# Targeting surface cell antigen 2 increases sensitivity of Rickettsia typhi detection

## TL;DR

A new qPCR test targeting the sca2 gene improves detection of Rickettsia typhi, which causes murine typhus, especially in cases with low bacterial levels.

## Contribution

A novel qPCR assay targeting the repetitive sca2 gene increases sensitivity for detecting Rickettsia typhi in patient blood.

## Key findings

- The sca2 qPCR assay showed 59.09% sensitivity compared to 36.36% for the ompB assay.
- DNA copy numbers detected by sca2 were 2.933 log units higher than those detected by ompB.
- The sca2 assay is more effective for detecting low concentrations of R. typhi in patient samples.

## Abstract

Murine typhus is a flea-borne disease caused by Rickettsia typhi that typically presents as an acute febrile illness. The diagnosis is often missed, leading to delays in appropriate treatment. A qPCR targeting a single gene (ompB) for R. typhi is widely used for diagnosis; however, it has low sensitivity for detecting bacterial DNA in patients’ blood.

We aimed to increase sensitivity of detection of R. typhi using qPCR by targeting a gene containing repetitive sequences, sca2 (surface cell antigen 2).

We compared diagnostic accuracy with the standard assay targeting single sequence ompB (outer membrane protein B). Specificity, sensitivity, and bacterial load measurement of both assays were compared using stored EDTA-anticoagulated buffy coat samples from 88 patients with febrile illness at Mahosot Hospital or provincial hospitals in Laos. Among these, 55 cases were confirmed as positive or negative by first culturing Rickettsia spp. from patients’ EDTA blood, followed by assessment with IFA and ompB PCR, and buffy coat from 6 additional cases was confirmed by ompB PCR. Ten further positive cases were confirmed by IFA using paired sera, and 17 cases classified as negative for scrub typhus and murine typhus based on Rapid Diagnostic Test (RDT) results were included in the evaluation.

The sca2 qPCR assay showed 59.09% sensitivity (95% CI, 38.73-76.74%) and 100% specificity (93.98-100%) for detection of R. typhi. In comparison, ompB assay demonstrated 36.36% sensitivity (95% CI, 19.73-57.05%) and 100% specificity (95% CI 93.98-100%). DNA copy number determined using the sca2 gene was approximately 2.933 log unit higher than that determined using ompB gene (median, 16,500 copies/μL; IQR, 13,045–40,000 versus median, 19.25 copies/μL; IQR, 11.11-56.62, P < 0.0001).

This study suggests qPCR targeting sca2 increases frequency of detection of R. typhi in patients with low bacterial DNA concentrations.

Rickettsia typhi is a bacterium that causes murine typhus. This disease is often overlooked because its symptoms are very similar to those of other infections. It can also be hard to detect because patients usually have a low blood bacterial concentration, making it difficult for current assays to detect. We focused on a part of the bacteria called sca2 (surface cell antigen 2), which has repeating sections, thinking that looking for these repeating sections could make molecular detection methods more sensitive, allowing them to detect even very low levels of the bacteria. We designed a new test that targets these repeating sections of the sca2 gene and tested it using different strains of R. typhi and related bacteria. Compared to the conventional test that looks for the ompB (outer membrane protein B) gene, the new sca2 test was able to detect lower amounts of the bacteria in patient samples. This means it could be a more effective tool for diagnosing the disease, especially when the amount of bacteria in a patient’s blood is very low.