Foundation for Innovative New Diagnostics

Effective Lassa fever management relies on diagnostics; however, diagnostic gaps have been documented, particularly for point-of-care (POC) tests for use in remote settings where Lassa fever virus (LASV) is endemic. We assessed the current LASV diagnostic landscape to determine whether these gaps persist. Commercially available and pipeline diagnostic tests for LASV diagnosis were identified via databases, news platforms, internet queries, and manufacturer outreach. Literature searches identified LASV development of molecular tests, immunoassays, and supportive sample management protocols, as well as independent performance evaluations, external quality assurance studies, and prior diagnostic landscapes. Twenty-four commercial LASV tests were found to be currently on the market, with only four of the molecular tests having documented regulatory approval. Two LASV molecular tests could be implemented at the near-POC level. Lateral flow LASV immunoassays for true-POC that had been in development or evaluation are now discontinued; available data indicate that POC performance failed to meet an acceptable diagnostic accuracy. Several innovative but early-stage LASV technologies were identified in the literature, none of which show a clear pathway to commercialization. Compared to prior reviews, this diagnostic landscape noted improvements for LASV lineage inclusivity but currently no diagnostic tools for true-POC settings. Key gaps revealed in this landscape impact the "diagnostic readiness" for LASV outbreak management in endemic areas, particularly for early detection in non-laboratory settings. Diagnostic testing at the true-POC level remains a critical unmet need.

Malaria remains a major global health challenge. Prompt, accurate diagnosis is crucial for effective case management. Current diagnostic approaches rely on invasive or minimally invasive sampling via venous or fingerpick blood draw, posing a risk to healthcare workers via the handling of potentially infectious body fluids. They may also be a barrier to recipients, particularly in malaria-endemic areas where there is routine testing of non-symptomatic individuals. Non-invasive tests based on saliva, exhaled volatile organic compounds (VOCs), and transdermal detection have the potential to increase case detection and linkage to care while reducing biosafety risks. Knowledge gaps exist regarding the feasibility and acceptability of these tools. This qualitative study, conducted in Indonesia, Peru, and Rwanda, aimed to generate evidence from end-users around the potential adoption of non-invasive diagnostic technologies, to determine whether these technologies are fit for purpose. Between October and November 2023, 24 semi-structured interviews were conducted with stakeholders and professionals working at borders, and 16 focus group discussions were conducted with caregivers of children under 5 years, pregnant women, healthcare workers, teachers and other community members (139 participants in total). The comfort non-invasive approaches offer to recipients of care was widely noted, especially compared with blood draw, which is considered painful for children. Ease of use and rapid diagnostic capabilities enabling real-time disease diagnosis were perceived as particularly beneficial in remote areas with limited healthcare infrastructure. Device portability was seen as a game-changer. Major concerns were the lack of information regarding the accuracy of non-invasive tools compared with established methods, the lack of trust in tests targeting specimens other than blood products, and their inability to differentiate between malaria species. Early engagement with stakeholders in the development of novel interventions is essential to ensure that end products meet the needs of communities.

Background: Gambian human African trypanosomiasis (gHAT) is a neglected tropical disease caused by Trypanosoma brucei gambiense transmitted by tsetse flies (Glossina). Humans are the major host and mass screening and treatment of the human population has contributed to the elimination of gHAT as a public health problem in many countries, including Uganda. There is some evidence that animals may act as hosts for T. b. gambiense which may undermine efforts to eliminate transmission. We undertook a study to determine whether cattle and pigs were harbouring T. b. gambiense in a region of north-western Uganda where efforts to eliminate transmission of gHAT were ongoing. Methodology: Blood was collected from 2,775 cattle and 571 pigs and examined for the presence of trypanosomes by the haematocrit centrifugation technique (HCT). In addition, polymerase chain reaction (PCR) was used to amplify the trypanosomal internal transcribed spacer (ITS1). Those whose ITS-1 results indicated the Trypanozoon group (T. brucei) were further tested by sub-species specific PCRs for Trypanosoma brucei gambiense and T. b. rhodesiense. The results were analysed by species of infecting parasite, geographic location and presence or absence of vector control activities in the area. Principal Findings: No samples were found positive for potentially human infective trypanosomes. Among the cattle samples, 2.2% were positive by HCT (95% CI = 1.7% - 2.8%), while 5% were positive by PCR (95% CI = 4.3% - 5.9%), but this varied depending on location with up to 26.5% positive in one village. Among pigs, 3 samples (0.6%; 95% CI 0.2% - 1.6%) were positive by HCT and none were positive by PCR. There was no significant association between trypanosome species and vector control activities (p > 0.05). Based on these results it was concluded with 95% confidence that human infective trypanosomes are not circulating in livestock at a prevalence greater than 1/1000, and when this is augmented with results of previous surveys the parasite is not circulating at a prevalence greater than 1/2734. At the level of the trypanosome species, there is no significant difference between the frequencies of species in vector controlled versus non-vector controlled areas (p > 0.05). Conclusions: These results support the evidence base that livestock are not an important reservoir and therefore not a threat to the elimination of Gambian human African trypanosomiasis in Uganda.

Introduction Recent public health emergencies of international concern (PHEIC) have highlighted the need to develop prophylactic treatments and effective diagnostics for pandemic preparedness. In particular, the 100 Days Mission, an initiative to respond to a PHEIC within the first 100 days after declaration, has put into focus the importance of the early availability of effective diagnostic tests. Reference materials are valuable to support the development, assessment and calibration of these tests. The use of World Health Organization (WHO) International Standards (IS), the highest order calibrant, allows for the harmonisation of data reporting which assists performance evaluation. WHO IS for emerging virus molecular testing are usually made by inactivating the pathogen of interest and they undergo extensive evaluation in multi-laboratory collaborative studies. However, the development of such reference material for Lassa virus (LASV) is challenging due to the requirement for high containment level facilities to handle LASV. Further, the development of molecular tests for the detection of LASV RNA is confounded by the high sequence diversity amongst circulating lineages and a need to evaluate performance against them. Methods To circumvent this, we have developed a low containment alternative using chimeric lentiviral particles packaging the RNA of five prototype LASV lineages. These were evaluated alongside the whole inactivated virus (Josiah strain) by 18 laboratories, across 25 methods, as part of an International multi-lab collaborative study. Results The data showed equivalent performance to the authentic virus in reducing interlaboratory variability; also, they highlighted the variability in detection across LASV lineages and supported the establishment of the chimeric particles as a WHO Reference Panel for Lassa virus RNA, alongside a WHO IS. This will greatly facilitate molecular test development and evaluation. Discussion Overall, the strategy has the flexibility to be applied to a range of high priority viral families and can rapidly be implemented to expand to new viral sequences of high consequence which may impact molecular test performance. This represents an important pandemic preparedness initiative to support the response to the next outbreak, including Disease X.

Background:Diagnosing tuberculosis in children remains challenging due to the paucibacillary nature of the disease, non-specific symptoms, and difficulty collecting samples. New tests are urgently needed. We evaluated the diagnostic accuracy of FujiLAM version 1 urine test in children under 5 years with presumptive tuberculosis in Uganda.Methods:This was a cross-sectional evaluation nested in the NOD-pedFEND diagnostic study. Children under 5 years with signs or symptoms of tuberculosis were recruited from three hospitals in Uganda. All participants underwent comprehensive baseline investigations, including culture and Xpert MTB/RIF Ultra on reference samples and chest X-ray. Culture and Xpert MTB/RIF Ultra results were used to define a microbiological reference standard. FujiLAM version 1 testing was performed on fresh urine at the baseline visit.Results:Seventy-nine children were included in the study. Fifteen participants (19%) were classified as having microbiologically confirmed TB, 44 (56%) as unconfirmed TB, thirteen (16%) as unlikely TB, and seven participants (9%) were unclassifiable. Only one participant (with unlikely TB) had a positive FujiLAM test. Culture and Xpert MTB/RIF Ultra on reference samples were negative in this child, indicating poor diagnostic accuracy with a sensitivity of 0% (95% CI: 0 – 21.8) of FujiLAM version 1 against the microbiological reference standard.Conclusion:The poor diagnostic accuracy of FujiLAM version 1 in children under five years of age makes it unsuitable as a diagnostic test in this age group. Further evaluation with the optimized version 2 of this test is needed.

Nucleic acid-based assays are the diagnostic gold standard for filoviruses, including Ebola (EBOV) and Sudan (SUDV) viruses. However, outbreaks in areas with limited laboratory infrastructure highlight the need for simpler diagnostic tests that can be rapidly and safely used in the field. We evaluated eight antigen rapid diagnostic tests (Ag-RDTs) for their ability to detect EBOV and SUDV. Analytical panels using virus cell slurries were used to assess limit of detection, and clinical samples were tested to determine sensitivity and specificity. Five Ag-RDTs detected EBOV and three detected SUDV, although clinical sensitivity was low (20-40% for EBOV, 33% for SUDV), improving only with higher viral loads. All assays demonstrated 100% clinical specificity with no cross-reactivity. Although none of the evaluated Ag-RDTs are suitable for routine diagnosis, some may be useful in high viral load contexts such as cadaver testing. Our findings highlight the need to improve Ag-RDT sensitivity or develop high-sensitivity point-of-care molecular diagnostics.

Assess Truenat MTB Ultima (Ultima) diagnostic accuracy, alongside standard-of-care Truenat MTB Plus (MTB Plus) and Xpert MTB/RIF Ultra (Ultra) tests, for pulmonary tuberculosis (TB) diagnosis in a high-burden setting. We secondarily evaluated Truenat MTB-RIF Dx (MTB-RIF Dx) on Ultima- or MTB Plus-positive samples for rifampicin susceptibility diagnosis. Adults (≥18 years; n = 498) with presumptive TB self-presenting to primary care clinics in Cape Town, South Africa (19/02/2016-22/02/2023) provided sputa. The microbiological reference standard was a single culture for TB and MTBDRplus on an isolate for rifampicin susceptibility. In total, 54% (n = 269) of the participants had HIV, and 42% (n = 210) had previous TB. The proportion of Ultima and MTB Plus unsuccessful results was 14% (95% CI 11, 16) and 20% (17, 23), respectively, with at least half resolving upon retesting the same eluate. In a three-way analysis, Ultima, MTB Plus, and Ultra had TB sensitivities of 90% (85, 93), 84% (78, 88), and 92% (87, 95) and specificities of 85% (80, 88), 95% (92, 97), and 95% (92, 97). Ultima specificity did not improve with Ultra in the reference standard. MTB-RIF Dx had high unsuccessful result rates that varied if done on the day of DNA extraction or on Ultima- [18% (10, 26) vs. 44% (35, 51) if after day of extraction] or MTB Plus-positive eluates [9% (3, 16) vs. 27% (18, 35)]. Same day rifampicin susceptibility testing was often unsuccessful in samples with the "very low" semiquantitation category reported by Ultima [75% (65, 86)] or MTB Plus [73% (58, 89)] but had 100% (40, 100) sensitivity and 99% (96, 100) specificity (on both MTB Plus- or Ultima-positive DNA). Reagent lot variation in unsuccessful and false positive results was observed. Ultima met the minimum sensitivity recommended by the WHO for TB detection, but specificity, reagent lot variation, and unsuccessful results were suboptimal.

A target specimen profile (TSP) corresponds to the required characteristics of the specimen panels needed to demonstrate that a diagnostic kit meets the target product profile (TPP). TSPs can guide biobanks in the prospective collection of sample panels to support the development and validation of diagnostics.

A critical impediment to efficient outbreak response is the availability of timely and complete data on cases and their linkage to care. To inform solutions to this issue, this study investigated the utility of self-testers reporting their coronavirus disease 2019 results using a mobile health platform. Our study has demonstrated that the mobile health platform is feasible; it achieved a 74.5% reporting rate, indicating a strong likelihood of data entry into the unstructured supplementary service data platform. Support from community health workers (CHWs) and healthcare professionals, particularly nurses, contributed to this success, especially among users with limited digital literacy. Specifically, 44.9% of self-test results were submitted by study participants themselves, 24.7% were submitted with the assistance of healthcare professionals, and 30.4% were submitted with the assistance of CHWs. The platform broadens the population base by increasing access and equity, allowing participation even among users without smartphones. Additionally, it integrates rapid antigen diagnostic tests with digital reporting, simplifying data processing and enabling standardized screening, real-time data capture, and effective patient follow-up. This technology also lays a foundation for pandemic preparedness in low- and middle-income countries by demonstrating the feasibility of fully integrating response loops for disease management and interventions. Future response loops could leverage artificial intelligence, machine learning, and integration with existing health surveillance systems, directly benefiting users through enhanced support.

Limited diagnostic access in resource-limited settings contributes to poor health outcomes among bacterial sepsis patients and the spread of antimicrobial resistance (AMR). Molecular diagnostic profiling of AMR may enable faster targeting of antibiotic therapies, improving clinical outcomes, reducing AMR development, and saving costs. We modeled the impact of a culture-dependent molecular test for pathogen identification and resistance testing among hospitalized bloodstream infection patients to guide effective and cost-efficient implementation of these tools. We evaluated patient mortality, antibiotic use, hospital-associated infections, hospital days, and costs under the standard of care (empiric therapy, blood culture, phenotypic susceptibility testing) compared with molecular diagnostics by varying culture and susceptibility testing coverage, culture turnaround time, and AMR prevalence. The greatest impact of the molecular test occurred with 100% diagnostic coverage, shorter culture turnaround time, and high AMR prevalence, reducing up to 6% of deaths (interquartile range [IQR], 0%-12.1%), 5% of hospital days (IQR, 0.1%-10.7%), and 21% of days on inappropriate antibiotic therapy (IQR, 18.2%-24.4%). The minimum cost per molecular test performed, offset by cost savings, ranged from $109 in India to $585 in South Africa across all modeled scenarios. In high AMR burden settings with blood culture infrastructure supporting fast turnaround times, a molecular test could improve health outcomes of bloodstream infection. Impact is limited by delayed turnaround times and the effectiveness of empiric therapy. Molecular diagnostics implemented at $100 or less can generate healthcare-system cost savings, supporting their adoption to improve health outcomes and reduce AMR while remaining cost-neutral.