Preheating of urine improves the specificity of urinary cryptococcal antigen testing using the lateral flow assay

Brito‐Santos, Fabio; Ferreira, Marcelle de Lima; Trilles, Luciana; Muniz, Mauro de Medeiros; Santos, Valdiléa Gonçalves Veloso dos; Carvalho-Costa, Filipe Aníbal; Meyer, Wieland; Wanke, Bodo; Lazéra, Márcia dos Santos

doi:10.1371/journal.pntd.0005304

Cited by 8 publications

(8 citation statements)

References 9 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Considering EIA as the reference standard, both POC had excellent sensitivity while specificity was slightly better in serum for Biosynex CryptoPS. Interestingly, though urine screening provided false positive results with the LFA IMMY as previously reported ( Longley et al, 2016 ; Brito-Santos et al, 2017 ), Biosynex CryptoPS was not associated with false positives in urine.…”

Section: Discussionsupporting

confidence: 69%

Cryptococcal Antigen Screening in Asymptomatic HIV-Infected Antiretroviral Naïve Patients in Cameroon and Evaluation of the New Semi-Quantitative Biosynex CryptoPS Test

et al. 2018

View full text Add to dashboard Cite

Background: Cryptococcal meningitis (CM) is a major cause of AIDS-related mortality in Africa. Detection of serum cryptococcal antigen (CrAg) predicts development of CM in antiretroviral (ART) naïve HIV-infected patients with severe immune depression. Systematic pre-ART CrAg screening and pre-emptive oral fluconazole is thus recommended. We postulated that a semi-quantitative CrAg screening approach could offer clinically relevant advantages.Methods: ART-naïve asymptomatic adult outpatients with <100 CD4 cells/mm3 presenting to the Yaoundé Central Hospital, Cameroon were screened for CrAg using the IMMY lateral flow assay (LFA). CrAg positive patients were consented for lumbar puncture and those with proven CM were treated with combination antifungal therapy and those with no CM were offered long-term oral fluconazole. Simultaneous on-site evaluation of CrAg detection using the new LFA Biosynex® CryptoPS test was performed and both tests were subsequently compared to a reference commercialized CrAg enzyme immunoassay (EIA).Results: Prevalence of serum CrAg in 186 screened adults was 7.5% (95%CI: 4.5–12.4). In CrAg positive patients, CM prevalence was 45.5% (95%CI: 18.3–75.7). IMMY and Biosynex CryptoPS strongly agreed in serum, plasma, and cerebrospinal fluid (Kappa: 98.4, 99.5, 100%, respectively, p < 0.001), and disagreed in urine (29 isolated positive CrAg in urine with IMMY, none with Biosynex and none of whom had proven CM). Compared to EIA, serum specificities were 96.6 and 98.3%, respectively. With Biosynex CryptoPS, all CM patients were serum T2-band positive compared to nonewithout CM. Median EIA reciprocal titre was 160 (IQR: 13.5–718.8) and titres >160 strongly correlated with proven CM and Biosynex CryptoPS T2-band positivity. During the 1-year follow up period, there was no incident case of CM among screened patients and overall incidence of all-cause mortality was 31.5 per 100 person-years-at-risk (95%CI: 23.0–43.1).Conclusion: HIV-associated asymptomatic cryptococcosis is common in Cameroon, warranting integrated systematic screening and treatment. Biosynex CryptoPS holds promise, at point of care, for rapidly stratifying CrAg positive patients for optimal management including lumbar puncture and combination antifungal therapy when needed.Summary findings: Prevalence of CrAg and meningitis (CM) is high in Cameroon. Biosynex CryptoPS is comparable to IMMY LFA in CrAg screening. Its T2-band correlates with high antigen titres and CM, thus promising for identifying patients requiring effective induction therapy.Note: This study was presented in part at the 10th International Conference on Cryptococcus and Cryptococcosis (ICCC) in Iguazu in Brazil from 26 to 30th March 2017 and won a prize oral presentation.

show abstract

Section: Discussionsupporting

confidence: 69%

Cryptococcal Antigen Screening in Asymptomatic HIV-Infected Antiretroviral Naïve Patients in Cameroon and Evaluation of the New Semi-Quantitative Biosynex CryptoPS Test

et al. 2018

View full text Add to dashboard Cite

show abstract

“…Also, preheating urine was found to reduce the activity of thermally labile biomolecules, thus decreasing the number of FPs obtained when detecting cryptococcal antigen by LFAs. 163 For DNA/RNA LFAs with a sample preamplification step, FPs can be produced by the primers/probes and side products from the preamplification step, such as primer dimers. 160,164,165 Such FPs can be reduced by optimizing the primer and probe sequences in the preamplification step 151,152,166,167 and the capture and detection sequences in the LFA.…”

Section: Improving Specificitymentioning

confidence: 99%

“…For example, when detecting analytes from whole blood, blood cells and other large particles are usually separated from the blood before the LFA is run by adding a filtration pad before the conjugation pad or by centrifuging the sample. Also, preheating urine was found to reduce the activity of thermally labile biomolecules, thus decreasing the number of FPs obtained when detecting cryptococcal antigen by LFAs …”

Section: Improving Specificitymentioning

confidence: 99%

Ultrasensitive and Highly Specific Lateral Flow Assays for Point-of-Care Diagnosis

et al. 2021

View full text Add to dashboard Cite

Lateral flow assays (LFAs) are paper-based point-of-care (POC) diagnostic tools that are widely used because of their low cost, ease of use, and rapid format. Unfortunately, traditional commercial LFAs have significantly poorer sensitivities (μM) and specificities than standard laboratory tests (enzyme-linked immunosorbent assay, ELISA: pM−fM; polymerase chain reaction, PCR: aM), thus limiting their impact in disease control. In this Perspective, we review the evolving efforts to increase the sensitivity and specificity of LFAs. Recent work to improve the sensitivity through assay improvement includes optimization of the assay kinetics and signal amplification by either reader systems or additional reagents. Together, these efforts have produced LFAs with ELISA-level sensitivities (pM−fM). In addition, sample preamplification can be applied to both nucleic acids (direct amplification) and other analytes (indirect amplification) prior to LFA testing, which can lead to PCR-level (aM) sensitivity. However, these amplification strategies also increase the detection time and assay complexity, which inhibits the large-scale POC use of LFAs. Perspectives to achieve future rapid (<30 min), ultrasensitive (PCR-level), and "sample-to-answer" POC diagnostics are also provided. In the case of LFA specificity, recent research efforts have focused on high-affinity molecules and assay optimization to reduce nonspecific binding. Furthermore, novel highly specific molecules, such as CRISPR/Cas systems, can be integrated into diagnosis with LFAs to produce not only ultrasensitive but also highly specific POC diagnostics. In summary, with continuing improvements, LFAs may soon offer performance at the POC that is competitive with laboratory techniques while retaining a rapid format.

show abstract

“…However, the heat-stable nature of the PD7 epitope both in the 18 kDa monomeric ribotoxin Asp f I and the 36 kDa dimeric ribotoxin mitogillin, enabled us to incorporate a 10-min heating step at 100 °C that led to aggregation of the 18 kDa species and subsequent detectability by mAb PD7 in the Afu -LFD ® test. Heating also served to eliminate non-specific binding in the test with normal human urine (results not shown), a phenomenon similarly shown to improve the specificity of the urinary cryptococcal LFA [ 77 ]. Heating combined with a urine concentration step resulted in a limit of detection of ~15 ng Asp f I/mL urine.…”

Section: Discussionmentioning

confidence: 99%

Aspergillus fumigatus and Its Allergenic Ribotoxin Asp f I: Old Enemies but New Opportunities for Urine-Based Detection of Invasive Pulmonary Aspergillosis Using Lateral-Flow Technology

Davies

Singh

Prattes

et al. 2020

JoF

View full text Add to dashboard Cite

Invasive pulmonary aspergillosis (IPA) caused by Aspergillus fumigatus is a life-threatening lung disease of immunocompromised patients. Diagnosis currently relies on non-specific chest CT, culture of the fungus from invasive lung biopsy, and detection of the cell wall carbohydrate galactomannan (GM) in serum or in BAL fluids recovered during invasive bronchoscopy. Urine provides an ideal bodily fluid for the non-invasive detection of pathogen biomarkers, with current urine-based immunodiagnostics for IPA focused on GM. Surrogate protein biomarkers might serve to improve disease detection. Here, we report the development of a monoclonal antibody (mAb), PD7, which is specific to A. fumigatus and related species in the section Fumigati, and which binds to its 18 kDa ribotoxin Asp f I. Using PD7, we show that the protein is secreted during hyphal development, and so represents an ideal candidate for detecting invasive growth. We have developed a lateral-flow device (Afu-LFD®) incorporating the mAb which has a limit of detection of ~15 ng Asp f I/mL urine. Preliminary evidence of the test’s diagnostic potential is demonstrated with urine from a patient with acute lymphoid leukaemia with probable IPA. The Afu-LFD® therefore provides a potential novel opportunity for non-invasive urine-based detection of IPA caused by A. fumigatus.

show abstract

Preheating of urine improves the specificity of urinary cryptococcal antigen testing using the lateral flow assay

Cited by 8 publications

References 9 publications

Cryptococcal Antigen Screening in Asymptomatic HIV-Infected Antiretroviral Naïve Patients in Cameroon and Evaluation of the New Semi-Quantitative Biosynex CryptoPS Test

Cryptococcal Antigen Screening in Asymptomatic HIV-Infected Antiretroviral Naïve Patients in Cameroon and Evaluation of the New Semi-Quantitative Biosynex CryptoPS Test

Ultrasensitive and Highly Specific Lateral Flow Assays for Point-of-Care Diagnosis

Aspergillus fumigatus and Its Allergenic Ribotoxin Asp f I: Old Enemies but New Opportunities for Urine-Based Detection of Invasive Pulmonary Aspergillosis Using Lateral-Flow Technology

Contact Info

Product

Resources

About