Tospoviridae is a single-stranded negative sense RNA virus known to cause disease symptoms in many crops and ornamental plant species. This virus is in the top 10 of most economically important plant viruses worldwide (Rybicki 2015; Scholthof et al. 2011). In May 2021, leaf material from three agapanthus (Agapanthus praecox) plants displaying chlorotic mottling, and yellow lesions (Supplementary material 1A) was collected in Mbombela, South Africa. One gram of symptomatic leaf material was used for total RNA extraction from each of the three samples using a CTAB extraction protocol (Ruiz-García et al. 2019). The three RNA extracts were pooled, and a sequencing library was constructed using the Ion Total RNA-Seq Kit v2.0 and RiboMinus™ Plant Kit for RNA-Seq (ThermoFisher Scientific) (Central Analytical Facility (CAF), Stellenbosch University). The RNA library was sequenced on an Ion Torrent Proton Instrument (CAF). A total of 34,392,939 single-end reads were obtained. Data was trimmed for quality with Trimmomatic (CROP:250, MINLEN:50). De novo assembly was performed on the remaining 32,281,645 trimmed reads (average readlength: 100 nt, range: 50-250 nt) using SPAdes 3.13.0 and resulted in 4,788 contigs. BLASTn analysis identified viral contigs longer than 1,000 nucleotides (nts) with high nucleotide (nt) identity to TSWV (6 contigs), as well as to the newly discovered viruses, agapanthus tungro virus (AgTV) (1 contig), and agapanthus velarivirus (AgVV) (4 contigs) (Read et al 2021). Read mapping was performed against the relevant reference sequence with the highest nt identity to the contigs. For TSWV, 4995, 21221 and 14574 reads mapped to segment L (KY250488), M (KY250489) and S (KY250490) of isolate LK-1, respectively resulting in 99.97%, 100.00% and 99.97% genome coverage of the reference accessions. The nt identity between the reference accessions and the consensus sequences generated (OP921761-OP921763) were 97.26%, 97.64% and 97.82% for segment L, M and S. The presence of TSWV was confirmed in the HTS sample using an RT-PCR assay (primers L1 and L2) targeting the L segment of TSWV (Mumford et al. 1994). In July 2022, additional leaf samples displaying symptoms of chlorotic mottling, streaking, and ringspots were collected from 31 symptomatic and 3 asymptomatic agapanthus plants in public gardens in Stellenbosch, South Africa. Using the above-mentioned RT-PCR assay, 13 of the symptomatic samples tested positive for TSWV. All six plants displaying ring spot symptoms (Supplementary material 1B) were infected with TSWV. However, plants that displayed yellow streaking (five samples) and chlorotic mottling (two samples) (Supplementary material 1C-D) were also positive for TSWV which could be due to the presence of other viruses, plant growth stage, infection time or just variable symptom expression in a single host species as reported previously (Sherwood et al. 2003). The 275 bp RT-PCR amplicons of the HTS sample and three additional positive samples were validated with bidirectional Sanger sequencing (CAF) and had 96% identity to accession KY250488. The pairwise nt identity between amplicons was 98.55-100%. This is the first report of TSWV infecting agapanthus in South Africa. This study contributes information towards the distribution and incidence of TSWV and highlights the need for nurseries to screen plant material before propagation.

Highly sensitive droplet digital PCR for detection of RET fusion in papillary thyroid cancer

BackgroundThyroid cancer is the most frequent malignancy of the endocrine system, of which papillary thyroid cancer (PTC) is the predominant form with a rapid increasing incidence worldwide. Rearranged during transfection (RET) fusions are common genetic drivers of PTC and the potent RET inhibitor selpercatinib has been recently approved for treating advanced or metastatic RET fusion-positive thyroid cancer. In this study we aimed to develop a droplet digital PCR (ddPCR) system to accurately detect RET fusion in PTC samples.MethodsThe frequency and distribution of RET fusions in PTC were analyzed using genomic data of 402 PTC patients in The Cancer Genome Atlas (TCGA) database. To establish the ddPCR system for detecting CCDC6::RET fusion, a plasmid containing CCDC6::RET infusion fragment was constructed as standard template, the annealing temperature and concentrations of primers and probe were optimized. The analytical performance of ddPCR and quantitative reverse transcription PCR (qRT-PCR) were assessed in standard templates and tissue samples from 112 PTC patients. Sanger sequencing was performed in all the RET fusion-positive samples identified by ddPCR.ResultsRET fusions were observed in 25 (6.2%) of the 402 TCGA samples, and 15 (60%) of the RET fusion-positive patients had the CCDC6::RET fusion. Compared with qRT-PCR, the ddPCR method showed a lower limit of detection (128.0 and 430.7 copies/reaction for ddPCR and qRT-PCR, respectively). When applying the two methods to 112 tissue samples of PTC, eleven (9.8%) CCDC6::RET fusion-positive samples were detected by qRT-PCR, while ddPCR identified 4 additional positive samples (15/112, 13.4%). All the CCDC6::RET fusion-positive cases identified by ddPCR were confirmed by Sanger sequencing except for one case with 0.14 copies/uL of the fusion.ConclusionThe accurate and sensitive ddPCR method reported here is powerful to detection CCDC6::RET fusion in PTC samples, application of this method would benefit more RET fusion-positive patients in the clinic.

Western blot using Trypanosoma cruzi chimeric recombinant proteins for the serodiagnosis of chronic Chagas disease: A proof-of-concept study.

Chagas disease (CD) is caused by Trypanosoma cruzi. The chronic phase of CD is characterized by the presence of IgG anti-T. cruzi antibodies; and diagnosis is performed by serological methods. Because there is no reliable test that can be used as a reference test, WHO recommends the parallel use of two different tests for CD serodiagnosis. If results are inconclusive, samples should be subjected to a confirmatory test, e.g., Western blot (WB) or PCR. PCR offers low sensitivity in the chronic phase, whereas few confirmatory tests based on the WB method are commercially available worldwide. Therefore, new diagnostic tools should be evaluated to fill the gap in CD confirmatory tests. In recent years, four chimeric recombinant antigens (IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4) have been evaluated in phase I, II and III studies using ELISA, liquid microarray and immunochromatography with 95-100% accuracy. Given the high diagnostic performance of these antigens, the present study investigated the ability of these molecules to diagnose chronic CD using a WB testing platform. In this study, we analyzed the diagnostic potential of four chimeric antigens using 40 T. cruzi-positive, 24-negative, and three additional positive samples for visceral leishmaniasis (i.e., potentially cross-reactive) using WB as the diagnostic platform. Checkerboard titration with different dilutions of antigens, conjugated antigens, and serum samples was performed to standardize all assays. All IBMP antigens achieved 100% sensitivity, specificity, and accuracy, with the exception of IBMP-8.3, which had 100% specificity despite lack of significance, but lower sensitivity (95%) and accuracy (96.9%). No cross-reactivity was observed in samples positive for leishmaniasis. The present phase I (proof-of-concept) study demonstrated the high diagnostic potential of these four IBMP antigens to discriminate between T. cruzi-positive and -negative samples, making them candidates for phase II and confirmatory testing with WB.

Evaluation of two different semi-automated homogenization techniques in microbiological diagnosis of periprosthetic joint infection: disperser vs. bead milling method

BackgroundIn microbiological diagnosis of periprosthetic joint infection (PJI) there is no consensus regarding the most suitable and optimal number of specimens to be cultured or the most effective technique of tissue processing. This comparative study analysed the accuracy of two semi-automated homogenization methods with special focus on the volume and exact origin of each sample.MethodsWe investigated a total of 722 periprosthetic tissue samples. PJI was defined according to the new scoring system for preoperative and intraoperative criteria. We compared the performance of our routinely used single tissue processing by disposable high-frequency disperser with the bead milling method.ResultsEighty patients were included. Among forty classified PJIs, 34 patients yielded positive culture results. In 23 cases (68%) exact concordant results were generated with both techniques. However, in seven cases (20%) processing by the disperser and in four cases (12%) by bead milling provided additional positive samples, but without significant difference since the major definition criteria were met in all cases. The percentage of positive results was influenced by the volume and origin of the tissue samples. Results for small tissue samples tended to be better using the bead milling method. This might lead to improved preoperative arthroscopic diagnosis, as the volume of biopsies is generally limited. Six patients had negative results due to previous antimicrobial therapy. Forty other patients were classified as aseptic failures. Neither procedure resulted in any contamination.ConclusionBoth methods enable reliable processing of tissue samples for diagnosis of PJI and are suitable for routine use.

Development of monoclonal antibody based IgG and IgM ELISA for diagnosis of severe fever with thrombocytopenia syndrome virus infection

IntroductionSevere fever with thrombocytopenia syndrome virus (SFTSV) is a newly emerged virus that poses a great threat to human health because of high fatality rate. MethodsTo develop sensitive and specific sero-diagnostic systems for SFTSV infections, monoclonal antibodies (MAbs) against recombinant SFTSV nucleocapsid (rSFTSV-N) protein were developed by immunizing BALB/C mice with rSFTSV-N protein and fusing the spleen cells with SP2/0 myeloma cells. Three hybridoma cell lines secreting MAbs against rSFTSV-N were obtained. MAb based IgG sandwich enzyme linked immunosorbent assay (ELISA) and IgM capture ELISA systems were established by using the newly developed MAbs. One hundred fifteen clinical suspected SFTS patients serum samples were used to evaluate the newly established systems by comparing with the total antibody detecting sandwich ELISA system and indirect ELISA systems. ResultsThe MAbs based sandwich IgG ELISA was perfectly matched with that of the total antibody sandwich ELISA and the indirect IgG ELISA. IgM capture ELISA results perfectly matched with that of the total antibody sandwich ELISA while detecting eight additional positive samples missed by the indirect IgM ELISA. ConclusionsThe MAbs against rSFTSV-N protein offer new tools for SFTSV studies and our newly developed MAb-based IgG and IgM capture ELISA systems would offer safe and useful tools for diagnosis of SFTS virus infections and epidemiological investigations.

Rapid detection of the widely circulating B.1.617.2 (Delta) SARS-CoV-2 variant

The emergence of the B.1.617.2 (Delta) variant of the severe acute syndrome coronavirus (SARS-CoV-2) that emerged in 2019 (COVID-19), resulted in a surge of cases in India and has expanded and been detected across the world, including in the United States. The B.1.617.2 (Delta) variant has been seen to be twice more transmissible coupled with potential increases in disease severity and immune escape. As a result, case numbers and hospitalisations are once again on the rise in the USA. On 16 July 2021, the Centers for Disease Control and Prevention (CDC) reported a 7-day average 69.3% increase in new cases and a 35% increase in hospitalisations. Although the gold standard for SARS-CoV-2 variants identification remains genomic sequencing, this approach is not accessible to many clinical laboratories. The main goal of this study was to validate and implement the detection of the B.1.617.2 (Delta) variant utilising an open reverse transcription polymerase chain reaction (RT-PCR) platform by explicitly detecting the S-gene target failure (SGTF) corresponding to the deletion of two amino acids (ΔE156/ΔF157) characteristic of B.1.617.2 (Delta) variant. This approach was conceived as a rapid screening of B.1.617.2 (Delta) variant in conjunction with CDC’s recommended N1 (nucleocapsid gene), N2, and RP (human RNase P) genes, as a pre-screening tool prior to viral genomic sequencing. We assessed 4,937 samples from 5 July to 5 September 2021. We identified the B.1.617.2 (Delta) variant in 435 of 495 positive samples (87.8%); the additional positive samples (7 samples, 1.4%) were found to belong to the B.1.1.7 (Alpha, UK) lineage and the remaining 53 samples (10.7%) were reported as ‘other’ lineages. Whole genome sequencing of 46 randomly selected samples validated the strains identified as positive and negative for the B.1.617.2 (Delta) variant and confirmed the S gene deletion in addition to B.1.617.2 characteristic mutations including L452R, T478K, P681R and D950N located in the spike protein. This modality has been used as routine testing at the Riverside University System Health (RUHS) Medical Center as a method for detection of B.1.617.2 (Delta) to pre-screen samples before genome sequencing. The assay can be easily implemented in clinical laboratories, most notably those with limited economic resources and access to genomic platforms.

Human astrovirus types 1, 4 and 5 circulating among children with acute gastroenteritis in a rural Brazilian state, 2010-2016.

This study combined conventional epidemiology of human astroviruses. From 2010 to 2016, 232 stool samples from children under 5 years of age were screened using NGS and conventional RT-PCR followed by genetic analysis in order to investigate the genotypic diversity of classical human astrovirus (HAstV) circulating in Tocantins State, Brazil. HAstV was detected in 16 cases (6.9%). Seven specimens (43.7%; 7/16) were positive according RT-PCR and next-generation sequencing (NGS) to investigate the molecular to both NGS and RT-PCR. NGS and RT-PCR individually revealed six (37.5%; 6/16) and three (18.8%; 3/16) additional positive samples, respectively. Sequencing of the HAstV-positive samples revealed HAstV-1a (9/16), HAstV-4c (3/16), and HAstV-5c (4/16) lineages.

Genetic diversity and Multilocus Sequence Typing Analysis of Bartonella henselae in domestic cats from Southeastern Brazil

Bartonella henselae is the causative agent for the infectious disease Cat Scratch Disease (CSD), which can be fatal. Domestic and wild felines are known to be its main mammal reservoirs. The present study aimed to investigate the occurrence and genetic diversity of Bartonella spp. in cats sampled in São Paulo (SP) and Minas Gerais (MG) States, Southeastern Brazil. Based on a quantitative real-time PCR (qPCR) assay, a Bartonella sp. nuoG gene fragment was detected in 39.9% (122/306) of the blood samples (46/151 cats of SP; 76/155 cats of MG). The blood samples were submitted to a pre-enrichment culture technique that allowed the detection of 12 additional positive samples, which showed to be negative in the qPCR using DNA blood samples as templates. Furthermore, five B. henselae isolates were obtained from qPCR-negative samples for both blood and pre-enrichment culture. Seven out of 24 Ctenocephalides felis fleas were positive for Bartonella spp. in the qPCR assay; 4/7 positive fleas were collected from Bartonella-negative cats. Twenty-three rpoB B. henselae cloned sequences were obtained from nine cats’ blood samples, showing the occurrence of 13 different genotypes. Median-joining network and SplitsTree distance analysis showed that the obtained sequences represented distinct B. henselae genotypes when compared to those previously deposited in GenBank. Intra-host diversity was found, since different rpoB genotypes of B. henselae were detected in individual single cats. Bartonella henselae isolates showed two allelic profiles (ST37 in cats from MG state and ST9 in SP state) by MLST (Multilocus Sequence Typing) based on sequencing of eight molecular markers. The present study is the first molecular report of Bartonella sp. in cats from Minas Gerais State. In summary, this body of work showed the occurrence of different B. henselae rpoB genotypes at an intra-reservoir host level. Based on qPCR from blood samples and pre-enrichment liquid culture and isolation, occurrence of 33.1% (50/151) and 56.8% (88/155) for Bartonella sp. was found in cats from SP and MG states, respectively. Two different allelic profiles of B. henselae were found in cats from the states of São Paulo (ST9) and Minas Gerais (ST37), suggesting a clonal evolution of Bartonellae in a certain geographical region.

Home-based HIV testing: Using different strategies among transgender women in Argentina

BackgroundIn Argentina, HIV prevalence among transgender women (TGW) has been reported at 34%. The stigma is one of the most important factors limiting their access to healthcare services. The aims of this study were to compare different HIV testing methodologies, to determine the factors associated with HIV diagnosis and to determine the feasibility of a home-based HIV testing service for TGW.MethodsA multidisciplinary team performed home-based HIV testing interventions in four cities of Argentina. Participants self-identified as TGW, older than 14 years and with a negative or unknown HIV status. Blood samples were screened by two rapid tests (RT), one based on antibodies (Determine™ HIV-1/2) and the other on antigen and antibodies (Determine™ HIV-1/2 Combo), and the subsequent blood processing via 4th generation ELISA (VIDAS HIV DUO). All reactive samples were confirmed with a viral load (VL). We compared the results of both RT with the ELISA. Samples were pooled in groups of 6 and a VL (Abbott Real Time) performed to identify acute HIV infections. Factors associated with HIV infection were evaluated with multivariate logistic regression analysis.ResultsA total of 260 TGW were tested, 51 tested positive (HIV prevalence 19.6%). There were no discordant results between both RTs nor between RTs and 4th generation ELISA, therefore the correlation was 100%. The VL identified 2 additional positive samples. The final analytic sample for positive cases consisted of 53 TGW. In the multivariate analysis, factors associated with a positive HIV result were history of other sexually transmitted infections (STIs) and not being previously tested for HIV. TGW tested for the first time were at 4 times greater risk of being HIV positive compared to those that were tested previously.ConclusionsA multidisciplinary home-based HIV testing service among TGW is feasible and effective to detect cases of HIV infection. The testing algorithm should start with an RT followed by molecular diagnosis. The history of STIs and never having been tested for HIV were the factors associated with HIV-positive results and should determine efforts to reach this population. Home-based testing reaches individuals that were not tested before and who have more risk of acquiring HIV.

HPV-negative Tumors in a Swedish Cohort of Cervical Cancer

Despite the common perception that the human papilloma virus (HPV) is a requirement for the development of cervical cancer (CC), a considerable number of CCs test HPV negative. Presently, many countries are shifting to HPV primary CC screening, and it is of importance to increase the knowledge about the group of CCs that test HPV negative. The aim of this study was to reinvestigate a proportion of cervical tumors with a primary negative or invalid test result. Reinvestigation with repeated genotyping (targeting L1) was followed by analysis with an alternative target method (targeting E6/E7) on existing or additional tumor material. Consistently negative tumors were histologically evaluated, and cases with low or lacking tumor cell content, consistent invalid test results, or with suspicion of other than cervical origin were excluded. HPV-negative cases were thereafter subjected to immunohistochemistry (Cytokeratin 5, pan cytokeratin, protein 63, P16, and P53). The HPV-negative proportion could after reinvestigation be reduced by one-half (14%–7%). Additional positive samples were often detected in late polymerase chain reaction cycles, with an alternative (E6/E7) or the same (L1) target, or with a method using shorter amplicon lengths. Confirmed HPV negativity was significantly associated with worse prognosis, high patient age, longer storage time, and adenocarcinoma histology. Some of the HPV-negative cases showed strong/diffuse p16 immunoreactivity, indicating some remaining false-negative cases. False HPV negativity in this cohort was mainly linked to methodological limitations in the analysis of stored CC material. The small proportion of presumably true HPV-negative adenocarcinomas is not a reason for hesitation in revision to CC screening with primary HPV testing.

Determination of cerebrospinal fluid leakage by selective deletion of transferrin glycoform using an immunochromatographic assay.

Cerebrospinal fluid (CSF) leakage can lead to brain and spine pathologies and there is an urgent need for a rapid diagnostic method for determining CSF leakage. Beta-2 transferrin (β2TF), asialotransferrin, is a specific CSF glycoprotein biomarker used to determine CSF leakage when distinguished from serum sialotransferrin (sTF).Methods: We detected β2TF using an immunochromatographic assay (ICA), which can be potentially developed as a point-of-care (POC) testing platform. Sialic acid-specific lectin selectively captures sTF in multiple deletion lines within an ICA test strip, enabling the detection of β2TF. A sample pre-treatment process efficiently captures excess sTF increasing sensitivity for CSF leakage detection.Results: An optimal cut-off value for determining the presence of CSF in test samples was obtained from receiver operating characteristic (ROC) analysis of the ratio of the test signal intensity and the deletion lines. On 47 clinical samples, ICA test strips discriminated CSF positive from negative samples with statistically significant (positive versus negative t-test; P =0.00027). Additional artificial positive samples, prepared by mixing CSF positive and negative clinical samples, were used as a further challenge. These positive samples were clearly discriminated from the negative samples (mixture versus negative t-test; P =0.00103) and CSF leakage was determined with 97.1% specificity and 96.2% sensitivity.Conclusions: ICA represents a promising approach for POC diagnosis of CSF leakage. While requiring 70 min assay time inconvenient for POC testing, our method was significantly shorter than conventional electrophoresis-based detection methods for β2TF.

HIV rapid screening tests and self-tests: Be aware of differences in performance and cautious of vendors.

BackgroundRapid tests for HIV testing are essential tools to achieve the 90-90-90 target of the World Health Organization. Many tests are available, some directly from websites. Evaluation of the performance of rapid tests, under close to real-life usage, is therefore needed to ensure accurate diagnosis in the context of the recommendation for their more widespread use.MethodNine third- (3G) or fourth-generation (4G) rapid screening tests or self-tests (two bought on websites), were evaluated on an extensive panel of 200 HIV-negative and 312 HIV-positive samples, representative of a wide variety of clinical situations and HIV genetic diversity. A whole blood reconstitution protocol was designed to simulate real-life usage of these tests in community-based and private settings.FindingsThe specificity was high (98.5–100%) and sensitivity excellent (100%) for samples from patients chronically infected with the pandemic strains. The performance for infrequent situations with a major epidemiological and clinical impact, such as infection with divergent viruses or primary infection, was highly variable, depending on the test. One of the two 4G tests allowed detection of additional positive samples from early stages of infection, whereas the second (sold as a 4G test on a website) corresponded in reality to a 3G test.InterpretationOur study showed that not all tests are equal for the detection of major HIV variants or early stages of HIV infection; adding the detection of specific p24Ag improved the latter point. This study also showed, for the first time, that buying through web-based vendors can be risky, due to the varying performance of the tests and questionable sales practices. Our results are of particular importance in the context of the increasing use of rapid tests in an “outside laboratory” settings.FundSanté Publique France, COREVIH – Normandie, and Rouen University Hospital.

Astrovirus MLB2, a New Gastroenteric Virus Associated with Meningitis and Disseminated Infection.

Next-generation sequencing has identified novel astroviruses for which a pathogenic role is not clearly defined. We identified astrovirus MLB2 infection in an immunocompetent case-patient and an immunocompromised patient who experienced diverse clinical manifestations, notably, meningitis and disseminated infection. The initial case-patient was identified by next-generation sequencing, which revealed astrovirus MLB2 RNA in cerebrospinal fluid, plasma, urine, and anal swab specimens. We then used specific real-time reverse transcription PCR to screen 943 fecal and 424 cerebrospinal fluid samples from hospitalized patients and identified a second case of meningitis, with positive results for the agent in the patient's feces and plasma. This screening revealed 5 additional positive fecal samples: 1 from an infant with acute diarrhea and 4 from children who had received transplants. Our findings demonstrate that astrovirus MLB2, which is highly prevalent in feces, can disseminate outside the digestive tract and is an unrecognized cause of central nervous system infection.

The first molecular genetic identification of the tularemia pathogen in Ixodes trianguliceps Bir. ticks in Russia

A real-time PCR (RT PCR) assay for Francisella tularensis DNA in ixodes ticks Ixodes trianguliceps (140 mature individuals and 211 nymph pools) found on small mammals from the Middle Ural forests (Chusovskoi district, Perm Region) has been performed for the first time. Francisella DNA was detected in 12 mature ticks and 4 nymph pools when the 16SrRNA gene (amplicon size 1165–1170 bp) was used as the target. Amplification of a shorter fragment of the same gene (221–222 bp) resulted in identification of additional positive samples among mature ticks (17 of 128) and nymph pools (16 of 89). All 49 RT-PCR positive samples were identified as F. tularensis DNA using primers and probes complementary to a fragment of the lpnA (tul4) gene and the ISFtu2 element. The data obtained are indicative of the possible involvement of I. trianguliceps ticks in tularemia pathogen circulation in natural foci of forest type.

Molecular detection of novel Anaplasmataceae closely related to Anaplasma platys and Ehrlichia canis in the dromedary camel (Camelus dromedarius)

Serological surveys have confirmed Anaplasma marginale and Anaplasma phagocytophilum infections in dromedary camels, but molecular surveys and genetic characterisation of camel-associated Anaplasma species are lacking. In this study, we detected tick-borne Anaplasmataceae in 30 of 100 (30%) healthy dromedary camels screened using a combined 16S rRNA–groEL PCR-sequencing approach. Nucleotide sequencing confirmed Anaplasmataceae genome presence in 28 of the 33 16S rRNA PCR-positive samples, with two additional positive samples, for which 16S rRNA sequence data were ambiguous, being identified by groEL gene characterisation. Phylogenetic analyses of a 1289nt segment of the 16S rRNA gene confirmed the presence of a unique Ehrlichia lineage and a discrete Anaplasma lineage, comprising three variants, occurring at an overall prevalence of 4% and 26%, respectively. Genetic characterisation of an aligned 559nt groEL gene region revealed the camel-associated Anaplasma and Ehrlichia lineages to be novel and most closely related to Anaplasma platys and Ehrlichia canis. Based on the confirmed monophyly, minimum pairwise genetic distances between each novel lineage and its closest sister taxon, and the inability to isolate the bacteria, we propose that Candidatus status be assigned to each. This first genetic characterisation of Anaplasmataceae from naturally infected, asymptomatic dromedary camels in Saudi Arabia confirms the presence of two novel lineages that are phylogenetically linked to two pathogenic canid species of increasing zoonotic concern.

Systematic comparison of drug-tolerant assays for anti-drug antibodies in a cohort of adalimumab-treated rheumatoid arthritis patients

Drug interference complicates assessment of immunogenicity of biologicals and results in an underestimation of anti-drug antibody (ADA) formation. Drug-tolerant assays have the potential to overcome such limitations. However, to which extent drug-tolerant assays provide an unbiased picture of the antibody response to a biological is unknown.In this study, we compared the measurement of ADA to adalimumab in 94 consecutive adalimumab-treated rheumatoid arthritis patients using the traditional antigen binding test (ABT) and four different drug-tolerant assays, the Ph-shift anti-Idiotype Antigen binding test (PIA) and three newly developed assays for this study: an acid-dissociation radioimmunoassay (ARIA), a temperature-shift radioimmunoassay (TRIA) and an electrochemoluminescence-based assay (ECL).Our results indicate that drug-tolerant assays provide a fairly consistent view on the antibody formation: quantitatively, the results from all four assays correlate well (Spearman r>0.9). However, the percentage of ADA-positive patients ranges from 51 to 66% between assays, with the ARIA identifying the highest number of patients as positive. These differences are largely due to patients making low amounts of ADA; if ADA levels were above ca. 100AU/ml, a patient was identified as positive in all four assays. Adalimumab concentrations were significantly lower in ADA-positive samples.Taken together, the results indicate that these different drug-tolerant assays provide a similar and reasonably consistent view on ADA responses, which however, breaks down at the lower end of the detectable range, and highlight that ADA is best reported quantitatively. Furthermore, if an even more sensitive drug-tolerant assay could be developed, one would probably find additional positive samples that will predominantly contain very low levels of ADA.