RAPID DETECTION OF ANTI-COCCIDIOIDES · PDF fileValley Fever, is a fungal agent endemic to the...
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Maddox, S. , Doherty, B. , Pelfrey, J. , Thompson, G. , Bauman, S. IMMY, Norman, Oklahoma Department of Internal Medicine, Division of Infectious Diseases, University of California Davis Medical Center, Davis, California INTRODUCTION Coccidioides, the causative agent of San Joaquin Valley Fever, is a fungal agent endemic to the arid regions of the Americas. Due to the difficulties associated with fungal culture, the dominant method of diagnosis is serology. Coccidioidomycosis serology can be a laborious, time consuming and expensive process. It is for this reason that many hospitals rely on send out diagnostic testing from reference laboratories. IMMY has developed a lateral flow assay designed to quickly rule out negative cases, near the point of care. Reduced turnaround time, cost-savings and enhanced anti-microbial stewardship are benefits of the assay, in addition to those provided by more rapid patient management. The IMMY sona™ Coccidioides Ab lateral flow could be an effective tool for ruling out potential Coccidioides infections. Figure 1: The CTA2003 LFA strips for the detection of antibodies against Coccidioides species. METHODS Ninety-two human serum samples (52 positive, 42 negative) were characterized by the University of California, Davis Coccidioidomycosis Serology Laboratory and IMMY. Specimens were considered positive if re- active in two of any of the regularly performed assays: complement fixation, immunodiffusion or quantitative immunodiffusion (performed at UC-Davis) and Premier® Coccidioides EIA manufactured by Meridian Bioscience (performed at IMMY). These specimens were kindly provided to IMMY for the evaluation of sona™ Coccidioides Ab lateral flow assay (CTA2003). Briefly, specimens were diluted to 1:441 in specimen diluent and tested on the sona™ Coccidioides Ab. After 20 minutes, results were interpreted by two operators. A ROC analysis was performed to determine assay sensitivity and specificity. CONCLUSIONS The sona™ Coccidioides Ab test is a rapid immunoassay that detects anti-Coccidioides antibodies. The high negative predictive value of the assay allows for a 20 minute screen to rule out patients suspected of having Valley Fever. Management of this population of patients can be significantly improved by ruling out Valley Fever, allowing physicians to better direct treatment. Patients positive on the sona™ Coccidioides Ab test can then be referred to more traditional serology techniques to confirm the presence of antibodies to Coccidioides. RAPID DETECTION OF ANTI-COCCIDIOIDES ANTIBODIES USING THE sona COCCIDIOIDES Ab LATERAL FLOW ASSAY TM 1 1 1 1 2 1 2 RESULTS The area under the ROC curve was 0.885 (95% confidence interval (CI) = 0.801,0.942, standard error 0.0377, ID EIA CF* IgG+ IgM+ Total+ 26 37 - 20 23 - 40 40 40 19 * CF does not distinguish IgG/IgM ABSTRACT Coccidioides, the causative agent of San Joaquin Valley Fever, is a fungal agent endemic to the arid regions of the Americas. Due to the difficulties associated with fungal culture, the dominant method of diagnosis is serology. Typical serological diagnosis methods require from 48 to 72 hours using technologies like immunodiffusion or complement fixation. IMMY has developed a new 20 minute lateral flow assay for the detection of antibodies against Coccidioides sp. and it was compared to these typical serological methods. Specimens were considered reactive for antibodies against Coccidioides if positive in two out of three different traditional assays: immunodiffusion, complement fixation and ELISA. A two by two diagnostic table and an ROC curve was developed comparing the assays. The area under the ROC curve was 0.885 (95% confidence interval (CI) = 0.801, 0.942, standard error 0.0377, p<0.001) for the sona™ Coccidioides Ab assay. The assay had 100% sensitivity (95% CI: 91.1 - 100) and 76.92% specificity (95% CI: 63.2 - 87.5) when compared to the reference method. In this study, the positive predictive value was 76.92% and the negative predictive value was 100%. Figure 3: ROC analysis of the CTA2003 kit vs characterized specimens p<0.001) for the sona™ Coccidioides Ab assay. The assay had 100% sensitivity (95% CI: 91.1 - 100) and 76.92% specificity (95% CI: 63.2 - 87.5) when compared to the EORTC criteria. The positive predictive value was 76.9% and the negative predictive value was 100% (Figure 3). Probable Negative Positive: Negative: Totals Totals 40 12 40 0 40 52 52 40 IMMY EORTC 29 Coccidioidomycosis Symposium 7th International Stanford, CA Aug. 10-13, 2017 1 Obtain 3 test tubes for each serum specimen. • 2 for the dilution • 1 flat-bottom tube for running the test Transfer Specimen Diluent to both tubes 200 μl diluent in tube 1 200 μl diluent in tube 2 SPECIMEN PREPARATION Dilute serum 1:441 with 1X Specimen Diluent: 3 1 2 Transfer 10 μl from tube 1 to tube 2. Mix thoroughly. 200 μl 200 μl 2 Transfer 10 μl of patient serum to tube 1. Mix thoroughly. 1 10 μl 10 μl 1 2 RUN TEST SAMPLE SAMPLE Cocci Ab Cocci Ab Cocci Ab Cocci Ab 5 Insert strip ( down) Incubate for 20 min. Read Test 1 line = negative 2 lines = positive 6 C T SAMPLE SAMPLE SAMPLE SAMPLE + - Cocci Ab Cocci Ab Cocci Ab Cocci Ab Cocci Ab Cocci Ab Cocci Ab Cocci Ab 4 Transfer 100 μL from tube 2 to tube 3 2 3 100 μl 3 20 min. PROCEDURE Table 1: Specimen Characterization Table Sensitivity Specificity Positive Likelihood Ratio Negative Likelihood Ratio Disease Prevalence Positive Predictive Value Negative Predictive Value ROC Area Under Curve Standard Error (p<0.001) 100.00% 76.92% 4.33 0.00 43.48% 76.92% 100% 0.885 0.0377 91.11 % to 100.00% 63.16% to 87.46% 2.64 to 7.12 33.17% to 54.22% 63.16% to 87.46% 91.11 % to 100.00% 0.801 to 0.942 95% CI REFERENCES Nguyen C, Barker BM, Hoover S, Nix DE, Ample NM, Frelinger JA, Orbach MJ, Galgiani JN 2013. Recent advances in our understanding of the environmental, epidemiological, immunological, and clinical dimensions of coccidioidomycosis. Clin Microbiol Rev 26:505-525. Tsang CA, Anderson SM, Imholte SB 2007. Enhanced surveillance of coccidioidomycosis, Arizona, USA. Emerg Infect Dis 2010; 16:1738-44. Figure 2: The CTA2003 LFA Prodecure Table 2: 2x2 Contingency Table and Statistical Analysis Positive C T Negative
Transcript of RAPID DETECTION OF ANTI-COCCIDIOIDES · PDF fileValley Fever, is a fungal agent endemic to the...
Maddox, S. , Doherty, B. , Pelfrey, J. , Thompson, G. , Bauman, S. IMMY, Norman, Oklahoma Department of Internal Medicine, Division of Infectious Diseases, University of California Davis Medical Center, Davis, California
INTRODUCTIONCoccidioides, the causative agent of San Joaquin Valley Fever, is a fungal agent endemic to the arid regions of the Americas. Due to the difficulties associated with fungal culture, the dominant method of diagnosis is serology. Coccidioidomycosis serology can be a laborious, time consuming and expensive process. It is for this reason that many hospitals rely on send out diagnostic testing from reference laboratories. IMMY has developed a lateral flow assay designed to quickly rule out negative cases, near the point of care. Reduced turnaround time, cost-savings and enhanced anti-microbial stewardship are benefits of the assay, in addition to those provided by more rapid patient management. The IMMY sona™ Coccidioides Ab lateral flow could be an effective tool for ruling out potential Coccidioides infections.
Figure 1: The CTA2003 LFA strips for the detection of antibodies against Coccidioides species.
METHODSNinety-two human serum samples (52 positive, 42 negative) were characterized by the University of California, Davis Coccidioidomycosis Serology Laboratory and IMMY. Specimens were considered positive if re-active in two of any of the regularly performed assays: complement fixation, immunodiffusion or quantitative immunodiffusion (performed at UC-Davis) and Premier® Coccidioides EIA manufactured by Meridian Bioscience (performed at IMMY). These specimens were kindly provided to IMMY for the evaluation of sona™ Coccidioides Ab lateral flow assay (CTA2003). Briefly, specimens were diluted to 1:441 in specimen diluent and tested on the sona™ Coccidioides Ab. After 20 minutes, results were interpreted by two operators. A ROC analysis was performed to determine assay sensitivity and specificity.
CONCLUSIONSThe sona™ Coccidioides Ab test is a rapid immunoassay that detects anti-Coccidioides antibodies. The highnegative predictive value of the assay allows for a 20 minute screen to rule out patients suspected of having Valley Fever. Management of this population of patients can be significantly improved by ruling out Valley Fever, allowing physicians to better direct treatment. Patients positive on the sona™ Coccidioides Ab test can then be referred to more traditional serology techniques to confirm the presence of antibodies to Coccidioides.
RAPID DETECTION OF ANTI-COCCIDIOIDES ANTIBODIES USING THE sona COCCIDIOIDES Ab LATERAL FLOW ASSAYTM
1 1 1 12
1 2
RESULTSThe area under the ROC curve was 0.885 (95% confidence interval (CI) = 0.801,0.942, standard error 0.0377,
IDEIACF*
IgG+IgM+ Total+2637
-
2023
-
4040
4019
* CF does not distinguish IgG/IgM
ABSTRACTCoccidioides, the causative agent of San Joaquin Valley Fever, is a fungal agent endemic to the arid regions of the Americas. Due to the difficulties associated with fungal culture, the dominant method of diagnosis is serology. Typical serological diagnosis methods require from 48 to 72 hours usingtechnologies like immunodiffusion or complement fixation. IMMY has developed a new 20 minutelateral flow assay for the detection of antibodies against Coccidioides sp. and it was compared to these typical serological methods. Specimens were considered reactive for antibodies againstCoccidioides if positive in two out of three different traditional assays: immunodiffusion, complementfixation and ELISA. A two by two diagnostic table and an ROC curve was developed comparing the assays. The area under the ROC curve was 0.885 (95% confidence interval (CI) = 0.801, 0.942,standard error 0.0377, p<0.001) for the sona™Coccidioides Ab assay. The assay had 100%sensitivity (95% CI: 91.1 - 100) and 76.92% specificity (95% CI: 63.2 - 87.5) when compared to thereference method. In this study, the positivepredictive value was 76.92% and the negativepredictive value was 100%.
Figure 3: ROC analysis of the CTA2003 kit vs characterized specimens
p<0.001) for the sona™ Coccidioides Ab assay. The assay had 100% sensitivity (95% CI: 91.1 - 100) and 76.92% specificity (95% CI: 63.2 - 87.5) when compared to the EORTC criteria. The positive predictive value was 76.9% and the negative predictive value was 100% (Figure 3).
Probable NegativePositive:
Negative:Totals
Totals40 12
40040 52
5240IMMY
EORTC
29 Coccidioidomycosis
Symposium
7th International
Stanford, CAAug. 10-13, 2017
1 Obtain 3 test tubesfor each serum specimen. • 2 for the dilution • 1 flat-bottom tube for running the test
Transfer Specimen Diluentto both tubes
200 µl diluentin tube 1
200 µl diluent in tube 2
SPECIMEN PREPARATIONDilute serum 1:441 with 1X Specimen Diluent:
3
1 2
Transfer 10 µlfrom tube 1 to tube 2.
Mix thoroughly.
200 µ
l
200 µ
l 2
Transfer 10 µlof patient serum to tube 1.
Mix thoroughly.
1
10 µl 10 µl
1 2
RUN TEST
SAM
PLE
SAM
PLE
Coc
ci Ab
Coc
ci Ab
Coc
ci Ab
Coc
ci Ab
5
Insert strip ( down)Incubate for 20 min.
Read Test1 line = negative2 lines = positive
6CT
SAM
PLE
SAM
PLE
SAM
PLE
SAM
PLE
+-
Coc
ci Ab
Coc
ci Ab
Coc
ci Ab
Coc
ci Ab
Coc
ci Ab
Coc
ci Ab
Coc
ci Ab
Coc
ci Ab
4
Transfer 100 µLfrom tube 2 to tube 3
2 3
100 µl
320 min.
PROCEDURE
Table 1: Specimen Characterization Table
SensitivitySpecificityPositive Likelihood RatioNegative Likelihood RatioDisease PrevalencePositive Predictive ValueNegative Predictive ValueROC Area Under CurveStandard Error (p<0.001)
100.00%76.92%
4.330.00
43.48%76.92%100%0.8850.0377
91.11 % to 100.00%63.16% to 87.46%
2.64 to 7.12
33.17% to 54.22%63.16% to 87.46%
91.11 % to 100.00%0.801 to 0.942
95% CI
REFERENCESNguyen C, Barker BM, Hoover S, Nix DE, Ample NM, Frelinger JA, Orbach MJ, Galgiani JN 2013. Recent advances in our understanding of the environmental, epidemiological, immunological, and clinical dimensions of coccidioidomycosis. Clin Microbiol Rev 26:505-525.
Tsang CA, Anderson SM, Imholte SB 2007. Enhanced surveillance of coccidioidomycosis, Arizona, USA. Emerg Infect Dis 2010; 16:1738-44.
Figure 2: The CTA2003 LFA Prodecure
Table 2: 2x2 Contingency Table and Statistical Analysis
Positive
C
T
Negative
