A team from Estonia's University of Tartu and molecular diagnostics startup Selfdiagnostics has published a peer-reviewed study describing and clinically validating a recently developed point-of-care molecular assay to diagnose chlamydia infections.
The new assay uses recombinase polymerase amplification, an isothermal amplification method, to detect Chlamydia trachomatis plasmid DNA directly from a patient urine specimen in about 20 minutes with 100 percent specificity and 83 percent sensitivity — better than rapid immune-based tests and comparable to real-time PCR-based tests that require expensive machinery in a central laboratory.
The study is the first peer-reviewed clinical validation of the assay and of Selfdiagnostics' general point-of-care molecular testing system, according to CSO and study co-author Indrek Tulp, who also noted that his company is currently developing a slew of POC molecular tests for sexually transmitted infections, such as chlamydia, using similar isothermal amplification-based direct detection.
Details of the assay were published today in the Journal of Molecular Diagnostics.
The goal of the research, the authors noted, was to develop a clinical diagnostic test for chlamydia that combined the ease of use, speed, and low cost of immunoassays with the accuracy of more complex molecular methods such as real-time PCR. Immunoassays, they noted, can yield a patient result at the time of visit — a crucial aspect, since chlamydia often has no easily discernible symptoms, and about half of tested patients fail to follow up a doctor's visit to receive test results or treatment. However, studies have shown that the mean sensitivities of these tests falls somewhere in the 40 percent to 60 percent range compared to nucleic acid amplification techniques.
Meantime, nucleic acid amplification assays — such as those offered by Roche, Abbott, Hologic, Becton-Dickinson, and Cepheid, among others — have been demonstrated as highly sensitive and specific, but are suitable only for centralized hospital facilities and require trained personnel and expensive equipment.
The new assay attempts to rectify these shortcomings by using isothermal amplification, which in general has shown the ability to amplify target nucleic acids directly from clinical specimens in a number of infectious disease diagnostic assays.
Specifically, the Estonian researchers chose to use recombinase polymerase amplification (RPA), which uses a recombinase complex from T4 bacteriophage to introduce primers to specific DNA sites to initiate an amplification reaction by the strand-displacing DNA polymerase.
RPA does not require template denaturation and can operate at a relatively low and constant temperature, between 38° and 42° C, and has achieved sensitivities comparable to PCR with as few as 10 template DNA copies, producing a detectable amplification signal in about 10 minutes. These characteristics have made the technology an increasingly popular choice for use in POC molecular assays, such as the rapid HIV-1 subtyping test being developed by Seattle's PATH Institute, and the near-patient testing system being developed by Alere, which owns the RPA technology through its acquisition of the method's developer, TwistDx.
The Estonian scientists' assay targets the CDS2 gene of a plasmid specific to sexually transmitted strains of C. trachomatis. Besides the RPA technique, the assay uses heat-based pre-treatment of patient urine samples at about 90° C and visual detection of amplification products on an immunochromatographic flow strip. In general, the researchers noted, it takes about 20 minutes to perform from sample to answer.
In their study, using purified DNA from C. trachomatis-positive urine samples, the scientists determined that their assay could detect CDS2-specific RPA product from as few as 50 copies of plasmid template or 0.2 picograms of C. trachomatis genomic DNA; and that 0.05 to 0.5 microliters of urine alone could contain enough DNA to obtain a positive signal. In addition, the assay was highly specific in terms of discerning purified C. trachomatis DNA from excess genomic DNA from human and other bacteria often found in human urine samples.
For clinical validation, the group tested its assay on urine samples from 70 patients — 51 female and 19 male — attending a sexual health clinic in Estonia. They compared the results of the RPA assay with Roche's Cobas Amplicor CT/NG assay, a real-time PCR-based test generally performed in a central laboratory.
All 58 C. trachomatis-negative samples tested negative using the RPA method, essentially translating to a 100 percent specificity. Of the 12 C. trachomatis-positive urine samples, 10 tested positive and two negative using RPA on a 5-microliter sample of heat-treated urine, translating to a sensitivity of about 83 percent.
When purified DNA was used, the RPA method was able to detect all 12 positive samples. Conversely, when a 10-microliter sample of heat-treated urine was used, the sensitivity of the RPA dropped significantly, primarily due to the copious amounts of PCR inhibitors found in urine, meaning that an optimal clinical sample size for the assay is 5 microliters or less.
The researchers noted that the test sensitivity could increase if sample collection can be optimized to use the first 5 milliliters of collected urine, which studies have shown is particularly rich in C. trachomatis genomic material in infected patients.
Commercial prospects
In an email to PCR Insider, Selfdiagnostics' Tulp, who is also a researcher at the University of Tartu, underscored the importance of the paper as the first publicly available clinical validation of the assay.
Founded in 2008, Selfdiagnostics is developing point-of-care molecular testing technology for a number of sexually transmitted infections, including chlamydia, and as such the paper also serves to generally validate its approach, Tulp said.
"The basic technology behind the platform contains three steps: lysis of cells (including pathogenic ones) in order to reveal target DNA material; detection of pathogen-specific DNA sequences with further nucleic acid amplification; and, finally, producing visual signal with convenient lateral flow technology," Tulp said. "These three key steps work in sequential and additive form meaning that no extraction or purification step is necessary. Conditions of the steps are optimized in a way that they do not interfere or inhibit following steps in any manner."
Tulp noted that RPA is only one of several isothermal amplification methods that could be used in Selfdiagnostics' approach, and that the company has been working with the University of Tartu to screen many of them.
"All of them have their own advantages and disadvantages; none of them is ultimately the best one," Tulp said. "Our [JMD] publication is based on the RPA method, the main advantage [of which] is considered low working temperature (37°C). But, bearing in mind that such devices will be used in various ambient conditions, it still requires [a] temperature-controlling mechanism. In this sense there is not a very big difference to control 37° or 60° to 65° C that is required by other [isothermal] amplification methods. Of course the big advantage is that all of them are isothermal in contrast to PCR, which requires thermal cycling."
Tulp said that Selfdiagnostics believes its approach could be applied to other sample types beyond urine, and that the company's ultimate vision is a non-instrumented, disposable device for screening, near-patient use, and in home settings. In the long term the company would also like to move beyond STIs into other infectious disease areas.
As RPA is only one of several isothermal amplification technologies that the company has evaluated, "because of technological and economical reasons we haven't decided yet the method we will utilize in our first device," Tulp said. "To date we do not have any binding agreements to use [third-party] technologies but we are open for propositions."
Selfdiagnostics hopes to have a prototype diagnostic device to present to investors in 2015, Tulp said.