NEW YORK – Infectious disease laboratory IGeneX has been a player in the tick-borne disease space since 1994, but recent strides in its technology have allowed for more sensitive and broad tests that don't require blood cultures.
Specializing exclusively in tick-borne diseases until the COVID-19 pandemic hit, when it began offering a SARS-CoV-2 test, IGeneX uses a wide range of modalities, including Western blot and PCR testing, as well as its patented recombinant protein ImmunoBlot tests.
While it has offered its ImmunoBlot tests for Borrelia, which causes Lyme disease and Tick-Borne Relapsing Fever, since 2017, the lab launched a new test for Bartonella at the end of July. Bartonella causes a variety of diseases in humans, including Cat scratch disease, endocarditis, Trench fever, and Carrion's disease.
The traditional method of testing for tick-borne diseases is two-tiered, according to Raphael Stricker, medical director at Union Square Medical Associates who has worked with IGeneX. An immunofluorescent antibody (IFA) test is performed as a screen, and if the antibodies are found in the sample, a confirmatory Western blot test is performed.
Stricker noted that the two-tiered system was originally developed for surveillance but has been used for diagnosis "inappropriately."
PCR can also be used to test for the bacteria, but the sensitivity is low "due to the low bacterial load present within most biological samples, the long time required for bacterial growth in enrichment culture, and the presence of PCR-inhibitory components" found in samples, according to a study published last year in the Journal of Microbiological Methods.
IGeneX's ImmunoBlot tests also use blotting, similar to Western blots, but have a special sauce – the specific recombinant protein antigens used in each test.
When testing with the ImmunoBlot assays, specific amounts of recombinant protein antigens are sprayed directly onto a nitrocellulose membrane at specific locations and cut into strips. Using such precise amounts mean the banding and fluorescent intensity of the antigens is less variable, more clearly displaying positive reactions.
Controlling both the location and concentration of antigens allows for more consistent results with more specificity, IGeneX consultant Joseph Burrascano, a specialist in tick-borne diseases and a founding board member of the International Lyme and Associated Diseases Society, said.
Those recombinant proteins are an essential component of the test and its performance. The proteins are "very exact" and specific to the organism being looked for, Burrascano said. They're created by going to a "known culture source" and extracting the exact genetic code of the organism, the proteins of which are then built back by the IGeneX team, he said.
He noted that a key issue with IFA and Western blot testing is that the results are dependent on the antigen source. For Bartonella, Burrascano said not many species are available to culture or purchase commercially, making scarcity a real concern for test developers. Bartonella cultures are particularly hard to come by because they don't grow in human serum, he added.
"Quality of [the] antigen will vary based upon quality and purity of the culture," he said. "Using a culture as the source for your proteins limits what you can test for and it limits the accuracy, the repeatability, and the sensitivity," of the test.
By using pure recombinant proteins created with IGeneX's proprietary genetic method, that potential issue is bypassed, since the antigens aren't grown from culture or provided by an outside source but rather are created in the lab.
IFA and Western blot testing can result in false positives from "other things in the patient serum" lighting up during the test or from finding proteins that are shared by other bacteria or autoimmune diseases, Burrascano said.
Sensitivity for the two-tier method recommended by the US Centers for Disease Control and Prevention is about 50 percent, Burrascano said, calling it a "coin toss." Sensitivity for IGeneX's Lyme ImmunoBlot, a combination of its Borrelia IgM and IgG tests, is about 91 percent. Meantime, sensitivity for the Bartonella IgM test is 75 percent and 89 percent for the IgG test.
IGeneX's technology also enables an expanded number of detectable species and can test a sample for species and genus of the bacteria, Burrascano said. If the lab doesn't have antigens for a specific organism present, the genus-level testing can detect that an infection is present.
The Bartonella test includes 22 Bartonella-specific antigens and two controls on each patient test strip, he added.
Other methods that use culture or commercial antigens can't track more than one or two pathogens, which "limits what you can test for," Burrascano said. At least 30 species of Bartonella have been recognized, but most labs that use culture or commercial antigens "have been focused on one species, maybe two," he said.
Traditional tests are also manual and dependent on the abilities of a laboratory technician, since techs need to look at the sample on a glass slide through a microscope.
The ImmunoBlot test, however, prints the quantity and location of the antigens onto a paper, automating the result and requiring less input and room for error than from a lab tech, Burrascano said.
USMA's Stricker said that in his experience as a provider for patients with tick-borne diseases, the IGeneX ImmunoBlot tests "are much better than what's available."
Currently, IGeneX offers six tests for Borrelia – one each for IgM and IgG antibodies for Lyme disease, along with a panel test combining the two, as well as IgM, IgG, and panel tests for Tick-Borne Relapsing Fever – and two for Bartonella, an IgM and an IgG test. The Borrelia tests are available across the country as laboratory-developed tests, while the Bartonella test is available in every state except New York, which requires a further approval process for LDTs.
The tests have been submitted for clearance from the US Food and Drug Administration, as well as regulatory clearance in Canada and Australia, Burrascano said. The individual antibody tests for Borrelia and Bartonella cost $225, while the Lyme ImmunoBlot panel retails for between $490 and $893, depending on the targets included, and the TBRF panel costs $686.
Joe Sullivan, IGeneX's head of marketing, said the company wants to expand its ImmunoBlot tests to other diseases, although he declined to comment on which ones the firm is targeting in development. However, he noted that the firm has other non-ImmunoBlot tests for diseases including babesiosis, bartonellosis, anaplasmosis, ehrlichiosis, and rickettsiosis, which could be the focus of future ImmunoBlot tests.
The firm has made strides in that direction, however, by offering its COVID-19 ImmunoBlot tests. Providing quantitative and qualitative results, its SARS-CoV-2 ImmunoBlot IgG test has a sensitivity of 82 percent and a specificity of 98 percent.
Its ImmunoBlot panel detects both IgM and IgG antibodies with an overall sensitivity of 94 percent. IgM sensitivity was 73 percent, while IgG sensitivity was 82 percent. Specificity for both antibodies was above 98 percent. The SARS-CoV-2 panel is included in the firm's comprehensive Vaccine Response Panel, which also includes a test for T cell response to monitor a patient's reaction to the vaccine.
IGeneX also offers PCR tests for COVID-19, which have made up the majority of the firm's COVID-19 sales, Sullivan said.