Diagnostics are fundamental for successful outbreak containment. In this supplement, ‘Diagnostic preparedness for WHO Blueprint pathogens’, we describe specific diagnostic challenges presented by selected priority pathogens most likely to cause future epidemics.Some challenges to diagnostic preparedness are common to all outbreak situations, as highlighted by recent outbreaks of Ebola, Zika and yellow fever. In this article, we review these overarching challenges and explore potential solutions. Challenges include fragmented and unreliable funding pathways, limited access to specimens and reagents, inadequate diagnostic testing capacity at both national and community levels of healthcare and lack of incentives for companies to develop and manufacture diagnostics for priority pathogens during non-outbreak periods. Addressing these challenges in an efficient and effective way will require multiple stakeholders—public and private—coordinated in implementing a holistic approach to diagnostics preparedness. All require strengthening of healthcare system diagnostic capacity (including surveillance and education of healthcare workers), establishment of sustainable financing and market strategies and integration of diagnostics with existing mechanisms. Identifying overlaps in diagnostic development needs across different priority pathogens would allow more timely and cost-effective use of resources than a pathogen by pathogen approach; target product profiles for diagnostics should be refined accordingly. We recommend the establishment of a global forum to bring together representatives from all key stakeholders required for the response to develop a coordinated implementation plan. In addition, we should explore if and how existing mechanisms to address challenges to the vaccines sector, such as Coalition for Epidemic Preparedness Innovations and Gavi, could be expanded to cover diagnostics.

The most performing techniques enabling early diagnosis of infectious diseases rely on nucleic acid detection. Today, because of their high technicality and cost, nucleic acid amplification tests (NAAT) are of benefit only to a small fraction of developing countries population. By reducing costs, simplifying procedures and enabling multiplexing, paper microfluidics has the potential to considerably facilitate their accessibility. However, most of the studies performed in this area have not quit the lab. This letter brings NAAT on paper closer to the field, by using clinical samples and operating in a resource-limited setting. We first performed isothermal reverse transcription and Recombinase Polymerase Amplification (RT-RPA) of synthetic Ribonucleic Acid (RNA) of Ebola virus using paper microfluidics devices. We further applied this method in Guinea to detect the presence of Ebola virus in human sample RNA extracts, with minimal facilities (carry-on detection device and freeze-dried reagents on paper). RT-RPA results were available in few minutes and demonstrate a sensitivity of 90.0% compared to the gold-standard RT-PCR on a set of 43 patient samples. Furthermore, the realization of a nine-spot multilayered device achieving the parallel detection of three distinct RNA sequences opens a route toward the detection of multiple viral strains or pathogens.

In the context of the coronavirus disease 2019 (COVID-19) pandemic there has been an increase of the use of antigen-detection rapid diagnostic tests (Ag-RDT). The performance of Ag-RDT vary greatly between manufacturers and evaluating their analytical limit of detection (LOD) has become high priority. Here we describe a manufacturer-independent evaluation of the LOD of 19 marketed Ag-RDT using live SARS-CoV-2 spiked in different matrices: direct culture supernatant, a dry swab, and a swab in Amies. Additionally, the LOD using dry swab was investigated after 7 days’ storage at − 80 °C of the SARS-CoV-2 serial dilutions. An LOD of ≈ 5.0 × 102 pfu/ml (1.0 × 106 genome copies/ml) in culture media is defined as acceptable by the World Health Organization. Fourteen of 19 Ag-RDTs (ActiveXpress, Espline, Excalibur, Innova, Joysbio, Mologic, NowCheck, Orient, PanBio, RespiStrip, Roche, Standard-F, Standard-Q and Sure-Status) exceeded this performance criteria using direct culture supernatant applied to the Ag-RDT. Six Ag-RDT were not compatible with Amies media and a decreased sensitivity of 2 to 20-fold was observed for eleven tests on the stored dilutions at − 80 °C for 7 days. Here, we provide analytical sensitivity data to guide appropriate test and sample type selection for use and for future Ag-RDT evaluations.

The diagnosis of infectious diseases is entering a new and interesting phase. Technologies based on paper microfluidics, coupled to developments in isothermal amplification of Nucleic Acids (NAs) raise opportunities for bringing the methods of molecular biology in the field, in a low setting environment. A lot of work has been performed in the domain over the last few years and the landscape of contributions is rich and diverse. Most often, the level of sample preparation differs, along with the sample nature, the amplification and detection methods, and the design of the device, among other features. In this review, we attempt to offer a structured description of the state of the art. The domain is not mature and there exist bottlenecks that hamper the realization of Nucleic Acid Amplification Tests (NAATs) complying with the constraints of the field in low and middle income countries. In this domain however, the pace of progress is impressively fast. This review is written for a broad Lab on a Chip audience.

In 2020, the World Health Organization (WHO) recommended two SARS-CoV-2 lateral flow antigen-detecting rapid diagnostics tests (Ag-RDTs), both initially with nasopharyngeal (NP) sample collection. Independent head-to-head studies are necessary for SARS-CoV-2 Ag-RDT nasal sampling to demonstrate comparability of performance with nasopharyngeal (NP) sampling. We conducted a head-to-head comparison study of a supervised, self-collected nasal mid-turbinate (NMT) swab and a professional-collected NP swab, using the Panbio™ Ag-RDT (distributed by Abbott). We calculated positive and negative percent agreement between the sampling methods as well as sensitivity and specificity for both sampling techniques compared to the reference standard reverse transcription polymerase chain reaction (RT-PCR). A SARS-CoV-2 infection could be diagnosed by RT-PCR in 45 of 290 participants (15.5%). Comparing the NMT and NP sampling the positive percent agreement of the Ag-RDT was 88.1% (37/42 PCR positives detected; CI 75.0–94.8%). The negative percent agreement was 98.8% (245/248; CI 96.5–99.6%). The overall sensitivity of Panbio with NMT sampling was 84.4% (38/45; CI 71.2–92.3%) and 88.9% (40/45; CI 76.5–95.5%) with NP sampling. Specificity was 99.2% (243/245; CI 97.1–99.8%) for both, NP and NMT sampling. The sensitivity of the Panbio test in participants with high viral load (> 7 log10 SARS-CoV-2 RNA copies/mL) was 96.3% (CI 81.7–99.8%) for both, NMT and NP sampling. For the Panbio supervised NMT self-sampling yields comparable results to NP sampling. This suggests that nasal self-sampling could be used for to enable scaled-up population testing.Clinical Trial DRKS00021220.

BackgroundYellow fever (YF) is an acute viral hemorrhagic disease transmitted by Aedes mosquitoes. The causative agent, the yellow fever virus (YFV), is found in tropical and subtropical areas of South America and Africa. Although a vaccine is available since the 1930s, YF still causes thousands of deaths and several outbreaks have recently occurred in Africa. Therefore, rapid and reliable diagnostic methods easy to perform in low-resources settings could have a major impact on early detection of outbreaks and implementation of appropriate response strategies such as vaccination and/or vector control.MethodologyThe aim of this study was to develop a YFV nucleic acid detection method applicable in outbreak investigations and surveillance studies in low-resource and field settings. The method should be simple, robust, rapid and reliable. Therefore, we adopted an isothermal approach and developed a recombinase polymerase amplification (RPA) assay which can be performed with a small portable instrument and easy-to-use lyophilized reagents. The assay was developed in three different formats (real-time with or without microfluidic semi-automated system and lateral-flow assay) to evaluate their application for different purposes. Analytical specificity and sensitivity were evaluated with a wide panel of viruses and serial dilutions of YFV RNA. Mosquito pools and spiked human plasma samples were also tested for assay validation. Finally, real-time RPA in portable format was tested under field conditions in Senegal.Conclusion/SignificanceThe assay was able to detect 20 different YFV strains and demonstrated no cross-reactions with closely related viruses. The RPA assay proved to be a robust, portable method with a low detection limit (<21 genome equivalent copies per reaction) and rapid processing time (<20 min). Results from real-time RPA field testing were comparable to results obtained in the laboratory, thus confirming our method is suitable for YFV detection in low-resource settings.

The emergence of novel SARS-CoV-2 variants of concern (VOCs) requires investigation of a potential impact on diagnostic performance, especially on Antigen-detecting rapid antigenic tests (Ag-RDT). Although anecdotal reports have been circulating that Omicron is in principle detected by several Ag-RDTs, no published data are a yet available for the newly emerged Omicron variant. Here, we have performed an analytical sensitivity testing with cultured virus in seven Ag-RDTs for their sensitivity to Omicron compared to data earlier obtained on VOCs Alpha, Beta Gamma and Delta and a pre-VOC isolate of SARS-CoV-2. Overall, we have found a tendency towards lower sensitivity for Omicron compared to pre-VOC SARS-CoV-2 and the other VOCs across tests. Importantly, while analytical testing with cultured virus may be a proxy for clinical sensitivity, is not a replacement for clinical evaluations which are urgently needed for Ag-RDT performance in Omicron-infected individuals.

Crimean-Congo hemorrhagic fever (CCHF) is a zoonosis caused by a Nairovirus of the family Bunyaviridae . Infection is transmitted to humans mostly by Hyalomma ticks and also by direct contact with the blood or tissues of infected humans or viremic livestock. Clinical features usually include a rapid progression characterized by hemorrhage, myalgia and fever, with a lethality rate up to 30%. CCHF is one of the most widely distributed viral hemorrhagic fevers and has been reported in Africa, the Middle East and Asia, as well as parts of Europe. There is no approved vaccine or specific treatment against CCHF virus (CCHFV) infections. In this context, an accurate diagnosis as well as a reliable surveillance of CCHFV infections is essential. Diagnostic techniques include virus culture, serology and molecular methods, which are now increasingly used. The European Network for the Diagnostics of “Imported” Viral Diseases organized the first international external quality assessment of CCHVF molecular diagnostics in 2011 to assess the efficiency and accurateness of CCHFV molecular methods applied by expert laboratories. A proficiency test panel of 15 samples was distributed to the participants including 10 different CCHFV preparations generated from infected cell cultures, a preparation of plasmid cloned with the nucleoprotein of CCHFV, two CCHFV RNA preparations and two negative controls. Forty-four laboratories worldwide participated in the EQA study and 53 data sets were received. Twenty data sets (38%) met all criteria with optimal performance, 10 (19%) with acceptable performance, while 23 (43%) reported results showing a need for improvement. Differences in performance depended on the method used, the type of strain tested, the concentration of the sample tested and the laboratory performing the test. These results indicate that there is still a need for improving testing conditions and standardizing protocols for the molecular detection of Crimean-Congo hemorrhagic fever virus.