A Lyme Disease Test with Results Too Good to be True?
The Lyme disease culture test was being developed by Dr. Eva Sapi and colleagues in Connecticut, who published a paper earlier this year outlining their success. The technique involved taking samples of serum from patients known to be positive for Lyme disease bacteria and using cultivating these samples to then test for the presence of Borrelia. Positive cultures were found in 47% of cases within six days of sampling, and in 94% of cases within 16 weeks. A control group of 48 people without Lyme disease was used in the study, comparing these to an initial group of 50 Lyme disease patients and a second group of 72 confirmed cases. The bacteria were identified using immunostaining, polymerase chain reaction (PCR), and direct DNA sequencing.
Caution Over Such Positive Results
The high success rate of this novel test was received enthusiastically by many Lyme disease patient advocates but scientists working in the field of infectious disease advised caution as the method was new and unconfirmed. Such caution appears wise in retrospect as the latest assessment of the method suggests that the high rate of seropositivity found may have been due to laboratory contamination.
DNA Matching of Lyme Disease Bacteria
When testing for Lyme disease using DNA sequencing it is common to see a variety of genetic signatures as Borrelia reproduce and mutate quite rapidly. Homogeneity amongst the results of such tests is unlikely, unless the test cases were deliberately infected in a controlled environment using a single source of bacteria. What the CDC researchers found in analyzing these results from Sapi’s study was that around 80% of the patients’ sample sequences were indistinguishable from those used in the development of the test. Just 10 of the 51 pyrG sequences identified were different to the investigators’ bacterial strains and the CDC scientists note that while they are unsure as to the relevance of these strains, validation of any new testing method is essential to prevent unnecessary antibiotic treatment following a false positive for Lyme disease.
New Test Finds Lyme Disease Bacterial Strains Not Present in US
Barbara J.B. Johnson, PhD, from the Division of Vector-Borne Disease in Fort Collins, Colorado, and colleagues at the CDC were particularly interested in working out why the DNA sequencing used in this study appeared to show that most patients were infected with strains of the Lyme disease bacteria that had not previously been detected in US or Canadian patients. These strains, Borrelia afzelii and Borrelia garinii, are more commonly found in Europe and Asia but are not known to be present in North America.
How the Test was Devised
The researchers developing the test used two Lyme disease bacterial strains commonly employed in laboratory tests for the disease in the US, B31 and 297, alongside the two Eurasian strains. These strains, when found in US patients, are assumed to be the result of exposure to ticks when travelling outside of the US, with no confirmed detection of these bacteria in ticks tested on US soil.
Bacterial Contamination and False Positives
As such, the CDC scientists began to wonder if the high degree of homogeneity between test samples and the presence of unlikely bacterial strains was not a result of exposure and infection but of accidental contamination in the laboratory from the testing protocol itself. Even the different strains of these bacteria would, if infection had arisen naturally, result in different DNA sequences amongst the patients’ samples and so the scientists compared the pyrG sequences for 51 patient isolates using the same primers as those used by Sapi, et al.
Almost Exact Matches for References Strains
What they found was the more than half (53%) of the patients’ samples were infected with B. garinii, with 20 of those 27 found to be identical clones to the strain used as a reference in the test’s development. Differences in the other samples were limited to just a single mutated nucleotide in five cases, two differences in one case, and three differences in the remaining case. These kinds of similarities are highly suggestive of something other than naturally occurring infection.
Less Than 10% of Samples Not Highly Suspect
In addition, 41% of the 51 patients had B. burgdorferi-related sequences and all but one of them had exact matches to the reference strain. Two patients’ samples contained strains of B. afzelii closely related to the reference sample, meaning that 80% (41/51) of the patients’ samples were identical to the test strains, with another 12% differing by a single nucleotide. That leaves just 4 patients whose tests were not an exact match or an almost identical match to the bacterial strains used in this novel, and seemingly highly successful test.
Further Validation Needed
Rather charitably, the CDC researchers note that “further validation of the proposed novel culture method is required,” despite what appears to be a pretty clear indication of something having gone wrong with the testing protocol leading to contamination with the reference strains. The suspected contamination could be due to the use of PCR methods which were employed despite having little merit when the culture contains enough bacteria to be seen under a microscope. As the control samples were not tested using PCR it may be that these avoided the contamination, thus resulting in seronegativity. Had they all been tested by PCR it may be that Sapi, et al, would have found an astonishingly high rate of positivity for Lyme disease amongst those with no other indication of the disease. This, itself, would have (hopefully) sounded the alert about the testing protocols.
Exercising Cautious Optimism Over New Lyme Disease Testing Protocols
Without carrying out the study again, eliminating the PCR testing step, it is impossible to say whether it was this that caused the suspected contamination and, indeed, whether the new test could be helpful in detecting Lyme disease. What the experience does tell us is that a single study presenting results that look too good to be true should lead to cautious optimism and healthy scepticism, rather than raising hopes ready to be dashed.
Barbara J.B. Johnson#, Mark A. Pilgard and Theresa M. Russell, Assessment of New Culture Method to DetectBorrelia species in Serum of Lyme Disease Patients, J Clin Microbiol. Published online August 14, 2013.
Sapi E, Pabbati N, Datar A, Davies EM, Rattelle A, Kuo BA. Improved Culture Conditions for the Growth and Detection of Borrelia from Human Serum. Int J Med Sci 2013; 10(4):362-376. Available from http://www.medsci.org/v10p0362.htm