Two-tier Testing Research Articles

P-2089. Transition of Lyme Serological Testing to the Modified Two Tiered-Testing Algorithm (MTTT): Observations from a Commercial Laboratory

Abstract Background 2019 FDA clearance of Lyme disease serologic assays with new indications for use in a modified two-tiered testing (MTTT) algorithm marked the first significant change in Lyme serologic testing in over 20 years. In 2022 Labcorp® fully transitioned Lyme serological testing from the standard two-tiered testing (STTT) algorithm to the MTTT algorithm. Methods Retrospective analysis of results reported for Lyme serological testing March through October to correlate with warmer months consistent with tick exposure, comparing samples received in 2019 and 2021 assessed with the STTT algorithm and samples received in 2022 and 2023 with the MTTT algorithm. Algorithm interpretation was based on APHL guidance. Results In 2019 and 2021, a combined total of 314,409 individuals were tested from March through October using the Lyme STTT algorithm while a combined total of 597,142 individuals were tested in 2022 and 2023 using MTTT during the same time frame. The populations of individuals tested by STTT and MTTT did not differ significantly in sex (STTT: 57.2% female, MTTT: 57.4%), age (STTT: mean 51 ± 29.0 years, MTTT: 51.0 ± 30.0 years) or geographic distribution (by U.S. census bureau region). An overall increase in positivity was observed with the MTTT (6.4%) compared to the STTT (3.2%) and was specifically observed in the percentage of individuals with results consistent with recent or remote past infection (tier 1+/tier 2 IgG+ or both IgG+ IgM+). A decrease in the percentage of results consistent with acute or recent infection (tier 1+/tier 2 IgM+) was observed with MTTT (1.1%) versus STTT (1.5%). 11.6% of Lyme MTTT orders included a concurrent order for Lyme immunoblot. MTTT and immunoblot results were discordant for 9.1% of samples. Of the discordant samples 43% had detectable IgG and/or IgM on the 2nd tier MTTT but were negative by immunoblot. Conclusion The transition from the STTT to MTTT algorithm revealed higher rates of serological positivity for Lyme disease. Analysis of samples tested by both MTTT and immunoblot indicates that MTTT assays exhibit greater sensitivity, aligning with findings in peer-reviewed literature. Some clinicians ordered both MTTT and immunoblot tests, suggesting a need for education on updated Lyme testing recommendations. Disclosures Laura Gillim, PhD, Labcorp: Employee|Labcorp: Stocks/Bonds (Public Company) Charles M. Walworth, MD, Labcorp: Employee|Labcorp: Employee|Labcorp: Stocks/Bonds (Public Company) David Alfego, PhD, Labcorp: Employee|Labcorp: Stocks/Bonds (Public Company)

P-2222. Optimization And Performance Assessment Of A Novel Assay In The Diagnosis Of Early Lyme Disease

Abstract Background There is a need for sensitive clinical diagnostics for early and acute infection as current serology-based tests have low sensitivities in the early phases of the disease and cannot discriminate between acute and past infections. In 2016, using a 33 peptide T-cell stimulating cocktail (QIAGEN prototype), Callister et al. provided the first published evidence showing that the integration of humoral and cell mediated immunity can result in earlier detection of Lyme Disease. Additional data have since shown that interferon γ (IFNγ) release is a marker of acute disease and decreases following antibiotic treatment of the patients. Methods A fully automated chemiluminescence immunoassay was developed that allows the detection of IgG, IgM, and an IFNγ release assay component (QFL) using a proprietary peptide-based technology; an algorithm combines the 3 results into a POS, NEG or IND call. The performance of the assay was compared to standard two-tiered testing (sTTT) in a prospective clinical trial (NCT05041595). Results In 2021, 54 patients with sign and symptoms consistent with Lyme disease were evaluated, as well as 200 healthy subjects from an area of low Lyme incidence. The assay showed significant sensitivity improvement over sTTT (79.6% vs 57.4%). However, suboptimal specificity was observed (89.5%) with some relatively high IFNγ values that required reassessment of the test. A troubleshooting process identified two decorin binding protein peptides as being responsible for most of the non-specific reactivity of QFL. The performance of an updated assay version with a 31-peptide QFL stimulation tube was evaluated in 2022 on 56 patients with erythema migrans, and 99 apparently healthy subjects from endemic areas. Sensitivity and specificity were assessed and compared to sTTT and found to be 73.2% vs 58.9%, and 100% for both, respectively. Conclusion The data presented support the operational utility of the assay as an aid in the diagnosis of early Lyme disease, while providing high specificity after the optimization of the stimulation peptides of the QFL assay component. A promising expanded clinical validation study for the assay is in process for submission to the U.S. Food and Drug Administration. Disclosures Fabrizio Bonelli, PhD, Diasorin SpA: employee Ashli Rode, MS, Diasorin Inc: employee Claudia Zierold, PhD, Diasorin SpA: Advisor/Consultant Jenny Howard, PhD, QIAGEN: Employee|QIAGEN: Stocks/Bonds (Public Company) Nadia Allen, PhD, QIAGEN: employee|QIAGEN: Stocks/Bonds (Public Company) Giorgio Ghignoni, MD, Diasorin: Employee

Seroprevalence of Borrelia burgdorferi sensu lato antibodies in English adult blood donors: A nationwide cross-sectional study, 2021-2022.

Estimates of Lyme disease incidence in England are based on reporting of cases with a laboratory-confirmed diagnosis only, underestimating total cases. In 2017 - 2018, two independent reviews commissioned by the UK Government highlighted the lack of official data on Lyme disease prevalence and incidence as a critical knowledge gap. To estimate the prevalence of IgG antibodies in the English adult population specific for Borrelia burgdorferi sensu lato (Bbsl), the causative agent of Lyme disease. The prevalence of Bbsl-specific antibodies in the English population was estimated in a cross-sectional cohort, selected from an archive of residual NHS blood donor plasma samples (age range 17 - 84, collected between 2021 - 2022). 10,000 samples were randomly selected proportionate to the population size of each of the nine English administrative regions. 9,994 samples were tested using a standard two-tiered testing strategy, with an IgG/IgM ELISA followed by an IgG immunoblot (array) test for any sera with positive or indeterminate reactivity in the ELISA. Out of the 9,994 samples tested, 482 were seroreactive by screening ELISA. After two-tier testing, 49 were confirmed positive. Regional and demographic differences in seroprevalence were observed after two-tier testing, but due to the low overall seroprevalence, were not significant upon multivariable analysis. The seroprevalence of Borrelia burgdorferi sensu lato-specific IgG in the English adult population (2021 - 2022), determined using two-tier testing was estimated at 0.49 % (95 % CI 0.36 - 0.65). This is lower than neighbouring UK nation Scotland and other northern European countries.

Diagnostic yield of cystic fibrosis from a South Australian monocentric cohort: a retrospective study

ObjectivesTo determine the diagnostic yield of cystic fibrosis (CF) using a two-tiered genetic testing approach. Although newborn screening includes CF, this typically only covers a selection of common genetic variants,...

Lyme Disease Confirmatory Western Blot Is Redundant for Screen Negative Samples in Low Endemic Areas, British Columbia, Canada.

Background: Borrelia burgdorferi sensu stricto is the causative agent of Lyme disease (LD). Possible early symptoms include flu-like symptoms and erythema migrans and later, the risk of disruption of the nervous system, joints, and heart. A two-tiered testing method is employed for serological diagnostics. The Public Health Agency of Canada guidelines recommend that samples tested negative on first-tiered test need not be confirmed by second-tiered test. Due to the challenging nature of diagnosis leading to misconceptions among physicians about false negatives, confirmatory testing is requested despite the initial negative result. Methods: Hundred screen-negative Lyme patient samples from 2007 to 2016 were tested by Western blot (WB) second-tiered confirmatory test upon physician's request in British Columbia to study the first-tiered screening test sufficiency. Results: Those negative for first-tiered enzyme-linked immunosorbent assay were also negative by WB. Conclusion: Results demonstrate that confirmatory testing is not necessary on screen-negative samples. Hence, first-tiered test is sufficient to rule out LD.

Evaluation of different standard and modified two-tier testing strategies for the laboratory diagnosis of lyme borreliosis in a European setting

BackgroundDiagnosis of Lyme borreliosis (LB) relies on clinical symptoms and detection of Borrelia-specific antibodies. Guidelines recommend a two-tier testing (TTT) strategy for disseminated LB: serological screening with a sensitive enzyme immunoassay (EIA) and confirmation with a specific immunoblot. Searching for the most sensitive and specific approach, this retrospective study evaluated standard (STTT) and modified (MTTT) strategies using a well-defined study population.MethodsCases included patients with active Lyme neuroborreliosis (LNB; n = 29) or Lyme arthritis (LA; n = 17). Controls comprised patients treated for LNB (n = 36) or LA (n = 8), healthy individuals who were either untreated (n = 75) or treated for LB (n = 15) in the past, and patients with potentially cross-reactive diseases (n = 16). Sera were subjected to three EIAs and two immunoblots. Reactive screening results were confirmed by immunoblot (STTT) or EIA (MTTT). Solitary IgM results in the screening assay and effects of antibiotic treatment on isotype-specific seropositivity rates were also assessed.ResultsSensitivities of STTT strategies ranged from 90%–97% for LNB and were 100% for LA. MTTT strategies were 100% sensitive. Specificities ranged from 89%–95% for STTT and from 88%–93% for MTTT strategies. Differences between STTT and MTTT strategies were not statistically significant. Solitary IgM reactivity was common among controls. Antibiotic treatment significantly reduced IgM/IgG positivity for LNB patients; for LA patients, a decline was only observed for IgM.ConclusionIn conclusion, MTTT strategies showed a slightly higher sensitivity and similar specificity compared to STTT strategies. Since EIAs are more time- and cost-efficient, MTTT strategies seem more favorable for clinical use. IgG testing enhances specificity with minimal sensitivity loss.

A-354 Evaluating Emergent POC Technology for Sickle Cell Testing in Resource Limited Settings

Abstract Background Sickle cell disease (SCD) represents a collection of inherited hematological disorders characterized by abnormal sickle-shaped erythrocytes resulting in an increased risk of morbidity and mortality. SCD represents a significant global health burden, affecting more than 300,000 newborns per year. Most of these births occur in resource limited areas, including sub-Saharan Africa, where the mortality rate before the age of five is estimated to be as high as 50-80% and the existing infrastructure and resources cannot support most current and robust methods that offer hemoglobin variant detection and quantification for the diagnosis and monitoring of SCD. While newborn screening programs (NBS) to identify individuals affected with SCD have demonstrated efficacy in reducing morbidity and early mortality, NBS programs are limited outside of the US and Europe and face many practical challenges for universal implementation in a resource limited setting. The need for inexpensive and reliable hemoglobin variant detection and quantification at the Point of Care is essential to the diagnosis and management of SCD in resource limited settings with a high prevalence of SCD, and the expansion of NBS programs. This study aims to evaluate an inexpensive testing strategy using two methodologies for POC screening and confirmation of SCD that could be supported in a low resource setting. Methods The two-step testing strategy for evaluation of POC testing for hemoglobin variant detection and quantification included testing residual specimens from normal and known sickle cell patients by first screening with the HemoTypeSC, a qualitative lateral flow immunoassay (LFIA) that has improved sensitivity and specificity from other available LFIA methodologies given the use of monoclonal antibodies for the detection of hemoglobin variants. Confirmatory testing and quantification of hemoglobin variants was accomplished with the use of the Gazelle POC test a miniaturized chip-based cellulose acetate electrophoresis device capable of identification and quantification of normal and variant hemoglobin. Statistical evaluation of device performance and clinical agreement between the device and known disease status was achieved in EP evaluator. Results The two-tier testing strategy demonstrated effective and inexpensive mechanism for SCD screening and confirmation. Overall, the HemoTypeSC and Gazelle demonstrated 100% qualitative agreement between methods, with the total cost of the two-tiered testing strategy estimated at under $6 per sample. Conclusions In conclusion, the two-tiered approach using the HemoTypeSC and Gazelle yielded a practical and cost-effective strategy for screening and confirmation of hemoglobin variants for the detection and monitoring of SCD. Both the strip and chip-based methodologies provide rapid results, are accessible at the point-of-care, and require minimal sample volume, a feature ideal for use in a pediatric population in low resource settings. Overall, these methods show promise for the use and expansion of NBS programs to improve public health initiatives for the diagnosis of SCD.

Seropositivity to tick-borne pathogens in nature management workers in the Netherlands

The incidence of tick-borne infections other than Lyme borreliosis and tick-borne encephalitis is rising in Europe, including the Netherlands. Nature management workers, being highly exposed to ticks, serve as valuable sentinels for seroprevalence studies on tick-borne pathogens (TBPs). This study assessed nature management workers’ seropositivity to TBPs including Anaplasma phagocytophilum, Babesia divergens, B. microti, Borrelia burgdorferi s.l., Rickettsia conorii and R. typhi in the Netherlands. In addition, the study examined coexposure to multiple TBPs and identified risk factors for B. burgdorferi s.l.- and A. phagocytophilum-seropositivity.The study included 525 nature management workers who donated serum and completed a questionnaire. Sera were analysed for exposure to A. phagocytophilum, B. divergens, B. microti, R. conorii and R. typhi using immunofluorescence assays. For B. burgdorferi s.l. antibody detection, the recommended two-tier testing strategy was used. Risk factor analysis was performed using logistic regression modelling.Seropositivity was 30.9 % for B. burgdorferi s.l.; 16.4 % for A. phagocytophilum; 6.5 % for R. conorii; 2.3 % for R. typhi; 4.2 % for B. divergens; and 0.4 % for B. microti. Almost half (49.3 %) of the participants demonstrated seropositivity for one or more pathogens. Risk factors for B. burgdorferi s.l.-seropositivity included being male, increasing age and tick bite frequency. For A. phagocytophilum-seropositivity, increasing age and working in North Holland province were significant risk factors.This study illustrates the exposure to TBPs in the Netherlands, emphasizing the need for ongoing vigilance and international collaborations to better understand and address the growing threat of TBPs in regions with demonstrated environmental TBP circulation.

Serological Assessment of Lyme borreliosis in Bulgaria: A Nationwide Study.

Lyme borreliosis (LB), a tick-borne infection caused by bacteria in the Borrelia burgdorferi sensu lato complex, is increasingly prevalent on the Balkan Peninsula, including Bulgaria, where it is the most common tick-borne disease. This study aimed to assess the seroprevalence of LB across Bulgaria by analyzing 1892 serum samples for specific IgG antibodies using a two-tier testing protocol involving an ELISA and immunoblot methods. The results revealed an overall seroprevalence rate of 5.4%, with significant variation based on age, sex, and residence. Seroprevalence increased with age, peaking at 8.4% in individuals over 65 years. Males had a seroprevalence of 8.4% compared to 3.3% in females, and rural residents showed higher seroprevalence (10.2%) compared to urban residents (4.4%). Regional analysis indicated that seroprevalence ranged from 0.0% to 20.0%, with higher rates in northern provinces such as Gabrovo (18.9%) and Targovishte (20.0%). This study highlights the importance of two-step testing protocols for accurate diagnosis and underscores the need for increased awareness and further research to enhance public health measures and the management of LB in Bulgaria.

Rapid single-tier serodiagnosis of Lyme disease

Point-of-care serological and direct antigen testing offers actionable insights for diagnosing challenging illnesses, empowering distributed health systems. Here, we report a POC-compatible serologic test for Lyme disease (LD), leveraging synthetic peptides specific to LD antibodies and a paper-based platform for rapid, and cost-effective diagnosis. Antigenic epitopes conserved across Borrelia burgdorferi genospecies, targeted by IgG and IgM antibodies, are selected to develop a multiplexed panel for detection of LD antibodies from patient sera. Multiple peptide epitopes, when combined synergistically with a machine learning-based diagnostic model achieve high sensitivity without sacrificing specificity. Blinded validation with 15 LD-positive and 15 negative samples shows 95.5% sensitivity and 100% specificity. Blind testing with the CDC’s LD repository samples confirms the test accuracy, matching lab-based two-tier results, correctly differentiating between LD and look-alike diseases. This LD diagnostic test could potentially replace the cumbersome two-tier testing, improving diagnosis and enabling earlier treatment while facilitating immune monitoring and surveillance.

Comparative Evaluation of Commercial Test Kits Cleared for Use in Modified Two-Tiered Testing Algorithms for Serodiagnosis of Lyme Disease.

Modified 2-tiered testing (MTTT) for Lyme disease utilizes automatable, high throughput immunoassays (AHTIs) in both tiers without involving western immunoblots, offering performance and practical advantages over standard 2-tiered testing (STTT; first-tier AHTI followed by immunoglobulin M (IgM) and immunoglobulin G (IgG) western immunoblots). For MTTT, Centers for Disease Control and Prevention recommends using AHTI test kits that have been cleared by Food and Drug Administration (FDA) specifically for this intended use. We evaluated performance of FDA-cleared MTTT commercial test kits from 3 manufacturers by comparing with STTT results. We performed MTTT (total antibody AHTI with reflex to separate IgM and IgG AHTIs) using test kits from Diasorin, Gold Standard Diagnostics (GSD), and Zeus Scientific on 382 excess serum samples submitted to the clinical laboratory for routine Lyme disease serologic testing in July 2018, measuring agreement between MTTT and STTT using the κ statistic. Overall agreement with STTT was 0.87 (95% confidence interval [CI], .77-.97) using Diasorin assays (almost perfect agreement), 0.80 (95% CI, .68-.93) using GSD assays (substantial agreement) and 0.79 (95% CI, .68-.90) using Zeus assays (substantial agreement). For detection of IgM reactivity, agreement between MTTT and STTT was 0.70 (.51-.90; substantial), 0.63 (95% CI, .44-.82; substantial) and 0.56 (95% CI, .38-.73; moderate), respectively. For detection of IgG reactivity, MTTT/STTT agreement was 0.73 (95% CI,.58-.88), 0.78 (95% CI, .62-.94), and 0.75 (95% CI, .60-.90), respectively (substantial agreement in all cases). MTTT results obtained using commercial test kits from 3 different manufacturers had substantial to almost perfect agreement with STTT results overall and moderate to substantial agreement for IgM and IgG detection independently. Commercial MTTT tests can be used broadly for the diagnosis of Lyme disease.

Evaluation of the Veterinary IDEXX SNAP 4Dx Plus Test for the Diagnosis of Lyme Disease in Humans.

Background: Lyme disease, caused by infection with Borrelia burgdorferi, is the most common vector-borne disease in the United States. The standard two-tier testing (STTT) algorithm suffers from low sensitivity, misinterpretation, and long turnaround time, preventing timely detection and treatment. To address these challenges, we hypothesized that the canine point-of-care (PoC) SNAP 4Dx Plus test used to detect Borrelia burgdorferi antibodies could be employed for human diagnosis. Materials and Methods: The SNAP 4Dx Plus testing was conducted in accordance with the manufacturer's instructions, with results read by manual inspection. All analyses were conducted using R version 4.3.1, and agreement between the PoC assay and the STTT was assessed using kappa statistics with GraphPad software. Results: We included 102 previously-tested human serum samples, of which 19 samples (18.6%) were STTT positive. Compared to the STTT, the SNAP 4Dx Plus test demonstrated a low sensitivity of 0.16 (95% CI 0.03 to 0.40). Conclusion: Overall, our results do not support the use of the SNAP 4Dx Plus LD assay for the diagnosis of human Lyme disease. Differences in antibody concentrations between human and canine samples may partly explain our findings.

Diagnostic Challenges of Lyme Neuroborreliosis in Inpatient Neurology: A Case Series.

Lyme disease is a multisystem disorder transmitted through the Ixodes tick and is most commonly diagnosed in northeastern and mid-Atlantic states, Wisconsin, and Minnesota, though its disease borders are expanding in the setting of climate change. Approximately 10%-15% of untreated Lyme disease cases will develop neurologic manifestations of Lyme neuroborreliosis (LNB). Due to varying presentations, LNB presents diagnostic challenges and is associated with a delay to treatment. We discuss three cases of LNB admitted to our referral center in a traditionally low-incidence state to highlight clinical pearls in LNB diagnosis. Three patients from low-incidence areas with prior diagnostic evaluations presented in August with neurologic manifestations of radiculoneuritis, cranial neuropathies, and/or lymphocytic meningitis. MRI findings included cranial nerve, nerve root, and leptomeningeal enhancement leading to broad differential diagnoses. Lumbar puncture demonstrated lymphocytic pleocytosis (range 85-753 cells/uL) and elevated protein (87-318mg/dL). Each patient tested positive for Lyme on two-tiered serum testing and was diagnosed with LNB. All three cases were associated with a delay to health care presentation (mean 20days) and a delay to diagnosis and treatment (mean 54days) due to under-recognition and ongoing evaluation. With the geographic expansion of Lyme disease, increasing awareness of LNB manifestations and acquiring detailed travel histories in low-incidence areas is crucial to prompt delivery of care. Clinicians should be aware of two-tiered serum diagnostic requirements and use adjunctive studies such as lumbar puncture and MRI to eliminate other diagnoses. Treatment with an appropriate course of antibiotics leads to robust improvement in neurological symptoms.

Performance of two modified two-tier algorithms for the serologic diagnosis of Lyme disease.

The laboratory diagnosis of Lyme disease relies upon the detection of antibodies to Borrelia species. Standard two tier testing (STTT) methods rely upon immunoblots which have clinical and technical limitations. Modified two-tier testing (MTTT) methods have recently become available and are being widely adopted. There are limited independent data available assessing the performance of MTTT and STTT methods.

Two-tiered serology in patients with Lyme neuroborreliosis

<p><strong>Introduction. </strong>Lyme borreliosis is a multisystem infectious disease caused by Borrelia burgdorferi sensu lato complex. It is transmitted by Ixodes ticks. Apart from erythema migrans, other manifestations require laboratory confirmation. The aim of our study was to analyze the results of Two-Tiered Testing in patients with possible Lyme neuroborreliosis (LNB).</p> <p><strong> Method.</strong> We conducted a prospective diagnostic study at the University Clinical Centre of Republic of Srpska from October 2017 to October 2021. The study included 51 patients exam ined and treated under suspicion of Lyme neuroborreliosis (LNB). We used Two-Tiered Testing through ELISA anti-Borrelia IgM/IgG and confirmatory Immunoblot anti-Borrelia test IgM/ IgG.</p> <p><strong> Results. </strong>ELISA anti-Borrelia IgM in serum was positive in 25.5% patients and IgG in 76.47%, while in cerebrospinal fluid (CSF) IgM was positive in 13.7% patients and IgG in 39.2%. Immuno blot test of anti-Borrelia IgM in serum was at the borderline in 7.84% patients, positive in 6.17% and no data for 35.29%, while IgG was positive in 54.9% of patients and no data for 37.25%. A strong correlation between results of ELISA anti-Borrelia IgM in serum and CSF was observed (ρ=0.802, p<0.001) and also between ELISA anti-Borrelia IgG and Immunoblot IgG in serum (ρ=0.787, p<0.001).</p> <p><strong>Conclusion.</strong> Two-Tiered Testing is very important in patients with possible LNB. False positive and false negative results are possible and test should be interpreted with caution and in correlation with the clinical manifestation and other avail able tests. More work needs to be done to develop new reli able diagnostic test for LNB, such as detection of CXCL13 in CSF, titrate the interleukin-6 (IL-6) in addition to the CXCL13 chemokine.</p>

Pediatric Lyme Disease in Northern Italy: An 18-Year Single-Center Case Series.

Tracing the profile of pediatric Lyme borreliosis (LB) in Europe is difficult due to the interregional variation in its incidence and lack in notifications. Moreover, the identification of LB can be challenging. This study is an 18-year case series of 130 children and adolescents aged under 18 years referred to the Pediatric Infectious Diseases Unit at L. Sacco Hospital, Milan, with suspicion of LB, between January 2005 and July 2023. The routine serological workup consisted of a two-step process: an initial screening test followed by Western blot (WB). Forty-four (34%) patients were diagnosed with LB. The median age was six years, and 45% were females. Of the children with erythema migrans (EM), 33 (57%) were confirmed as having true EM, and, of these, 4 (12%) were atypical. Ten (23%) patients had early disseminated/late diseases, including facial nerve palsy (n = 3), early neuroborreliosis (n = 1), arthritis (n = 3), relapsing fever (n = 2), and borrelial lymphocytoma (n = 1). No asymptomatic infections were documented. Over seventy percent of confirmed LB cases (n = 31/44) recalled a history of tick bites; in this latter group, 19 (61%) were from the area of the Po River valley in Lombardy. Almost half of the children evaluated for LB complained of non-specific symptoms (fatigue, musculoskeletal pain, skin lesions/rash, and persistent headache), but these symptoms were observed in only two patients with confirmed LB. Most LB cases in our study were associated with EM; two-tier testing specificity was high, but we found frequent non-adherence to international recommendations with regard to the timing of serology, application of the two-step algorithm, and antibiotic over-prescription. Most children were initially assessed for a tick bite or a skin lesion suggestive of EM by a family pediatrician, highlighting the importance of improving awareness and knowledge around LB management at the primary healthcare level. Finally, the strengthening of LB surveillance at the national and European levels is necessary.

Use of Specific Borrelia Phages as a New Strategy for Improved Diagnostic Tests.

The high failure rate of tick-borne infection (TBI)-related testing underscores the need for novel approaches that do not rely on serology and two-tier testing. Delayed diagnosis of TBIs, especially Borrelia infections, results in high healthcare costs and great suffering. There is a significant need for a reliable blood test that can aid in the diagnosis of Lyme disease, particularly when the current FDA-approved serological test is not sensitive enough to detect early Lyme patients who have not yet produced antibodies against Borrelia. Bacteriophages are viruses that specifically associate with their bacterial hosts, particularly prophages, bacteriophages residing in bacteria, and have proven to be tightly correlated with their bacterial hosts. They are poised to have wider applications as markers to detect bacteria, particularly in infectious disease. The gene of choice depends on the prevalence of phages within a particular group of bacteria. Phage genes that have been used as molecular markers to examine phage diversity include structural genes encoding the major capsid protein, the portal protein, the DNA polymerase, and the terminase. Borrelia species carry specific phage sequences that can be used as a proxy to identify the bacteria. Using phages as a proxy for bacteria is beneficial, as phages can be detected more easily than bacteria and can be used to bypass the cryptic and tissue-bound feature that typifies human Borrelia infections.We explored a completely new way of detecting Borrelia using Borrelia-specific bacteriophages as a diagnostic tool. Our detection method, patented by Phelix R&D and Leicester University (WO2018083491A1), could potentially transform infectious disease diagnostics through the innovative use of real-time PCR to target circulating bacteriophage DNA in blood from patients with Lyme disease. Firstly, this bacteriophage-based approach offers increased sensitivity since bacteriophages are typically present in five- to tenfold excess over bacterial cells, making it more accurate and sensitive than conventional bacteria-targeting PCR tests. One of the reasons bacteria-based PCR tests are frequently negative is due to the low bacterial concentration in the blood. Bacteriophage-based PCR surpasses this barrier and offers a direct test, as phages are part of bacteria's own genetic material, in contrast to all existing indirect tests (ELISA, Western BLOT, LTT/ELISPOT test). Secondly, a phage-based test can differentiate between different Lyme disease-causing and relapsing fever-causing Borrelia subtypes (B. burgdorferi s.l., B. miyamotoi, etc.), given that bacteriophages are indicators of bacterial identity. Finally, this test can detect Lyme disease in both early and late stages.

Diagnostic Precision in Lyme borreliosis: Assessing VlsE and C6 Antigens in a Pediatric Cohort

(1) Background: Lyme borreliosis (LB) is a tick-borne disease known for its diagnostic challenges. Conventional two-tiered testing (CTTT) for antibodies is time-consuming, has low sensitivity in the early stages of disease, and sometimes generates false-positive IgM immunoblots. To tackle this issue, modified two-tiered testing (MTTT) was introduced, incorporating recombinant VlsE and C6 antigens to enhance diagnostic accuracy. (2) Methods: In this prospective study, we enrolled children exhibiting symptoms indicative of LB. We collected serum samples at various intervals and subjected them to analysis using standard enzyme immunoassays. We then compared these results with the outcomes from the VlsE and C6 assays. (3) Results: In our study, all 33 patients displaying erythema migrans (EM), a characteristic symptom of LB, exhibited positive responses to the C6 antigen. This finding underscores the potential utility of the C6 antigen as a reliable diagnostic tool for LB. Additionally, we observed a significant reduction in anti-VlsE antibody levels following antibiotic treatment in EM patients. (4) Conclusions: The utilization of recombinant VlsE and C6 antigens in LB diagnostics and monitoring has yielded promising results. Nonetheless, it is imperative for clinicians to exercise caution and interpret results in conjunction with clinical findings, considering the dynamic nature of medical guidelines. Even with recombinant antigen tests, some children with EM tested negative, highlighting the importance of clinical diagnosis for treatment decisions. Furthermore, clinicians should be mindful of the possibility of persistently positive VlsE/C6 test results during LB treatment monitoring.

861. Development and Clinical Validation of a Custom Modified Two-Tier Testing Algorithm for Serodiagnosis of Lyme Disease

Abstract Background Diagnosis of Lyme Disease has relied on measuring the serological response to Borrelia sp. Over the years the rigor of serological diagnosis has evolved to meet the needs of the clinic and vaccine manufacturers, where the latter require especially sensitive and specific tests to measure efficacy. Likewise, current US guidelines recommend a two-tier testing approach to diagnose Lyme Disease. Among a constellation of commercially available in vitro diagnostics, two highly sensitive immunoassays with Borrelia sp. antigenic diversity were characterized for utility as a custom Modified Two-Tier Testing (MTTT) algorithm. This custom algorithm consists of a first tier assay from Bio-Rad and a second tier assay from Zeus Scientific, and was developed to capture cases of Lyme Disease. Methods The BioPlex 2200 Lyme Total assay from BioRad and VlsE1/pepC10 IgG/IgM ELISA from Zeus Scientific were evaluated as the first and second tier assays in the custom MTTT, respectively. The MTTT was clinically assessed for diagnostic accuracy and compared to the highly characterized CDC Pre-Marketing Panel, which is composed of samples from US donors diagnosed with Lyme Disease, “look-alike” diseases or healthy individuals. Other clinically relevant sample populations were benchmarked off the predicate FDA-approved MTTT, also from Zeus Scientific. Results The custom MTTT, described here, was more clinically specific than either test alone and was more clinically sensitive than the only other FDA-approved MTTT. When matched against the FDA-approved predicate method, sensitivity and specificity was 100%. The custom MTTT also detected fewer “look-alike” disease samples than either test alone, or the FDA-approved MTTT. Furthermore, detection of early Lyme Disease in the CDC Pre-Marketing panel was 80% sensitive. In addition, the custom MTTT identified Lyme disease in 98.6% of Lyme Disease samples collected from samples that originated in Europe. Conclusion The clinically validated custom MTTT method, described here, has utility as a Lyme Disease diagnostic and is a highly specific tool for assessing efficacy of Lyme Disease vaccines currently in development. Disclosures Michelle A. Gaylord, PhD, Pfizer Inc.: Stocks/Bonds Danielle Baranova, PhD, Pfizer Inc: Stocks/Bonds Michael W. Pride, PhD, Pfizer: Stocks/Bonds Raphael Simon, PhD, Pfizer: Stocks/Bonds Annaliesa S. Anderson, PhD, Pfizer: Employee|Pfizer: Stocks/Bonds Warren Vincent. Kalina, PhD, Pfizer: Stocks/Bonds

Possible effect of mutations on serological detection of Borrelia burgdorferi sensu stricto ospC major groups: An in-silico study.

The outer surface protein C (OspC) of the agent of Lyme disease, Borrelia burgdorferi sensu stricto, is a major lipoprotein surface-expressed during early-phase human infections. Antibodies to OspC are used in serological diagnoses. This study explored the hypothesis that serological test sensitivity decreases as genetic similarity of ospC major groups (MGs) of infecting strains, and ospC A (the MG in the strain B31 used to prepare antigen for serodiagnosis assays) decreases. We used a previously published microarray dataset to compare serological reactivity to ospC A (measured as pixel intensity) versus reactivity to 22 other ospC MGs, within a population of 55 patients diagnosed by two-tier serological testing using B. burgdorferi s.s. strain B31 as antigen, in which the ospC MG is OspC A. The difference in reactivity of sera to ospC A and reactivity to each of the other 22 ospC MGs (termed 'reactivity difference') was the outcome variable in regression analysis in which genetic distance of the ospC MGs from ospC A was the explanatory variable. Genetic distance was computed for the whole ospC sequence, and 9 subsections, from Neighbour Joining phylogenetic trees of the 23 ospC MGs. Regression analysis was conducted using genetic distance for the full ospC sequence, and the subsections individually. There was a significant association between the reactivity difference and genetic distance of ospC MGs from ospC A: increased genetic distance reduced reactivity to OspC A. No single ospC subsection sequence fully explained the relationship between genetic distance and reactivity difference. An analysis of single nucleotide polymorphisms supported a biological explanation via specific amino acid modifications likely to change protein binding affinity. This adds support to the hypothesis that genetic diversity of B. burgdorferi s.s. (here specifically OspC) may impact serological diagnostic test performance. Further prospective studies are necessary to explore the clinical implications of these findings.