Vector-borne disease impact and detection challenges
In one hand, the emergence of Hyalomma ticks in regions previously dominated by Ixodes and Dermacentor species has transformed the epidemiological landscape of tick-borne diseases across the Mediterranean Basin and parts of Central Europe. On the other hand, urbanisation associated with greener city favor movement of potential host for ticks, leading to an increase of host-vector-pathogen interactions. Medicalentomologist Dr. Eva Krupa from the Institut Pasteur in Paris focuses urbanization effect on ticks density and TBP prevalence and operational side of detection. Vector-borne diseases account for >17% of infectious diseases globally and over 700,000 deaths annually, yet a substantial portion of their microbial diversity remains uncharacterised or undetected. In this context, ticks are second vector of importance in human health and primary vector in veterinary health.
Ticks are particularly challenging matrices for molecular assays due to:
- A highly sclerotized cuticle.
- High levels of PCR inhibitors (e.g. heme, digested blood components).
- Large, highly variable blood meals, especially in engorged females (up to 200x their unfed weight).
A stepwise workflow: from field to result
Dr. Krupa structured the surveillance workflow into the following modular steps:
Collect – Clean – Lyse – Extract – Detect – Interpret
Key determinants of assay performance include:
- Collection mode: questing ticks collected by flagging or dragging behave differently from host-derived ticks in terms of pathogen load and inhibitor profile.
- Storage conditions: flash-freezing tends to preserve RNA integrity substantially better than prolonged storage in ethanol. Washing steps reduce contamination from environmental pathogens.
- Mechanical disruption: both insufficient and excessive homogenisation can compromise downstream detection.
Typical RNA yields from Ixodes ricinus range from 5–30 ng/µL, with engorged females exceeding 100 ng/µL, but such measurements may overestimate functional template due to inhibitors.
Choice of molecular platform
Dr. Krupa outlined the advantages and limitations of several methodological classes.
Targeted PCR / qPCR
Remain the reference standard for specificity and robustness. Universal Borrelia primers can detect >20 species simultaneously, but genus-level multiplexing can complicate interpretation in co-infection settings. Additional steps like sequencing may be require for a better interpretation of PCR results. High-throughput multiplex or microfluidic platforms allow screening of dozens of pathogens across tens of samples, but require continuous primer-panel maintenance and are cost-intensive.
Isothermal amplification (LAMP, RPA)
LAMP: operates at 60–65 °C; has demonstrated utility for spotted fever group (SFG) Rickettsia sp. and Borrelia sp. detection in field-collected ticks.
RPA: runs at 37–42 °C with turnaround times of 20–30 minutes; in PREPARE-TID consortium testing, it performed well on cultured tick-borne pathogens but was more sensitive to inhibitors in direct tick extracts.
Next-generation sequencing (NGS)
Essential for pathogen discovery, detection of unexpected agents, and characterization of microbiome–pathogen–symbiont interactions. A typical tick metagenome yields 10⁶–10⁷ reads, with >95% often attributable to host or commensal organisms; pathogen reads can be as low as 0.01%, requiring stringent bioinformatic filtering.
Lab-in-a-suitcase: field-deployable molecular workflows
Dr. Krupa presented the PREPARE-TID “lab-in-a-suitcase” concept:
- Approx. 15 kg, operable on a 12 V power supply.
- Includes manual grinders or bead-beaters, heat blocks, portable centrifuges, pipettes, and isothermal detection devices.
- For detection of pathogen in emergency or in remote areas
In comparative studies on natural Ixodes ricinus samples infected with Borrelia sp., three extraction configurations were assessed:
Protocol | Grinding method | Extraction chemistry | Yield | PCR/qPCR outcome |
1 | Bead-beating | Commercial RNA kit | High | Positive in all samples |
2 | Manual grinding | Reverse-purification (magnetic) | Moderate, but field-ready | Concordant with Method 1 |
3 | Bead-beating | Moderate, but field-ready | Negative in some replicates |
The reverse-purification method, produced Ct values within ±3 cycles of the reference kit, indicating comparable sensitivity and demonstrating that inhibitor-resistant chemistries can support field-level extractions.
Parallel experiments using Anopheles coluzzii mosquitoes fed with Plasmodium falciparum confirmed equivalent diagnostic performance relative to standard laboratory kits.
Extending the toolkit: sequencing and advanced detection
Dr. Krupa highlighted several technologies that complement core PCR and isothermal workflows:
- Portable nanopore sequencers (e.g. MinION) for near real-time species or strain identification.
- CRISPR-Cas–based diagnostics with sequence-specific fluorescent readouts.
- MALDI-TOF mass spectrometry for profiling infection-associated protein signatures.
The strategic aim is to align technology choice with contextual constraints (infrastructure, throughput, sample type) while maximizing sensitivity and preserving fragile nucleic acids.
Bring your diagnostics to the field
We work with ecologists, surveillance programs, and molecular laboratories to integrate ecological forecasts into diagnostic strategies.
- Contact us to integrate SwiftX toolbox for your virus detection and analysis in ticks and beyond
- Recommended further readings from Dr. Krupa:
- Watch the full PREPARE-TID Ep. 4 webinar