Sex- and Age-Specific Lyme Disease Testing Patterns in the United States, 2019 and 2022

Abstract

Objectives:

Serologic testing is a useful adjunct for the diagnosis of Lyme disease, a major public health problem in certain US regions. We aimed to determine whether Lyme disease serologic testing and results differed by sex and age group.

Methods:

We identified 2 cohorts of individuals across all ages who underwent serologic testing for Lyme disease at a national reference laboratory in 2019 (cohort 1) and 2022 (cohort 2). If an individual had multiple tests in the same year, we included only the first test. We excluded individuals who had been tested in the previous 5 years.

Results:

Cohorts 1 and 2 consisted of 578 052 and 550 674 people, respectively. Fewer males than females were tested in cohort 1 (42.7% vs 57.3%) and cohort 2 (42.3% vs 57.7%), although similar numbers were tested for both sexes among nonadults. More males than females had a positive test result in cohort 1 (53.9% more males) and cohort 2 (52.9% more males). The odds ratio of receiving a positive test result among males versus females was 2.09 (95% CI, 2.01-2.17) in cohort 1 and 2.12 (95% CI, 2.05-2.19) in cohort 2. Among people with positive test results, females (except children) were more likely than males to have positive immunoglobulin M and negative immunoglobulin G results, which can serve as a marker of early infection (odds ratio = 1.43 [95% CI, 1.31-1.55] in cohort 1 and 1.38 [95% CI, 1.29-1.47] in cohort 2).

Conclusions:

Further studies are needed to understand whether the observed differences in Lyme disease testing and positivity result from sex- and age-associated disparities in social behavior, health care seeking, clinical practice, or other factors.

Lyme disease is the most common tick-borne disease in the United States and is primarily caused by infection from the bacterium Borrelia burgdorferi sensu stricto. ¹ According to public health surveillance, most cases of Lyme disease are reported among residents of the Northeast, Upper Midwest, and Mid-Atlantic states, with higher frequency in the summer months. ² The highest incidences of Lyme disease are among children, adults aged >45 years, and males. ² Early signs of Lyme disease typically include a rapidly expanding rash called erythema migrans and general flu-like symptoms. When not promptly diagnosed and treated, infection can disseminate in the body to cause arthritis, neuropathy, meningitis, or cardiac conduction abnormalities.^3,4

The erythema migrans rash is diagnostic for Lyme disease, when tick exposure likely occurred in areas endemic for the disease. However, serologic testing is a useful adjunct for the diagnosis of Lyme disease in people with atypical skin lesions and for people with extracutaneous manifestations of the disease. For these cases, a 2-tier serologic test for detection of antibodies to B burgdorferi is generally recommended by the Infectious Diseases Society of America, American Academy of Neurology, and American College of Rheumatology. ³

A specific antibody response to B burgdorferi is not typically detectable when serologic testing occurs in the first days to weeks after infection, the so-called window period. For infections among people who have not previously had Lyme disease, immunoglobulin M (IgM) antibodies develop first, followed by immunoglobulin G (IgG) antibodies in the subsequent days to weeks. IgG antibodies are reliably present after 1 to 2 months of active infection. However, the timing of antibody transition and detection will vary according to when effective antimicrobials are administered. Antimicrobial treatment that begins early in infection can abrogate the specific immune response if the treatment is microbiologically curative.^5-10 Accordingly, the longevity of IgM and IgG antibody detection can depend on the duration and severity of the disease before treatment. Variations in antibody evolution and persistence might depend on other underlying pathologic or physiologic processes, including those that may be associated with age and sex.

Disparities between men and women are known to exist with respect to diagnosis and treatment of certain health conditions.^11,12 Men are less likely than women to use health care in general (as shown by underrepresentation of men in primary care visits) and tend to delay seeking care for certain, though not all, health problems. ¹³ In an evaluation of commercial health insurance claims in the United States, the annual average incidence of Lyme disease diagnoses between 2010 and 2018, based on the presence of diagnosis codes and antibiotic treatment, was similar among males and females across all ages, although the incidence of Lyme disease according to public health surveillance is higher among males than among females.^2,14

In this cohort study, we investigated serologic testing and test results in males and females across all ages who were tested for Lyme disease in 2019 or 2022.

Methods

The Western Institutional Review Board deemed this retrospective analysis of deidentified data exempt from board approval and the need for informed consent. We followed the STROBE reporting guideline (Strengthening the Reporting of Observational Studies in Epidemiology; https://www.strobe-statement.org/checklists).

Study Design

We obtained retrospective and deidentified test results for Lyme disease from Quest Diagnostics, a major independent testing laboratory with patient service centers in every US state and the District of Columbia. Quest Diagnostics performs routine clinical testing of patient specimens for health care providers outside its facilities. We used test codes (laboratory Logical Observation Identifiers Names and Codes) for B burgdorferi to identify individuals who were tested for Lyme disease from January 2014 through December 2022. We limited the test codes to tests that corresponded to current serologic testing recommendations for clinicians. ³

To investigate Lyme disease serology testing and results, we constructed 2 study cohorts from eligible test data: cohort 1 (2019) and cohort 2 (2022) (Figure 1). We selected these 2 years because they covered the periods before and during the COVID-19 pandemic. We included individuals who were tested in 2019 or 2022 and who were not tested in the previous 5 years. Specifically, if a person had multiple tests in 2019 or 2022, we included only the first test in that year. In addition, we applied a 5-year “washout” to ensure that individuals had not been tested in the 5 years before 2019 for cohort 1 and before 2022 for cohort 2. For positivity analysis, we excluded individuals who were not tested under the standard 2-tier test (STTT) or modified 2-tier test (MTTT) protocols. This study design differed from a previous study ¹⁵ in which all tests performed in a given year for analysis had been included and in which results had been presented on a test basis rather than on a patient basis, as in our study. We thus designed our cohorts to assess patterns of initial testing and test results by increasing the likelihood of including people with possible acute Lyme disease, rather than including people who received nonrecommended periodic testing, such as that during routine health screens or for test of cure.

Figure 1.

Flow diagram showing selection of 2 cohorts of individuals across all ages who had specimens submitted by health care providers to Quest Diagnostics for serologic testing of Lyme disease in 2019 and 2022, United States. Abbreviations: MTTT, modified 2-tier test; STTT, standard 2-tier test.

Measurements

Quest Diagnostics performed serologic testing for evidence of Lyme disease on serum specimens that had been submitted at the request of an ordering clinician who ordered either a 2-tier reflex test or a stand-alone test. The typical testing schema used the STTT protocol, which consisted of a first-tier enzyme immunoassay (EIA) screening test, followed by a second-tier confirmatory IgM and/or IgG immunoblot for specimens yielding first-tier positive or equivocal results. In some instances, Quest Diagnostics performed immunoblot testing only, which may have occurred when an outside referring laboratory had previously performed screening EIAs or when ordering clinicians requested only the immunoblot test for other unknown reasons. In 2022, MTTT was performed on some specimens tested and was conducted with a first-tier EIA screening test, followed by a second-tier confirmatory EIA for specimens yielding first-tier positive or equivocal results. For STTT assays, Quest Diagnostics used a chemiluminescent immunoassay (LIAISON Lyme Total Antibody Plus; DiaSorin) that reflexed to IgG and IgM immunoblots from Gold Standard Diagnostics. Quest Diagnostics used MTTT assays from Zeus Scientific with separate confirmatory EIA tests for IgM and IgG.

Data Analysis

We performed descriptive analyses of testing and test positivity in the overall study population and for each cohort, stratified by age group, sex, state of residence, and calendar month when testing was performed. We defined test positivity in STTT and MTTT protocols when screening and confirmatory tests showed test positivity (vs a previous study that did not define all test positivity by the STTT protocol ¹⁵ ). We assessed associations between sex and (1) test positivity and (2) IgM/IgG antibodies with Pearson χ² analysis by using Prism version 9.4.1 (GraphPad Software, LLC) and/or with logistic regression models adjusted for age by using SAS Studio 3.6 on SAS version 9.4 (SAS Institute Inc). For the 2022 cohort, we assessed associations between IgM-positive and IgG-negative (IgM+IgG−) antibodies and sex and associations between IgM-negative and IgG-positive (IgM−IgG+) antibodies and age (≥65 vs <65 y) for each STTT and MTTT protocol separately, which we then pooled by using the Mantel–Haenszel method. We considered P < .05 to be significant. We performed data analyses from January 2023 through March 2024.

Results

From the Quest Diagnostics database, we identified 758 854 Lyme disease serology tests in cohort 1 (all tests by STTT) and 733 165 in cohort 2 (95.2% by STTT and 4.8% by MTTT). After excluding repeat tests performed in the same year (n = 45 274 in cohort 1 and 48 812 in cohort 2), we identified 713 580 tests from unique individuals in cohort 1 and 684 353 in cohort 2. After further exclusion of those who had a serology test for Lyme disease in the previous 5 years (n = 135 528 in cohort 1 and 133 679 in cohort 2), the study cohorts included 578 052 people who were tested for the first time in 2019 but not in the previous 5 years and 550 674 people who were tested for the first time in 2022 but not in the previous 5 years. For positivity analyses in which individuals who had test results from the STTT and MTTT protocols were analyzed, most serology testing for Lyme disease followed these protocols (n = 437 686 in cohort 1 and 426 451 in cohort 2); however, some testing did not follow these protocols per the request of ordering clinicians (n = 140 366 in cohort 1 and 124 223 in cohort 2). The number of positive tests according to the STTT and MTTT protocols was 11 899 of 437 686 (2.7%) in cohort 1 and 15 330 of 426 451 (3.6%) in cohort 2.

Cohorts 1 and 2 had similar age distributions; the median (IQR) age was 50 (33-63) years and 51 (33-64) years, respectively. In both cohorts, testing pattern and test positivity showed the expected geographic distribution and seasonality of Lyme disease (eFigures 1 and 2 in the Supplement); for example, 81% of positive test results in cohort 1 and 82% in cohort 2 occurred among residents of 5 Northeastern states (Connecticut, Massachusetts, New Jersey, New York, and Pennsylvania), and the number peaked in June and July.

In cohorts 1 and 2, test orders increased steadily with age, peaking among adults who were in their late 50s and early 60s (Figure 2). Fewer males than females were tested in both cohorts: 246 576 males (42.7%) versus 330 819 females (57.2%) of the 578 052 tested in cohort 1 and 232 809 males (42.3%) versus 317 810 females (57.7%) of the 550 674 tested in cohort 2, corresponding to 25.5% and 26.7% fewer males than females, respectively. However, although the number of females versus males tested was similar among children and adolescents, more adult females than adult males were tested. In contrast, more males than females had a positive test result: 7202 males versus 4680 females (53.9% more) in cohort 1 and 9267 males versus 6062 females (52.9% more) in cohort 2. The odds ratio of males (vs females) testing positive was 2.09 (95% CI, 2.01-2.17; P < .001) in cohort 1 and 2.12 (95% CI, 2.05-2.19; P < .001) in cohort 2. The number of individuals with positive test results was consistently higher among males than among females at all ages and peaked among children aged 5 to 9 years and adults aged 60 to 69 years. The distribution of percentage positives (number of positive test results per 100 tests performed) by age group showed the highest positivity rate among children and the lowest positivity rate among young and middle-aged adults, regardless of sex.

Figure 2.

Test frequency and test positivity by age and sex in individuals across all ages who had specimens submitted by health care providers to Quest Diagnostics for serologic testing of Lyme disease in 2019 (cohort 1; 3 panels on the left side) and 2022 (cohort 2; 3 panels on the right side), United States. For each cohort, the top panel shows the number of people tested; the middle panel, the number of people who had a positive test result; and the bottom panel, the test positivity rate. A positive test result required test positivity for immunoglobulin M and/or immunoglobulin G according to the standard and modified 2-tier test protocols.

Distributions of IgM and IgG antibodies present in males versus females were similar in cohort 1 and cohort 2. Among individuals with positive test results, males had a lower proportion of IgM+IgG− antibodies and a higher proportion of IgM+IgG+ and IgM−IgG+ antibodies than females (Figure 3). The distribution of IgM and IgG antibodies detected was not consistently significantly different between male and female children and adolescents aged <18 years in the 2 cohorts, but males were significantly less likely than females to have IgM+IgG− antibodies detected in all other age groups (range, P < .001 to .008; eFigures 3 and 4 in the Supplement). In 5 Northeastern endemic states (Connecticut, Massachusetts, New Jersey, New York, and Pennsylvania), males were also significantly less likely than females to have IgM+IgG− antibodies detected (eFigure 5 in the Supplement). The odds ratio for detection of the IgM+IgG− antibodies in females versus males was 1.43 (95% CI, 1.31-1.55; P < .001) in cohort 1 and 1.38 (95% CI, 1.29-1.47; P < .001) in cohort 2. In addition, adults aged ≥65 years consistently had significantly higher proportions of IgM−IgG+ antibodies than individuals aged <65 years (eFigure 6 in the Supplement). The odds ratio for detection of IgM−IgG+ antibodies among people aged ≥65 versus <65 years was 1.67 (95% CI, 1.54-1.82; P < .001) in cohort 1 and 1.82 (95% CI, 1.70-1.96; P < .001) in cohort 2.

Figure 3.

Distribution of immunoglobulin M (IgM) and immunoglobulin G (IgG) detection by sex in individuals across all ages who tested positive in 2019 (cohort 1) and 2022 (cohort 2) for Lyme disease, per serologic testing by Quest Diagnostics, United States. The odds ratio for detection of the IgM+IgG– antibodies in males versus females was 0.70 (95% CI, 0.64-0.76; P < .001) in cohort 1 and 0.73 (95% CI, 0.68-0.78; P < .001) in cohort 2 (Mantel–Haenszel test). Abbreviations: MTTT, modified 2-tier test; STTT, standard 2-tier test.

Discussion

In this study, we constructed 2 cohorts of >1.1 million people who had serologic tests for possible Lyme disease, as performed by a large national reference laboratory, and no evidence of testing in the previous 5 years, and we reported patterns for test results by age and sex. We found that serologic testing for Lyme disease varied significantly by age, with relatively few tests performed for children and more tests performed for every year of age until older adulthood (aged approximately ≥50 y). Variability in test frequency by age might reflect different practices by health care provider type, where blood draws are ordered for children only when suspicion of Lyme disease is high. A lower number of tests in children (vs other age groups) might also be explained by the high proportion of pediatric Lyme disease cases that involve erythema migrans, where serologic testing is generally neither needed nor recommended. Adults might be tested more when the clinical picture is less straightforward (vs not less straightforward), such as when Lyme disease has an atypical presentation or occurs in the presence of comorbidities. Adults may also be tested more at their own request, which could reflect inappropriate testing of adults who have only nonspecific symptoms—with a low a priori likelihood of infection. In addition, fewer adult males than females were tested, even when including those tested in the preceding 5 years, which is consistent with proportionally fewer commercial health insurance claims among males than among females. ¹⁴ However, significantly more males than females had a positive test result for Lyme disease (P < .001), possibly because more males than females were participating in outdoor activities; for example, in 2023, men accounted for 53.9% of those who participated in outdoor activities in the United States versus 46.1% for women. ¹⁶ Testing for children and adolescents was similar according to sex, although more males than females had positive test results.

For both cohorts, adult males were less likely than adult females to have positive test results for IgM+IgG− antibodies, some of which might represent false-positive results. This difference might be linked to lower serum IgM levels demonstrated for men as compared with women. ¹⁷ Alternatively, because IgM is typically an early antibody response to infection, the difference shown between men and women might represent a delay in health care seeking, including diagnostic testing, by men experiencing symptoms of Lyme disease. ¹⁸ A delay in diagnosis and treatment provides more time not only for dissemination of bacteria but also for an evolution of the antibody response, including the potential for switching of isotype class. Conversely, higher proportions of IgM+IgG− antibodies in women might be associated with women seeking health care earlier or more often for Lyme disease than men, possibly as a result of nonspecific symptoms such as fatigue and pain.^19,20 If the clinician threshold for Lyme disease testing is effectively lower for women than for men, then this difference would simultaneously explain the greater numbers of tests performed for women and the lower positivity rate. The lower threshold would result in a lower positive predictive value for the test and, concordantly, a higher rate of false-positive results among women being tested than among men being tested. In our study, we also found a higher percentage of individuals with IgG antibodies to Lyme disease among older adults aged ≥65 years versus <65 years. Explanations for this observation could include a higher ratio of memory to naive B cells and a longer history of underlying conditions, infections (including previously unrecognized or even asymptomatic Lyme disease cases and others), or malignancies. ²¹

Testing and positivity patterns according to age, sex, and geography were similar in our 2 study cohorts and were consistent with trends reported through public health surveillance, ² although bias in testing cannot be completely ruled out. More males than females had positive test results, just as more males than females constituted the confirmed cases reported to the Centers for Disease Control and Prevention from 1992 through 2016. ²² Similarly, the age distribution of people who tested positive for Lyme disease was similar to that among confirmed Lyme disease cases, with peaks among younger (aged 5-9 y) and older (aged 55-64 y) groups. However, of note, surveillance data have been likely influenced in recent years by testing trends, given that (1) reporting of Lyme disease to public health authorities most often begins with positive laboratory reports and (2) reporting of cases directly from health care providers is somewhat rare. ²³ In our study, laboratory test data showed that much testing occurred in states with large populations and in some areas with large proportions of older adults who may have relocated from Lyme disease–endemic areas (eg, Florida). For every 10 positive test results in the United States, 8 were in 5 Northeastern states known to have a high incidence of Lyme disease.

Limitations and Strengths

Our study had several limitations. First, our data lacked information on (1) symptom onset and clinical presentation in relation to timing of when serum samples were collected and (2) history of Lyme disease and other illnesses among individuals in the cohorts. Timing, disease course, and history are important clinical considerations for understanding the development and expected occurrence of IgM and IgG antibodies. ¹⁷ In this regard, a large number of people with acute Lyme disease may have been tested during the window period or after early antimicrobial treatment and did not have detectable antibodies. Thus, some results (eg, IgM/IgG distributions) may not represent the population with acute Lyme disease. However, by excluding follow-up tests and people who had any tests in the previous 5 years, we took steps to increase the likelihood that the cohorts represented those with acute disease. Second, we used testing data from a single reference laboratory. Although Quest Diagnostics has patient service centers in every state, Quest Diagnostics may have only a portion of the market share of Lyme disease testing, and the proportion of samples tested by Quest Diagnostics could vary by region. Yet, testing and positivity patterns in our study were consistent with epidemiologic observations, ² suggesting that results in our 2 cohorts do provide a representative snapshot for the general population. Third, some individuals in our cohorts might have had a test done at another laboratory in the 5-year washout window and, thus, might not represent an acute case of Lyme disease.

Our study also had some strengths. First, we had a large sample size, comprising >1.1 million individuals tested from all 50 states and the District of Columbia. The utility of these data is supported by the consistency of testing and positivity patterns with epidemiologic observations and surveillance data trends. Second, the use of 2 cohorts in different years showing similar results provided evidence for the reproducibility of the different testing patterns between males and females and across age groups. Because cohort 1 covered the period before the COVID-19 pandemic outbreak and cohort 2 covered the period after the outbreak, their consistent findings suggest that the pandemic had a negligible effect on Lyme disease test volume in 2022, which is in contrast to conditions such as cancer, with screening and diagnosis affected by COVID-19. ²⁴

Conclusion

Our findings reflect characteristics of people receiving diagnostic testing for possible Lyme disease and clarify the occurrence of Lyme disease test positivity by sex and age group. Further studies are needed to understand whether observed differences in Lyme disease testing and positivity between males and females and by age are due to sex- and age-associated disparities in social behavior, health care seeking, clinical practice, or other factors.