Multisite validation of cryptococcal antigen lateral flow assay and quantification by laser thermal contrast

David R Boulware, 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, David B Meya, David R Boulware, 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, David B Meya

Abstract

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.

Keywords: Cryptococcus spp.; HIV; India ink microscopy; South Africa; Uganda; cerebrospinal fluid; cryptococcal antigen; cryptococcal antigen latex agglutination; cryptococcal meningitis; culture; diagnostic techniques; fungi; laser thermal contrast measurement; lateral flow immunochromatographic assay; point-of-care systems; sensitivity; specificity; validation studies.

Figures

Figure 1
Figure 1
Five steps of the cryptococcal antigen lateral flow assay.
Figure 2
Figure 2
Venn diagram of distribution of 393 cryptococcal meningitis cases tested by 4 diagnostic assays, Uganda and South Africa. CSF, cerebrospinal fluid; CRAG LFA, cryptococcal antigen lateral immunochromatographic flow assay; India ink, India ink microscopy of 1 mL of concentrated CSF specimen. Numbers at the bottom right indicate 2 scenarios in which the Venn diagram does not overlap visually.
Figure 3
Figure 3
A) Prediction of cryptococcal antigen titer based on laser thermal contrast measurement and concept of lateral flow immunochromatographic assay (LFA) thermal contrast measurement in which a laser irradiates the test line in the LFA (19). The test line is formed by gold–monoclonal antibody–antigen sandwich complex with a monoclonal antibody affixed at the test line. When irradiated by a green laser (532 nm), any gold present absorbs light from the laser and generates heat in direct proportion to the amount of gold (and thereby antigen) present at the test line. This temperature change can be measured by using an infrared camera. B) Association of measured semiquantitative LFA cryptococcal antigen (CRAG) titer starting at a 1:250 dilution by the predicted CRAG titer based on thermal contrast measurement. Measurements on the negative portion of the x-axis are beyond the visual range when specimens were diluted 1:250, yet still detectable by thermal contrast. The Pearson correlation coefficient was r = 0.91 (p<0.001, R2 = 0.84) among 115 positive specimens quantified. A total of 58 LFA CRAG–negative specimens established background levels of heat radiation.

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