# A Set of Rapid Diagnostic Tool for Babesia microti Infection

## TL;DR

A new low-cost and rapid diagnostic test for Babesia microti infection is developed, offering high sensitivity and specificity for use in resource-limited areas.

## Contribution

The CPA-VF assay is a novel, low-cost, and instrument-free diagnostic method for detecting B. microti with high sensitivity and specificity.

## Key findings

- The CPA-VF assay detects 2.56 fg/reaction with 95.5% sensitivity and 95.5% specificity compared to nested PCR.
- The assay costs $3.8 per test, significantly lower than traditional methods like PCR.
- It shows no cross-reactivity with related parasites like B. duncani, B. divergens, or Plasmodium.

## Abstract

Human babesiosis caused by Babesia microti is an emerging tick‐borne zoonosis, with a global pooled prevalence of 2.23% and regional peaks in Europe (4.17%) and North America (1.54%). Traditional diagnostics like microscopy and polymerase chain reaction (PCR) suffer from low sensitivity in low‐parasitemia cases or high costs ($230/test), necessitating accessible, rapid assays for resource‐limited regions.

A cross‐priming amplification combined with vertical flow visualization (CPA‐VF) assay, a straightforward molecular method targeting the 18S rRNA gene of
B. microti
, requires minimal equipment and facilitates rapid detection.

Sensitivity/Specificity: The CPA‐VF assay detected 2.56 fg/reaction (equivalent to 0.000004% parasitic red blood cells), with a sensitivity of 95.5% matching that of RT‐PCR but at a 60‐fold lower cost ($3.8/test). It showed no cross‐reactivity with
B. duncani, B

. divergens

, or Plasmodium. Clinical Validation: Testing 49 positive samples (19 experimentally infected mice +30 artificially spiked) and 492 field samples, CPA‐VF demonstrated 95.5% sensitivity (95% CI: 88.2–98.7) and 95.5% specificity compared to nested PCR (nPCR). Intra‐assay coefficients of variation (CV) was 2.1%–7.2% and inter‐assay kappa coefficient was 0.94, confirming reliability.

CPA‐VF is a rapid, low‐cost ($3.8/test), and instrument‐free diagnostic tool for
B. microti
, particularly suitable for endemic regions where timely diagnosis reduces mortality risks from misdiagnosis as malaria. Its portability and visual readout address critical gaps in resource‐constrained settings.

This study develops a cross‐priming amplification combined with vertical flow visualization (CPA‐VF) assay for rapid, low‐cost detection of Babesia microti, achieving a detection limit of 2.56 fg/reaction (equivalent to 0.000004% parasitic red blood cells) with 95.5% sensitivity and specificity compared to nested PCR. The assay, costing $3.8 per test and yielding results in 70 min, shows no cross‐reactivity with related parasites, making it particularly suitable for resource‐limited endemic regions to address diagnostic gaps in human babesiosis.