NEW YORK (360Dx) – An international group of researchers led by the University of Chicago has described their use of a low-cost, rapid serotyping assay to diagnose toxoplasmosis in patients just before or during pregnancy.
According to the scientists, the assay demonstrated high accuracy and clinical potential as a diagnostic tool for use in the intensive care unit or in obstetricians' offices. Additionally, LDBIO Diagnostics, which developed the lateral flow immunochromatography assay, called Toxoplasma ICT IgG IGM, is seeking US Food and Drug Administration approval to market the test here.
In a study published yesterday in PLOS Neglected Tropical Diseases, the researchers described their work using LDBIO Dx's toxoplasmosis assay to interrogate 180 sera samples from 116 infected patients. Based on their determination that the assay showed 100 percent sensitivity and 100 percent specificity for distinguishing immunoglobulin G (IgG)/immunoglobulin M (IgM)-positive from IgG/IgM-negative sera, they wrote "Use of such reliable [point-of-care] tests can be cost-saving and benefit patients."
'Not … without consequences'
For most people, toxoplasmosis is relatively benign and disappears on its own without need for any treatment. For expectant mothers, however, the repercussions can be serious. If untreated, congenital toxoplasmosis can cause visual and brain damage in a baby, heightening the need for early diagnosis.
"This is not a disease without consequences," said UChicago School of Medicine ophthalmology professor Rima McLeod, the senior author on the PLOS Neglected Tropical Diseases study. "While the disease is treatable … in its earlier version with medicine, its latent form can cause vision loss in children by the time they're teenagers."
Risk factors for Toxoplasma gondii include eating raw or undercooked meat from an infected animal. Another culprit is infected cats, who can pass on the parasite to their owners through their feces.
Current diagnosis of T. gondii relies on methods that include expensive serological tests that measure the levels of IgG and IgM. In addition, clinicians can directly diagnose the parasite by extracting cerebrospinal fluid or by analyzing tissue samples by microscopy. As well as being expensive, these approaches can be painful for patients, however.
During a pregnancy, if the mother is diagnosed with toxoplasmosis, an ultrasound scan or sampling of amniotic fluid can determine if the baby is infected, as well. While an ultrasound scan doesn't diagnose toxoplasmosis, it can indicate certain signs such as fluid buildup that indicates presence of the parasites.
The LDBIO Dx assay works by flowing a patient's sample through a paper matrix that detects IgG and IgM antibodies to T. gondii. For the study, the researchers chose sera samples with T. gondii types that are representative of strains that infect mothers of congenitally infected children in the US, Morocco, and Panama.
In previous work published in 2017 in PLOS Neglected Tropical Diseases, McLeod collected serum samples from the National Collaborative Chicago-Based Congenital Toxoplasmosis Study and used the assay to diagnose patients with the condition. While the assay accurately detected T. gondii IgG or IgM antibodies in patients, McLeod explained that the tool was not practical outside the laboratory because in order to produce near-perfect results, it required expensive lab equipment such as a centrifuge and a biosafety hood.
McLeod needed to simplify the assay so that clinicians could perform the POC assay in low-resource areas, especially in developing countries that lack access to expensive tools.
In the current study, the team modified the assay by using whole-blood samples instead of serum. Following a finger prick sample from patients, the team collected 30 microliters of whole blood using a small capillary tube and placed the solution into a well within the microcassette. Adding three drops of eluent into the well, the team ran the assay on the blood samples. The addition of the eluent caused the solution to bind and migrate together to form a colored band, McLeod said.
The team then examined whether the test was positive — indicated by a pink, positive line and a blue, positive control line on the test — or negative, which lacked the pink line.
According to McLeod, the blood-based assay quickly detected signs of toxoplasmosis within 20 minutes of running the samples on the microcassette
Once the researchers interpreted the samples as positive or negative, they looked at the monthly prenatal screening costs during pregnancy in the US for the assay as a POC test versus commercially available tests. They found that the assay was dramatically cheaper than standard methods, costing as low as $4 to $6 per test, compared to $650 for the standard lab-based toxoplasma test based on IgG and IgM. McLeod noted that the dramatic shift was due to the cheaper cost in instrument development, as well as the high costs that US hospitals charge to perform the standard assay.
The high sensitivity and specificity of the assay, in particular, is an important feature of the test, the researchers said. "Given the genetic diversity that characterizes the US population of T. gondii, we felt it was important to assess ease of performance, sensitivity, specificity and positive and negative predictive value of the test for US patients," they wrote. McLeod further noted that she was surprised to see the assay perform so well despite the genetic difference between the parasites from the US and European regions.
She also highlighted that that the assay could be used for monthly prenatal testing of T. gondii for women in low- and middle-income areas.
Limitations
The researchers acknowledged that the assay could not distinguish between acute and chronic cases in suspected patients and pregnant women. The team also noted that the assay lacks reliability for testing whole blood when patients have low levels of antibodies, as whole blood obscured the pink indicator for positive results. Researchers, therefore, plan to develop an assay with a dark-colored indicator in future studies.
Christelle Pomares, an associate professor of parasitology mycology at the University of Nice Sophia Antipolis, took particular note of the assay’s inability to tell an acute infection apart from a chronic infection. "The limitation of this assay is that it is a qualitative test without differentiation between IgG and IgM. In case of an acute infection, the assay will be positive as well as in case of a chronic infection, she said. "In pregnant women, this is an issue as the workup and treatment is totally different. When the assay is positive, confirmatory testing is required in order to detect IgG and IgM."
However, she said she is not surprised by the test's clinical sensitivity and specificity. She noted that the US study improved the tool's accuracy from a previous study published in July 2017 in the Journal of Clinical Microbiology, which compared the ICT assay to Abbott’s Toxo IgG and IgM assays.
McLeod envisions the test being used as part of a larger, multiplexed infectious disease panel — including Hepatitis B, syphilis, HIV, and rubella — on expectant mothers at a low cost. In addition, the multiplex assay could include biomarkers for Zika virus or Chagas disease in areas where the infections are prevalent.
"Women who are at risk and have a positive test can undergo additional, conventional screening tests," McLeod explained. Meanwhile, "patients with negative results could be screened monthly, and if they seroconvert, then infection is detected, allowing them to be treated without delay and [ensure] the infants are [healthy]."
McLeod noted that her team's next step is to have nurses offer the test for pregnant women when they visit the hospital or clinic for standard appointments.
"Assuming the hospital [at UChicago] IRB approves the assay, we hope to [eventually] integrate it as part of the standard checkup," McLeod explained. "We've been talking with several obstetrical groups [at UChicago] to have the nurse, while checking urine for glucose and other issues, to use the finger prick test, bring it to the obstetrician, enter it into the chart, and become a regular part of the obstetrician practice."
She plans to apply for funding from the National Institutes of Health to perform a validation study with collaborators at Stanford University.
Meanwhile, Lyon, France-based LDBIO Diagnostics, which developed the assay five years ago, has submitted to the FDA in order to potentially market the test in the US. In an email, LDBIO Cofounder Denis Limonne explained that the Toxoplasma ICT IgG IGM assay was developed several years ago as part of a suite of reference confirmatory ICT assays that uses whole-blood samples and serum to detect infectious diseases. The toxoplasmosis assay is CE marked and is currently used as a reference test.
Working with Limonne, McLeod performed clinical validation studies in the US to demonstrate the assay's potential in the US clinical space. According to Limonne, the firm is now pursuing large investments and "building an automatic and robotic line to supply customers" with its ICT tests.
While LDBIO Dx is currently applying for FDA approval for its ICT assays — starting with the Toxoplasma ICT IgG IGM test — Limonne has not decided whether the firm will commercialize the assay for primary diagnosis of toxoplasmosis or as a reference confirmatory test.
If the company fails to get FDA approval for the assay, LDBIO Dx will focus on commercializing the tool as a reference confirmatory assay in African, European, and Asian markets, Limonne said. He added, however, that LDBIO has been contacted by undisclosedUS distributors interested in the ICT assays.