Assessment of Rapid Diagnostic Tests for Typhoid Diagnosis and Assessment of Febrile Illness Outbreaks in Fiji.

Typhoid is an endemic in Fiji with increases observed since the early 2000s and frequent outbreaks reported. We assessed the diagnostic accuracy of currently available typhoid rapid diagnostic tests (RDTs) (TUBEX, Typhidot Rapid, and Test-It assay) to establish their performance against blood culture in Fiji and to examine their suitability for rapid typhoid outbreak identification. The performance of RDTs was assessed in the public health reference laboratory in Suva, Fiji, according to the manufacturers' instructions. A simulation was used to examine the potential use of RDTs for attribution of a febrile illness outbreak to typhoid. For the diagnostic evaluation, 179 patients were included; 49 had blood culture-confirmed typhoid, 76 had fever as a result of non-typhoid etiologies, and 54 were age-matched community controls. The median (interquartile range) age was 29 (20-46) years. Of the participants, 92 (51.4%) were male and 131 (73.2%) were indigenous Fijians. The sensitivities of the tests were 77.6% for TUBEX, 75.5% for Typhidot Rapid, and 57.1% for Test-It assay. The Test-It assay had the highest specificity of 93.4%, followed by Typhidot Rapid 85.5% and TUBEX 60.5%. Typhidot Rapid had the best performance in the simulation for attribution of a febrile illness outbreak to typhoid. Typhoid RDTs performed suboptimally for individual patient diagnosis due to low sensitivity and variable specificity. We demonstrate that RDTs could be useful in the field for rapid attribution of febrile illness outbreaks to typhoid. Typhidot Rapid had the best combination of sensitivity, specificity, positive and negative predictive values, cost, and ease of use for this purpose.

Multicenter Comparative Assessment of the TIB MolBiol Toxoplasma gondii Detection Kit and Four Laboratory-Developed PCR Assays for Molecular Diagnosis of Toxoplasmosis.

Toxoplasmosis can be a life-threatening infection, particularly during pregnancy and in immunocompromised patients. The biological diagnosis of toxoplasmosis is challenging and has been revolutionized by molecular detection methods. This article summarizes the data of a multicenter study involving four centers to assess the performances of a commercial PCR assay as compared with four in-house PCR assays using Toxoplasma gondii standards, 20 external quality control specimens, and 133 clinical samples. This clinical cohort includes well-characterized clinical samples corresponding to different clinical situations: confirmed congenital toxoplasmosis (44 samples), toxoplasmosis in immunocompromised patients (25 samples), and chorioretinitis (5 samples). Furthermore, 59 samples from patients without toxoplasmosis were included as negative controls. The analytical sensitivities of the five methods tested were very similar; and the limit of Toxoplasma DNA detection was around 0.01 T. gondii genome per reaction for all the methods. The overall concordance between the commercial PCR and the four in-house PCR assays was 97.7% (130/133). The clinical sensitivity and specificity were >98% and could be increased for the commercial kit when PCR was performed in multiplicate to detect low parasitic loads. In conclusion, the commercial PCR assay shows suitable performances to diagnose the different clinical forms of toxoplasmosis.

Implementation outcomes of HIV self-testing in low- and middle- income countries: A scoping review.

INTRODUCTION: HIV self-testing (HIV-ST) is an effective means of improving HIV testing rates. Low- and middle-income countries (LMIC) are taking steps to include HIV-ST into their national HIV/AIDS programs but very few reviews have focused on implementation in LMIC. We performed a scoping review to describe and synthesize existing literature on implementation outcomes of HIV-ST in LMIC. METHODS: We conducted a systematic search of Medline, Embase, Global Health, Web of Science, and Scopus, supplemented by searches in HIVST.org and other grey literature databases (done 23 September 2020) and included articles if they reported at least one of the following eight implementation outcomes: acceptability, appropriateness, adoption, feasibility, fidelity, cost, penetration, or sustainability. Both quantitative and qualitative results were extracted and synthesized in a narrative manner. RESULTS AND DISCUSSION: Most (75%) of the 206 included articles focused on implementation in Africa. HIV-ST was found to be acceptable and appropriate, perceived to be convenient and better at maintaining confidentiality than standard testing. The lack of counselling and linkage to care, however, was concerning to stakeholders. Peer and online distribution were found to be effective in improving adoption. The high occurrence of user errors was a common feasibility issue reported by studies, although, diagnostic accuracy remained high. HIV-ST was associated with higher program costs but can still be cost-effective if kit prices remain low and HIV detection improves. Implementation fidelity was not always reported and there were very few studies on, penetration, and sustainability. CONCLUSIONS: Evidence supports the acceptability, appropriateness, and feasibility of HIV-ST in the LMIC context. Costs and user error rates are threats to successful implementation. Future research should address equity through measuring penetration and potential barriers to sustainability including distribution, cost, scale-up, and safety.

Laboratory and Field Evaluation of the Crystal VC-O1 Cholera Rapid Diagnostic Test.

Cholera is a severe acute, highly transmissible diarrheal disease which affects many low- and middle-income countries. Outbreaks of cholera are confirmed using microbiological culture, and additional cases during the outbreak are generally identified based on clinical case definitions, rather than laboratory confirmation. Many low-resource areas where cholera occurs lack the capacity to perform culture in an expeditious manner. A simple, reliable, and low-cost rapid diagnostic test (RDT) would improve identification of cases allowing rapid response to outbreaks. Several commercial RDTs are available for cholera testing with two lines to detect either serotypes O1 and O139; however, issues with sensitivity and specificity have not been optimal with these bivalent tests. Here, we report an evaluation of a new commercially available cholera dipstick test which detects only serotype O1. In both laboratory and field studies in Kenya, we demonstrate high sensitivity (97.5%), specificity (100%), and positive predictive value (100%) of this new RDT targeting only serogroup O1. This is the first field evaluation for the new Crystal VC-O1 RDT; however, with these high-performance metrics, this RDT could significantly improve cholera outbreak detection and improve surveillance for better understanding of cholera disease burden.

Assessment of 12 qualitative RT-PCR commercial kits for the detection of SARS-CoV-2.

The emergence of the novel coronavirus, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the late months of 2019 had the officials to declare a public health emergency leading to a global response. Public measurements rely on an accurate diagnosis of individuals infected with the virus by using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The aim of our study is to relate the fundamental clinical and analytical performance of SARS-CoV-2 (RT-PCR) commercial kits. A total of 94 clinical samples were selected. Generally, 400 µl of each respiratory specimen was subjected to extraction using ExiPrep 96 Viral RNA Kit. All kits master mix preparation, cycling protocol, thermocycler, and results interpretation were carried out according to the manufacturer's instructions of use and recommendations. The performance of the kits was comparable except for the LYRA kit as it was less sensitive (F = 67, p < .001). Overall, four kits scored a sensitivity of 100% including: BGI, IQ Real, Sansure, and RADI. For specificity, all the tested kits scored above 95%. The performance of these commercial kits by gene target showed no significant change in CT values which indicates that kits disparities are mainly linked to the oligonucleotide of the gene target. We believe that most of the commercially available RT-PCR kits included in this study can be used for routine diagnosis of patients with SARS-CoV-2. We recommend including kits with multiple targets in order to monitor the virus changes over time.

Assessment of three commercial ELISAs for the detection of antibodies against Porcine epidemic diarrhea virus at different stages of the immune response.

Three commercial ELISAs -two based on spike (E1 and E3) and one on nucleocapsid protein (E2)-were used to analyze the development and persistence of antibodies against Porcine epidemic diarrhea virus (PEDV). Seventy-five four-week-old PEDV-negative piglets were inoculated orally with a European G1b PEDV (INOC) and fourteen were kept as controls (CTRL). After the inoculation, E3 detected positive animals as soon as 7 days post inoculation (dpi), while the earliest detection with E1 and E2 was at 14 dpi. All samples were positive at 21 and 28 dpi using E1 and E3, respectively, while E2 failed to detect 23.3 % of the inoculated pigs at any time point. The percentages of positive samples were different through the study: E1 and E3 > E2 from 14 to 56 dpi; and E3 > E1 > E2 from 56 to 154 dpi (P < 0.05). Five months after the inoculation, E3 still detected 92.0 % (IC95 % = 85.1-98.8 %) of pigs as positive, while E1 and E2 detected only 27.0 % (IC95 % = 16.0-37.9 %) and 0%, respectively. The sensitivity for E2 never exceeded 0.62. Specificity was 1 for all ELISAs. These different outcomes could be related to the ELISA strategies (indirect versus competition), the antigens used, the cut-off, or to other intrinsic factors of each test. The observed differences could be of importance when assessing whether older animals, such as fatteners or gilts, had previously been in contact with PEDV.

Evaluation of two automated low-cost RNA extraction protocols for SARS-CoV-2 detection.

BACKGROUND: Two automatable in-house protocols for high-troughput RNA extraction from nasopharyngeal swabs for SARS-CoV-2 detection have been evaluated. METHODS: One hundred forty one SARS-CoV-2 positive samples were collected during a period of 10-days. In-house protocols were based on extraction with magnetic beads and designed to be used with either the Opentrons OT-2 (OT-2in-house) liquid handling robot or the MagMAXTM Express-96 system (MMin-house). Both protocols were tested in parallel with a commercial kit that uses the MagMAXTM system (MMkit). Nucleic acid extraction efficiencies were calculated from a SARS-CoV-2 DNA positive control. RESULTS: No significant differences were found between both in-house protocols and the commercial kit in their performance to detect positive samples. The MMkit was the most efficient although the MMin-house presented, in average, lower Cts than the other two. In-house protocols allowed to save between 350€ and 400€ for every 96 extracted samples compared to the commercial kit. CONCLUSION: The protocols described harness the use of easily available reagents and an open-source liquid handling system and are suitable for SARS-CoV-2 detection in high throughput facilities.

Nationwide External Quality Assessment of SARS-CoV-2 Molecular Testing, South Korea.

External quality assessment (EQA) is essential for ensuring reliable test results, especially when laboratories are using assays authorized for emergency use for newly emerging pathogens. We developed an EQA panel to assess the quality of real-time reverse transcription PCR assays being used in South Korea to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). With the participation of 23 public health organization laboratories and 95 nongovernmental laboratories involved in SARS-CoV-2 testing, we conducted qualitative and semiquantitative performance assessments by using pooled respiratory samples containing different viral loads of SARS-CoV-2 or human coronavirus OC43. A total of 110 (93.2%) laboratories reported correct results for all qualitative tests; 29 (24.6%) laboratories had >1 outliers according to cycle threshold values. Our EQA panel identified the potential weaknesses of currently available commercial reagent kits. The methodology we used can provide practical experience for those planning to conduct evaluations for testing of SARS-CoV-2 and other emerging pathogens in the future.

Sensitivity and specificity of commercially available rapid diagnostic tests for viral hepatitis B and C screening in serum samples.

Early diagnosis of chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections is pivotal for optimal disease management. Sensitivity and specificity of 19 rapid diagnostic test (RDT) kits by different manufacturers (ABON, CTK Biotech, Cypress Diagnostics, Green Gross, Human Diagnostic, Humasis, InTec, OraSure, SD Bioline, Wondfo) were assessed on serum samples of 270 Mongolians (90 seropositive for hepatitis B surface antigen (HBsAg), 90 seropositive for hepatitis C antibody (HCV-Ab), 90 healthy subjects). All tested RDTs for detection of HBsAg performed with average sensitivities and specificities of 100% and 99%, respectively. Albeit, overall sensitivity and specificity of RDTs for detection of HCV-Ab was somewhat lower compared to that of HBsAg RDTs (average sensitivity 98.9%, average specificity 96.7%). Specificity of RDTs for detection of HCV-Ab was dramatically lower among HBsAg positive individuals, who were 10.2 times more likely to show false positive test results. The results of our prospective study demonstrate that inexpensive, easy to handle RDTs are a promising tool in effective HBV- and HCV-screening especially in resource-limited settings.

[Assessment of the possibility of application in laboratory practice of reagent kit for diagnosis of glanders and melioidosis by real-time polymerase chain reaction.]

The reagent kit AmpligenBurk-mallei/pseudomallei-RT PCR is designed for detecting in vitro diagnostics and differentiate the DNA of glanders and melioidosis pathogens by real-time multiplex PCR in biological (clinical) material and cultures of microorganisms, as well as environmental objects and solid food products (rice). During clinical testing diagnostic value of reagent kit AmpligenBurk-mallei/pseudomallei-RT PCR has been studied. Based on the results obtained, a high analytical sensitivity (1×103 microbe cells/ml) and specificity (100%) of PCR-RT with the developed reagent kit were established, regardless of the type of material being studied. The diagnostic sensitivity of PCR-RT using a set of reagents was at least 98.0% and specificity at least 99%. The stages of state examination have been completed, a registration certificate has been obtained at Roszdravnadzor, production, sale and use of reagent kit in medical laboratory practice have been permitted.

A comparison of Lyse-It to other cellular sample preparation, bacterial lysing, and DNA fragmentation technologies.

The ability for safe and rapid pathogenic sample transportation and subsequent detection is an increasing challenge throughout the world. Herein, we describe and use bead-beating, vortex, sonication, 903 protein saver cards, and Lyse-It methods, aiming to inactivate Gram-positive and -negative bacteria with subsequent genome DNA (quantitative Polymerase Chain Reaction) qPCR detection. The basic concepts behind the four chosen technologies is their versatility, cost, and ease of use in developed and underdeveloped countries. The four methods target the testing of bacterial resilience, cellular extraction from general and complex media and subsequent DNA extraction for qPCR detection and amplification. These results demonstrate that conventional high temperature heating, 903 protein saver cards, and Lyse-It are all viable options for inactivating bacterial growth for safe shipping. Additionally, Lyse-It was found to be particularly useful as this technology can inactivate bacteria, extract cells from 903 protein saver cards, lyse bacterial cells, and additionally keep genomic DNA viable for qPCR detection.

Impact of Rapid On-demand Molecular Diagnosis of Pediatric Seasonal Influenza on Laboratory Workflow and Testing Costs: A Retrospective Study.

BACKGROUND: Seasonal influenza imposes a considerable burden worldwide. We aimed to evaluate impact of rapid pediatric seasonal influenza diagnosis on laboratory workflow and cost using a rapid antigen detection-based test combined with either a reverse transcriptase polymerase chain reaction (RT-PCR) or the Alere i Influenza A and B (Alere i) assay for confirmation of negative results as well as single Alere i testing on nasopharyngeal aspirates. A secondary objective was assessing performance of Alere i against RT-PCR. METHODS: Effects of implementing the 3 diagnostic algorithms were assessed in the Emergency Department of Hospital Sant Joan de Déu (Barcelona, Spain) across the 2014-2015, 2015-2016 and 2016-2017 influenza seasons. Alere i performance against RT-PCR was determined during the 2015-2016 epidemic period. RESULTS: Median time to result decreased when using Alere i as a confirmatory test of previous antigen detection and RT-PCR results or alone (9.7vs. 3.5/2.0 and 0.7 hours, P < 0.001) along with mean testing costs (&OV0556;87.3 vs. &OV0556;38.2 and &OV0556;25.0, P < 0.001). Results available before patient discharge from the emergency department increased from 42.7% for sequential testing by antigen detection and RT-PCR to 80.0% when Alere i was utilized as a stand-alone test. Alere i sensitivity and specificity values were 96.6% (95% confidence interval: 82.8%-99.4%) and 94.4% (95% confidence interval: 86.6%-97.8%), respectively. CONCLUSIONS: Rapid Alere i testing facilitated efficient laboratory workflow near the patient during influenza epidemics while contributing cost savings when compared with serial testing by antigen and RT-PCR assays.

Improvement in serological diagnosis of pertussis by external quality assessment.

PURPOSE: Serological analysis is an essential tool for the diagnosis of pertussis or whooping cough, disease surveillance and the evaluation of vaccine effectiveness against Bordetella pertussis. Accurate measurement of anti-pertussis toxin (anti-PT) IgG antibody levels in sera is essential. These measurements are usually performed using immunological methods such as ELISA and multiplex immunoassays. However, there are a large number of different assay systems available, and therefore standardization and harmonization between the methods are needed to obtain comparable data. METHODOLOGY: In collaboration with ECDC, the EUPert-LabNet network has organized three External Quality Assessment (EQA) schemes (2010, 2012 and 2016), which initially identified the diverse range of techniques and reagents being used throughout Europe. This manuscript discusses the findings of each of the EQA rounds and their impact on the participating laboratories. RESULTS: The studies have shown an increasing number of laboratories (from 65% to 92%) using only the recommended coating antigen, purified PT, in immunoassays, as this allows exact quantification of serum anti-PT IgG and since PT is only produced by Bordetella pertussis this prevents cross-reactivity with other species. There has also been an increase in the numbers of laboratories (from 59% to 92%), including a WHO reference serum in their assays, which allows anti-PT IgG concentrations to be measured in International Units, thus enabling the comparison of results from different methods and laboratories. In addition, manufacturers have also considered these recommendations when they produce commercial ELISA kits. CONCLUSION: The three EQA rounds have resulted in greater harmonization in methods among different laboratories, showing a significant improvement of the ELISA methods used for serodiagnosis of pertussis.

A direct comparison of four high-risk human papillomavirus tests versus the cobas test: Detecting CIN2+ in low-resource settings.

Low-cost, accurate high-risk human papillomavirus (HR-HPV) tests are needed for cervical cancer screening in limited-resource settings. More than 200 cervical cytological specimens from hospital patients were collected and analyzed for a real-world study. We evaluated the analytical and clinical performance of four widely used HR-HPV test (Tellgen, Hybribio, Liferiver, and Sansure) based on real-time polymerase chain reaction technology platforms, compared with the cobas test. Cervical intraepithelial neoplasia grade 2 or worse lesions (CIN2+) were set as the disease endpoint, and all the five HPV tests were performed with equal sensitivity (McNemar's test; P = 0.971) and specificity (McNemar's test; P = 0.953). All genotyping using the INNO-LiPA HPV test showed that HPV-16, -52, and -54 were the most common types among CIN2+ cases. Overall, the four HR-HPV tests analyzed appear to be as effective as the cobas HPV test in both agreement and clinical performance. Therefore, each of these low-cost HPV test kits could be implemented in limited-resource settings to accelerate the control of cervical cancer. However, we suggest that there is a need to further standardize and optimize testing around clinical sensitivity and specificity.

Two-tailed RT-qPCR panel for quality control of circulating microRNA studies.

Circulating cell-free microRNAs are promising candidates for minimally invasive clinical biomarkers for the diagnosis, prognosis and monitoring of many human diseases. Despite substantial efforts invested in the field, the research so far has failed to deliver expected results. One of the contributing factors is general lack of agreement between various studies, partly due to the considerable technical challenges accompanying the workflow. Pre-analytical variables including sample collection, RNA isolation, and quantification are sources of bias that may hamper biological interpretation of the results. Here, we present a Two-tailed RT-qPCR panel for quality control, monitoring of technical performance, and optimization of microRNA profiling experiments from biofluid samples. The Two-tailed QC (quality control) panel is based on two sets of synthetic spike-in molecules and three endogenous microRNAs that are quantified with the highly specific Two-tailed RT-qPCR technology. The QC panel is a cost-effective way to assess quality of isolated microRNA, degree of inhibition, and erythrocyte contamination to ensure technical soundness of the obtained results. We provide assay sequences, detailed experimental protocol and guide to data interpretation. The application of the QC panel is demonstrated on the optimization of RNA isolation from biofluids with the miRNeasy Serum/Plasma Advanced Kit (Qiagen).

Field performance of the INSTI HIV-1/-2 antibody test in two South African antenatal clinics.

This was a prospective study that assessed field performance of the INSTI HIV-1/-2 antibody test (INSTI test) in two antenatal clinics in South Africa (SA). INSTI test was evaluated against rapid tests used at these clinics, and pooled nucleic acid amplification testing (NAAT) performed for individuals with negative rapid tests. Three hundred and eighty-six pregnant women were enrolled; 334 (86.5%) with negative results on the screening rapid test, and 52 (13.5%; 95% confidence interval [CI]: 10.2-17.3%) with positive results on screening and confirmatory rapid tests. INSTI test yielded the same results as other rapid tests in all participants, thus showing a 100% sensitivity (95% CI: 93.2-100.0%) and specificity (95% CI: 98.9-100.0%). Pooled NAAT was performed for 290 participants who had negative rapid tests, and yielded negative results in all pools. These data show excellent field performance of the INSTI test, and highlight that this test can be implementedat SA clinics.

Multiplex STR panel for assessment of chimerism following hematopoietic stem cell transplantation (HSCT).

Short tandem repeat (STR) analysis is used in chimerism monitoring after allogeneic hematopoietic stem cell transplantation (HSCT) for patients with various hematologic malignancies. Commercial forensic STR kits often contain loci with huge differences in power of discrimination (PD) across populations, causing some loci to be less informative for chimerism analysis in certain populations. This study aimed to construct a new STR multiplex panel with highly informative loci for efficient chimerism analysis. Thirteen STR markers which exhibit high PD (> 0.9) in at least 80% of 50 populations globally were selected to form a new panel and used in STR analysis of 253 Malaysian subjects. Cumulative power of discrimination (CPD) and combined power of exclusion (CPE) were determined from 253 Malaysian individuals. Loci informativity was assessed and compared to the commercial AmpFLSTR Identifiler PCR Amplification kit in 14 donor-recipient pairs. The new panel had detected 202 unique alleles including five novel alleles from the 253 individuals with high CPD and CPE (> 0.99999999999999999 and > 0.999999997 respectively). All loci from the new panel in the donor-recipient pair analysis showed higher than 50% informativity, while five loci from the commercial kit demonstrated lower than 50% informativity. Four loci from the new panel ranked the highest informativity. A sequenced allelic ladder which consists of 202 unique alleles from the 253 subjects was also developed to ensure accurate allele designation. The new 13-loci STR panel, thus, could serve as an additional powerful, accurate, and highly informative panel for chimerism analysis for HSCT patients.

A review of the atomoRapid HIV self-testing device: an acceptable and easy alternative to facilitate HIV testing.

Introduction:HIV testing is the gateway to both HIV prevention and treatment, and increased HIV testing and linkage to services is vital for an effective HIV response. HIV testing has progressed significantly from a lengthy laboratory process conducted by specialist medical staff to rapid point of care testing performed by trained lay staff. Despite HIV testing services being widely available, testing rates remain suboptimal among young people and men. Alternative delivery strategies that complement conventional testing services are needed to reach these priority groups. Areas covered:This article reviewed the AtomoRapid HIV self-testing (HIVST) device as an innovative alternative to conventional testing. Expert commentary:HIVST complements traditional HIV testing options and can be used to overcome major barriers to testing by catering for testing outside of conventional settings and by allowing individuals to test themselves privately, and at their own discretion and frequency. We conclude that the high sensitivity, specificity, acceptability, usability, and fidelity of this device makes it an appropriate option for the enhancement of HIV testing strategies for harder to reach populations, such as young people and men.

In vitro medical devices: how businesses can successfully comply with the new European regulation.

The European parliament finally approved the new European in vitro diagnostic regulation (IVDR) on 5 April 2017. This new regulation is shaking up the industry as it has a wider scope than its predecessor, meaning manufacturers of in vitro diagnostic medical devices must revise their compliance strategies exhaustively. In order to help manufacturers begin the process of compliance, this article highlights the principal changes in the regulation, providing a starting point for industry players. Furthermore, the article draws attention to other obstacles to conformity, in particular the shortage of notified bodies, the organisations designated by member states to carry out compliance evaluations. In addition to the commercial stakes for businesses, it is essential to bear in mind the ultimate objective of this overhaul of the regulatory framework, namely, to improve patient safety.