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Multisite Validation of Cryptococcal Antigen Lateral Flow Assay and Quantification by Laser Thermal Contrast - Volume 20, Number 1—January 2014 - Emerging Infectious Disease journal - CDC

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Multisite Validation of Cryptococcal Antigen Lateral Flow Assay and Quantification by Laser Thermal Contrast - Volume 20, Number 1—January 2014 - Emerging Infectious Disease journal - CDC

January 2014 Issue Now Available Online

Volume 20, Number 1—January 2014


Multisite Validation of Cryptococcal Antigen Lateral Flow Assay and Quantification by Laser Thermal Contrast

Article Contents

David R. BoulwareComments to Author , Melissa A. Rolfes, Radha Rajasingham, Maximilian von Hohenberg, Zhenpeng Qin, Kabanda Taseera, Charlotte Schutz, Richard Kwizera, Elissa K. Butler, Graeme Meintjes, Conrad Muzoora, John C. Bischof, and David B. Meya
Author affiliations: University of Minnesota, Minneapolis, Minnesota, USA (D.R. Boulware, M.A. Rolfes, R. Rajasingham, M. von Hohenberg, Z. Qin, E.K. Butler, J.C. Bischof, D.B. Meya)Makerere University, Kampala, Uganda (R. Rajasingham, R.K. Kwizera, D.B. Meya)Mbarara University of Science and Technology, Mbarara, Uganda (K. Taseera, C. Muzoora)G.F. Jooste Hospital, Cape Town, South Africa (C. Schutz, G. Meintjes)University of Cape Town, Cape Town (C. Schutz, G. Meintjes)


Cryptococcal meningitis is common in sub-Saharan Africa. Given the need for data for a rapid, point-of-care cryptococcal antigen (CRAG) lateral flow immunochromatographic assay (LFA), we assessed diagnostic performance of cerebrospinal fluid (CSF) culture, CRAG latex agglutination, India ink microscopy, and CRAG LFA for 832 HIV-infected persons with suspected meningitis during 2006–2009 (n = 299) in Uganda and during 2010–2012 (n = 533) in Uganda and South Africa. CRAG LFA had the best performance (sensitivity 99.3%, specificity 99.1%). Culture sensitivity was dependent on CSF volume (82.4% for 10 μL, 94.2% for 100 μL). CRAG latex agglutination test sensitivity (97.0%–97.8%) and specificity (85.9%–100%) varied between manufacturers. India ink microscopy was 86% sensitive. Laser thermal contrast had 92% accuracy (R = 0.91, p< 0.001) in quantifying CRAG titers from 1 LFA strip to within < 1.5 dilutions of actual CRAG titers. CRAG LFA is a major advance for meningitis diagnostics in resource-limited settings.
Cryptococcal meningitis is the most frequent form of meningitis among adults in sub-Saharan Africa (14) and accounts for 20%–25% of AIDS-related deaths in Africa (57). Although culture is the standard method for definitive diagnosis, detection of cryptococcal antigen (CRAG) in serum or cerebrospinal fluid (CSF) is used for presumptive diagnosis. CRAG screening in peripheral blood is also recommended for HIV-infected persons with CD4 cell counts < 100/μL to reduce early deaths while receiving antiretroviral therapy (ART) (810). CRAG is usually detected by latex agglutination (CRAG latex), which has a sensitivity and specificity > 99% (11,12). However, CRAG latex testing requires laboratory infrastructure and expertise, electricity, heat inactivation, cold-chain shipping, and refrigeration of reagents. Unfortunately, required infrastructure is usually unavailable in resource-constrained settings in which cryptococcal incidence is greatest (5). Thus, India ink microscopy is the primary diagnostic modality, despite having lower sensitivity (1214).
In July 2011, a lateral flow immunochromatographic assay (LFA) (Immy, Inc., Norman, OK, USA) was approved by the US Food and Drug Administration for detection of CRAG in CSF and serum. This assay is a rapid diagnostic test that provides a definitive result in ≤10 min. With its low cost (currently $2/test for resource-limited settings and $5/test for high-income settings), shelf stability at room temperature, and ease of use, this rapid, point-of-care test might expedite diagnosis. Field testing data are needed for this assay.
Quantification of CRAG incidence can be determined by determining CRAG titer. Quantification is clinically useful because higher CSF CRAG titers are predictive of risk for death (1517), and higher serum CRAG titers predict risk for immune reconstitution inflammatory syndrome (IRIS) (18). Titers can be determined by using CRAG latex or LFA, but these procedures are rarely used in resource-limited settings because of additional incremental cost per titer dilution. Thus, novel methods of quantification are needed.
In a multisite cohort of HIV-infected persons with suspected meningitis in sub-Saharan Africa, we compared CRAG LFA performance with that of traditional diagnostic tests. We demonstrated excellent diagnostic performance by the CRAG LFA in the laboratory and as a point-of-care bedside test. In addition, we demonstrated within this clinical cohort a novel method of CRAG titer quantification for an LFA by using laser thermal contrast measurement (19), enabling CRAG titer quantification without traditional serial dilutions.

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