Optimizing use of multi-antibody assays for Lyme disease diagnosis: A bioinformatic approach

Multiple different recombinant and peptide antigens are now available for serodiagnosis of Lyme disease (LD), but optimizing test utilization remains challenging. Since 1995 the Centers for Disease Control and Prevention (CDC) has recommended a 2-tiered serologic approach consisting of a first-tier whole-cell enzyme immunoassay (EIA) for polyvalent antibodies to Borrelia burgdorferi followed by confirmation of positive or equivocal results by IgG and IgM immunoblots [standard 2-tiered (STT) approach]. Newer modified 2-tiered (MTT) approaches employ a second-tier EIA to detect antibodies to B. burgdorferi rather than immunoblotting. We applied modern bioinformatic techniques to a large public database of recombinant and peptide antigen-based immunoassays to improve testing strategy. A retrospective CDC collection of 280 LD samples and 559 controls had been tested using the STT approach as well as kinetic-EIAs for VlsE1-IgG, C6-IgG, VlsE1-IgM, and pepC10-IgM antibodies. When used individually, the cutoff for each kinetic-EIA was set to generate 99% specificity. Utilizing logistic-likelihood regression analysis and receiver operating characteristic (ROC) techniques we determined that VlsE1-IgG, C6-IgG, and pepC10-IgM antibodies each contributed significant diagnostic information; a single-tier diagnostic score (DS) was generated for each sample using a weighted linear combination of antibody levels to these 3 antigens. DS performance was then compared to the STT and to MTT models employing different combinations of kinetic-EIAs. After setting the DS cutoff to match STT specificity (99%), the DS was 22.5% more sensitive than the STT for early-acute-phase disease (95% CI: 11.8% to 32.2%), 16.0% more sensitive for early-convalescent-phase disease (95% CI: 7.2% to 24.7%), and equivalent for detection of disseminated infection. The DS was also significantly more sensitive for early-acute-phase LD than MTT models whose specificity met or exceeded 99%. Prospective validation of this single-tier diagnostic score for Lyme disease will require larger studies using a broader range of potential cross-reacting conditions.

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Multiplex Immunoassay for Lyme Disease Using VlsE1-IgG and pepC10-IgM Antibodies: Improving Test Performance through Bioinformatics

Clinical and Vaccine Immunology ◽

10.1128/cvi.00409-10 ◽

2011 ◽

Vol 18 (5) ◽

pp. 851-859 ◽

Cited By ~ 36

Author(s):

Richard B. Porwancher ◽

C. Greg Hagerty ◽

Jianqing Fan ◽

Lisa Landsberg ◽

Barbara J. B. Johnson ◽

...

Keyword(s):

Lyme Disease ◽

Western Blotting ◽

Test Performance ◽

Disease Diagnosis ◽

Multiplex Assay ◽

Content Type ◽

Diagnostic Score ◽

Igm Antibodies ◽

Convalescent Phase ◽

Second Tier

ABSTRACTThe Centers for Disease Control and Prevention currently recommends a 2-tier serologic approach to Lyme disease laboratory diagnosis, comprised of an initial serum enzyme immunoassay (EIA) for antibody toBorrelia burgdorferifollowed by supplementary IgG and IgM Western blotting of EIA-positive or -equivocal samples. Western blot accuracy is limited by subjective interpretation of weakly positive bands, false-positive IgM immunoblots, and low sensitivity for detection of early disease. We developed an objective alternative second-tier immunoassay using a multiplex microsphere system that measures VlsE1-IgG and pepC10-IgM antibodies simultaneously in the same sample. Our study population comprised 79 patients with early acute Lyme disease, 82 patients with early-convalescent-phase disease, 47 patients with stage II and III disease, 34 patients post-antibiotic treatment, and 794 controls. A bioinformatic technique called partial receiver-operator characteristic (ROC) regression was used to combine individual antibody levels into a single diagnostic score with a single cutoff; this technique enhances test performance when a high specificity is required (e.g., ≥95%). Compared to Western blotting, the multiplex assay was equally specific (95.6%) but 20.7% more sensitive for early-convalescent-phase disease (89.0% versus 68.3%, respectively; 95% confidence interval [95% CI] for difference, 12.1% to 30.9%) and 12.5% more sensitive overall (75.0% versus 62.5%, respectively; 95% CI for difference, 8.1% to 17.1%). As a second-tier test, a multiplex assay for VlsE1-IgG and pepC10-IgM antibodies performed as well as or better than Western blotting for Lyme disease diagnosis. Prospective validation studies appear to be warranted.

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2‐Tiered Antibody Testing for Early and Late Lyme Disease Using Only an Immunoglobulin G Blot with the Addition of a VlsE Band as the Second‐Tier Test

Clinical Infectious Diseases ◽

10.1086/648674 ◽

2010 ◽

Vol 50 (1) ◽

pp. 20-26 ◽

Cited By ~ 59

Author(s):

John A. Branda ◽

Maria E. Aguero‐Rosenfeld ◽

Mary Jane Ferraro ◽

Barbara J. B. Johnson ◽

Gary P. Wormser ◽

...

Keyword(s):

Lyme Disease ◽

Immunoglobulin G ◽

Antibody Testing ◽

Second Tier

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A comparative trial of anti-glycoconjugate antibody assays: IgM antibodies to GM1

Journal of Neurology ◽

10.1007/bf00919708 ◽

1994 ◽

Vol 241 (8) ◽

pp. 475-480 ◽

Cited By ~ 25

Author(s):

J. Zielasek ◽

G. Ritter ◽

S. Magi ◽

H. P. Hartung ◽

K. V. Toyka

Keyword(s):

Comparative Trial ◽

Igm Antibodies ◽

Antibody Assays

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Laboratory Diagnostics Performance in Uganda: A Survey of Test Availability and Constraints Across 100 Laboratories

10.21203/rs.3.rs-842323/v1 ◽

2021 ◽

Author(s):

Noel Namuhani ◽

Suzanne N Kiwanuka ◽

Martha Akulume ◽

Simeon Kalyesubula ◽

William Bazeyo ◽

...

Keyword(s):

Clinical Laboratory ◽

Disease Diagnosis ◽

Sub Saharan Africa ◽

Essential Medicines ◽

Full Blood Count ◽

Laboratory Diagnostics ◽

Cross Sectional ◽

Laboratory Services ◽

Control And Prevention ◽

Sub Saharan

Abstract Background Clinical laboratory services are a critical component of the health system for effective disease diagnosis, treatment, control and prevention. However, many laboratories in Sub Saharan Africa remain dysfunctional. The high costs of tests in the private sector also remain a hindrance to accessing testing services. This study aimed at assessing the functionality of laboratories based on test menus and the associated constraints in Uganda. Methods This cross sectional quantitative study involved an assessment of 100 laboratories randomly selected in 20 districts from four regions of the country. Sixteen percent of the studied laboratories were regional hub laboratories. Laboratory in charges and managers in each of the selected laboratories were interviewed. A checklist for laboratory supplies adapted from the Essential Medicines and Health supplies list for Uganda, (2012) was used to assess availability of testing supplies. Data was analyzed using excel and STATA 14. Results At the point of assessment, generally, all laboratories were able to perform malaria tests and HIV tests. All the hub laboratories conducted malaria tests and TB screening. Less than half had electrolytes tests due to lack of equipment, nonfunctioning equipment and lack of reagents. Full blood count tests were missing in 25% of the hub laboratories mainly due to lack of equipment. The lack of reagents (66.7%) and the lack of equipment (58.3%) caused the majority 10/16 of the hubs to routinely referred specimens for tests that are supposed to be carried out in these laboratories due to lack of reagents (66.7%) and non-functional equipment (58.3%). Although officially recognized as an operational structure, Hub laboratories lacked a list of essential and vital supplies. Conclusions Most laboratories performed well for the common tests. However, many laboratories did not meet testing requirements especially for the advanced tests according to standard testing menus for Uganda due to non-functioning equipment, lack of equipment and reagents. Hubs lack list of essential supplies. Therefore, there is need to provide equipment to laboratories, repair the non-functional ones and develop an essential list of supplies for the hub laboratories.

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Evaluation of bioMérieux's Dissociated Vidas Lyme IgM II and IgG II as a First-Tier Diagnostic Assay for Lyme Disease

Journal of Clinical Microbiology ◽

10.1128/jcm.02407-16 ◽

2017 ◽

Vol 55 (6) ◽

pp. 1698-1706 ◽

Cited By ~ 15

Author(s):

Claudia R. Molins ◽

Mark J. Delorey ◽

Adam Replogle ◽

Christopher Sexton ◽

Martin E. Schriefer

Keyword(s):

United States ◽

Lyme Disease ◽

Enzyme Immunoassay ◽

The United States ◽

Statistical Analyses ◽

Diagnostic Approach ◽

Diagnostic Assay ◽

Western Immunoblotting ◽

Testing Strategies ◽

Second Tier

ABSTRACTThe recommended laboratory diagnostic approach for Lyme disease is a standard two-tiered testing (STTT) algorithm where the first tier is typically an enzyme immunoassay (EIA) that if positive or equivocal is reflexed to Western immunoblotting as the second tier. bioMérieux manufactures one of the most commonly used first-tier EIAs in the United States, the combined IgM/IgG Vidas test (LYT). Recently, bioMérieux launched its dissociated first-tier tests, the Vidas Lyme IgM II (LYM) and IgG II (LYG) EIAs, which use purified recombinant test antigens and a different algorithm than STTT. The dissociated LYM/LYG EIAs were evaluated against the combined LYT EIA using samples from 471 well-characterized Lyme patients and controls. Statistical analyses were conducted to assess the performance of these EIAs as first-tier tests and when used in two-tiered algorithms, including a modified two-tiered testing (MTTT) approach where the second-tier test was a C6 EIA. Similar sensitivities and specificities were obtained for the two testing strategies (LYT versus LYM/LYG) when used as first-tier tests (sensitivity, 83 to 85%; specificity, 85 to 88%) with an observed agreement of 80%. Sensitivities of 68 to 69% and 76 to 77% and specificities of 97% and 98 to 99% resulted when the two EIA strategies were followed by Western immunoblotting and when used in an MTTT, respectively. The MTTT approach resulted in significantly higher sensitivities than did STTT. Overall, the LYM/LYG EIAs performed equivalently to the LYT EIA in test-to-test comparisons or as first-tier assays in STTT or MTTT with few exceptions.

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Clinical and diagnostic manifestations of tickborne mixed infection in combination with COVID-19

Russian Clinical Laboratory Diagnostics ◽

10.51620/0869-2084-2021-66-11-689-694 ◽

2021 ◽

Vol 66 (11) ◽

pp. 689-694

Author(s):

A. L. Shutikova ◽

G. N. Leonova ◽

A. F. Popov ◽

M. Yu. Shchelkanov

Keyword(s):

Lyme Disease ◽

Erythema Migrans ◽

Clinical Manifestations ◽

Underlying Disease ◽

Laboratory Diagnostics ◽

Igg Antibodies ◽

Serum Samples ◽

Igm Antibodies ◽

Chronic Encephalitis ◽

Tbev Strain

The coexistence of various pathogens inside the patient’s body is one of the poorly studied and current issues. The aim of the study is to identify the relationship between the indicators of complex laboratory diagnostics and the clinical manifestations of a mixed disease during subsequent infection with the SARS-CoV-2 virus using the example of a case of chronic encephalitis-borreliosis infection. Seven blood serum samples were collected from the patient over the course of a year. For the etiological verification of the causative agents of TBE, Lyme disease and COVID-19, the methods of ELISA and PCR diagnostics were used. The patient was diagnosed with Lyme disease on the basis of the detection of IgG antibodies to Borrelia 5 months after the onset of the disease, since she denied the tick bite. In the clinical picture, there was an articular syndrome and erythema migrans. Later, IgG antibodies to the TBEV were found in the blood. Throughout the study, IgM antibodies to Borrelia were not detected. The exacerbation of Lyme disease could be judged by the clinical manifestations of this disease and by the growth of specific IgG antibodies. A feature of this case was that during an exacerbation of the Lyme disease, an infection with the SARS-CoV-2 virus occurred. Treatment (umifenovir, hydroxychloroquine, azithromycin, ceftriaxone) was prescribed, which improved the condition of the underlying disease, decreased joint pain, decreased IgG levels to borrelia. However, during this period, serological markers of TBEV appear: antigen, IgM antibodies, and the titer of IgG antibodies increases. Most likely, this was facilitated by the switching of the immune system to the SARS-CoV-2 virus, with the simultaneous suppression of borrelia with antibiotics and the appointment of hydroxychloroquine, which has an immunosuppressive effect. Despite the activation of the virus, clinical manifestations of TBE were not observed in the patient, which is most likely associated with infection with a weakly virulent TBEV strain. The further course of tick-borne infections revealed the dominant influence of B. burgdorferi in relation to TBEV. Laboratory studies have shown that suppression of the activity of the borreliosis process by etiotropic treatment subsequently led to the activation of the persistent TBEV.

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Antibodies against Specific Proteins of and Immobilizing Activity against Three Strains of Borrelia burgdorferi Sensu Lato Can Be Found in Symptomatic but Not in Infected Asymptomatic Dogs

Journal of Clinical Microbiology ◽

10.1128/jcm.38.7.2611-2621.2000 ◽

2000 ◽

Vol 38 (7) ◽

pp. 2611-2621 ◽

Cited By ~ 26

Author(s):

Joppe W. R. Hovius ◽

K. Emil Hovius ◽

Anneke Oei ◽

Dirk J. Houwers ◽

Alje P. van Dam

Keyword(s):

Borrelia Burgdorferi ◽

Acute Phase ◽

Bactericidal Activity ◽

Borrelia Burgdorferi Sensu Lato ◽

Enzyme Linked Immunosorbent Assay ◽

Western Blots ◽

Cell Enzyme ◽

Specific Proteins ◽

Different Strains

In an area where Lyme disease is endemic in The Netherlands all dogs had positive titers by whole-cell enzyme-linked immunosorbent assay and appeared to be naturally infected by Borrelia burgdorferi sensu lato. To compare the antibody responses of symptomatic dogs and asymptomatic controls, we performed Western blots and in vitro immobilization assays to study antibody-dependent bactericidal activity. Strains from three different genospecies were employed as the antigen source: B. burgdorferi strain B31,Borrelia garinii strain A87S, and Borrelia afzelii strain pKo. Antibodies against flagellin (p41) and p39 for three strains were found in sera from both symptomatic and asymptomatic dogs and were therefore considered to be markers of exposure. Antibodies against p56 and p30 of strain B31, against p75, p58, p50, OspC, and p<19 of strain A87S, and against p56, p54, p45, OspB, p31, p26, and p<19 of strain pKo were found significantly more frequently in sera from symptomatic dogs younger than 8 years when the first symptoms were observed than in those from age-matched controls (P < 0.01). These antibodies were not found in preclinical sera and appeared during development of disease. Antibodies against OspA of strains B31 and A87S were only seen in acute-phase and convalescent sera from three dogs that recovered from disease. Incubation with 25% normal canine serum did not result in the immobilization of strains B31 and pKo, but partial immobilization of strain A87S (61% ± 24% [standard deviation] at 5 h) occurred. Seven of 15 sera from symptomatic dogs but none of the sera from 11 asymptomatic dogs had antibody-dependent immobilizing activity against one of the strains. Consecutive sera from one of these dogs immobilized two different strains. Antibody-mediated bactericidal serum was not seen before onset of disease, was strongest in the acute phase of disease, and fluctuated during chronic disease. From seven out of eight symptomatic dogs Borrelia DNA was amplified by PCR; in three of them the bactericidal activity was directed against one of the genospecies amplified from that dog; however, four PCR-positive dogs lacked bactericidal activity. In conclusion, dogs with symptomatic canine borreliosis have more-extensive antibody reactivity against Borrelia, as shown by both Western blotting and immobilization assays.

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