# Development and evaluation of fluorescent recombinase polymerase amplification (RPA)-based method for rapid detection of Necator americanus

## TL;DR

A new fluorescent RPA method was developed to quickly and accurately detect the hookworm Necator americanus, offering better sensitivity and portability for field use.

## Contribution

A novel fluorescent RPA assay targeting the ITS2 gene of N. americanus with high sensitivity and specificity for field diagnostics.

## Key findings

- The fluorescent RPA assay achieved 100% sensitivity and 100% specificity in lab validation against Kato-Katz and semi-nested PCR.
- In field validation, the assay showed 90.0% sensitivity and 91.1% specificity for N. americanus detection.
- The method outperformed semi-nested PCR in sensitivity and is suitable for on-site testing in endemic regions.

## Abstract

Necator americanus is the predominant species causing hookworm infections in humans. Despite advancements in prevention strategies, mild cases of infection still occur, highlighting the need for improved detection technology. Recombinase Polymerase Amplification (RPA) is an isothermal molecular diagnostic known for its sensitivity, speed, portability, and widespread application in detecting various pathogens. Although several molecular assays are available for N. americanus, they have limitations in detecting mild N. americanus infections.

Fluorescent RPA primers and probes targeting the N. americanus internal transcribed spacer 2 (ITS2) gene were developed. The method’s detection limit was assessed via serial dilution of genomic DNA. Specificity was confirmed against Clonorchis sinensis, Schistosoma japonicum, Fasciola hepatica, Ascaris lumbricoides, Enterobius vermicularis and Ancylostoma duodenale. Thirty samples identified as positive by Kato-Katz, along with 11 samples identified as negative by the method, were tested to evaluate the sensitivity and specificity of fluorescent RPA. Additionally, 287 field samples were tested for validation with these methods. All positive samples were identified as either N. americanus or A. duodenale.

This study successfully developed a fluorescent RPA assay targeting the ITS2 gene of N. americanus. The length of the amplified fragment was 237 bp. Optimized conditions were achieved, resulting in a minimum detection limit of 1fg/µL, with no cross-reactivity with other pathogens. In laboratory validation, the fluorescent RPA assay demonstrated 100% sensitivity (30/30) and 100% specificity (11/11) compared to the Kato-Katz, and 100% sensitivity (29/29) and 91.7% specificity (11/12) when compared to the semi-nested PCR. In field validation using human fecal samples, the fluorescent RPA assay showed a sensitivity of 90.0% (36/40) and a specificity of 91.1% (225/247) compared to the Kato-Katz. And the sensitivity of the fluorescent RPA method compared to the semi-nested PCR method was 100% (34/34), while the specificity was 90.5% (229/252).

The fluorescent RPA assay presents a rapid and dependable method for detecting N. americanus in fecal samples. Its high sensitivity and specificity provide significant utility for field surveillance and early identification of N. americanus infections. This advancement could facilitate the rapid molecular diagnosis of N. americanus disease in hookworm-endemic regions.

The fluorescent recombinase polymerase amplification (RPA) technique is a state-of-the-art method for isothermal DNA amplification, utilizing a portable instrument and lyophilized reagents. This innovative approach allows for on-site testing using a portable fluorescence detector, significantly enhancing its utility in field settings. In our study, we evaluated 287 samples obtained directly from the field. The conventional semi-nested PCR assay demonstrated a sensitivity of 62.5% and a specificity of 96.4%. In contrast, the newly developed fluorescent RPA assay exhibited superior performance, with a sensitivity of 90.0% and a specificity of 91.1% for detecting N. americanus infection. These findings highlight the potential of fluorescent RPA as a more sensitive method for identifying N. americanus. Moreover, our research suggests that the established fluorescent RPA could play a crucial role in various applications, such as surveillance and epidemiological and clinical investigations related to N. americanus. Further studies are warranted to explore its full potential and optimize its implementation in practical settings. This advancement represents a significant step forward in the field of diagnostic techniques for parasitic infections.