B-224 Rapid, Extraction-Free, and Portable Molecular Detection of HIV-1 and HCV Directly From Whole Blood
Bibliographic record
Abstract
Abstract Background Early diagnosis of human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV) is key to preventing viral transmission and improving linkage to treatment. Isothermal amplification methods, such as reverse transcription looped-mediated amplification (RT-LAMP), have enabled molecular diagnostics to expand beyond centralized laboratories. Despite the advances in molecular point-of-care (POC) testing, many of these tests are costly and still depend on a reliable power-source, specialized equipment, and cold-chain storage, making them impractical for resource-limited settings. To overcome these barriers, there is a need for innovative molecular diagnostics for blood-borne pathogens. Here, we propose a simple, extraction-free, portable workflow for detecting HIV-1 and HCV from whole blood within 40 minutes. Methods We developed singleplex RT-LAMP-based tests using modified published primer sets. Contrived whole blood samples containing HIV-1 or HCV virions were diluted in equal parts water and loaded directly into optimized RT-LAMP master mixes. To mitigate cold-chain storage dependence, RT-LAMP reactions were performed using a lyophilized master mix. The reactions were heated for 30 minutes using a hand-held, battery-powered heating device for simultaneous virion lysis and amplification. For simple visual detection, amplification was coupled with a lateral flow-based dipstick test. The analytical sensitivity of each detection test was evaluated using contrived whole blood samples ranging in HIV-1 and HCV 1a viral loads. Additionally, HCV detection was evaluated on genotypes 1b, 2b, and 3a. For result confirmation, a custom CRISPR-Cas12a-based assay was used to verify the presence or absence of pathogen-specific amplicons following amplification. Results The diagnostic workflow can be completed within 40 minutes, including less than 10 minutes of hands-on time. At optimal conditions, the HIV-1 and HCV 1a singleplex tests had lower limits of detections of 4.89 log10 cp/mL and 3.77 log10 IU/mL, respectively. Evaluation of the published literature showed that these viral loads are within the ranges observed during acute HIV-1 and HCV infection. HCV detection was also observed for genotypes 1b and 3a, albeit at a lower sensitivity than genotype 1a. No detection was observed for genotype 2b, presumably due to the lower viral loads of the samples. No cross-reactivity between the two tests was observed. Conclusions Our proposed workflow allows for rapid detection of blood-borne pathogens without the need for complex equipment or cold-chain storage, showing potential for application in resource-limited settings. Additional validation within a clinical setting using fingerstick blood remains to be tested.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".