1. Cryptococcal Antigen (CrAg) Essential In Vitro Diagnostic Device
David R Boulware MD, MPH, CTropMed
Associate Professor
University of Minnesota, USA

2. Overview
Need
Existing CrAg LFAs
Existing Validation Data
Discussion

3. Meningitis Etiologies in Adults in Africa
Hospital(s)
Country
Sample Size
HIV infected
Meningitis Prevalence
Bacterial / Pyogenic
Tuberculous
Cryptococcal
Aseptic / Viral Meningitis
Mulago and Mbarara1
Uganda
416
98% 4% 8%
59%
29%
GF Jooste, Cape Town2
South Africa
1,737
96%
19%
13%
30%
38%
Queen Elizabeth Central3
Malawi
263
77%
20%
17%
43%
Harare Central and Parirenyatwa4
Zimbabwe
200
90%
16%
12%
45%
28%
Univ. Teaching Hospital5
Zambia
331
100% 3%
14.5%
27.5%
55%
Pooled Average
2947
95%
(94-96%)
15.0%
( %)
12.8%
( %)
36.0%
( %)
36.3%
( %)
1 Durski K et al. J AIDS 2013; 63: e101-8.
2 Jarvis JN, et al. BMC Infect Dis 2010; 10: 67.
3 Cohen DB, et al. Trop Med Int Health 2010; 15:
4 Hakim JG et al. AIDS 2000; 14:
5 Siddiqi OK et al. Clin Infect Dis 2014; 58:

4. Lancet Infectious Diseases 2017; 17: 873-881.
2014 Estimated Annual Global Burden
Average CrAg prevalence 6.0% (95%CI 5.8–6.2) in CD4 <100 cells/μL
CrAg+ (95%CI, – )
cryptococcal meningitis cases (95%CI – ).
global deaths (95% CI – ),
~15% of AIDS-related deaths (95%CI 10–19%)
Plateau in Deaths since 2014, likely similar burden in 2018

5. CrAg LFAs, 2018 Manufacturer Year Country Approval Immy CrAg LFA 2011
USA
FDA, CE
Biosynex CryptoPS
2016
France CE
StrongStep, Liming Bio
2017
China ?
FungiXpert® Genobio Pharmaceutical
2018

6. Validation Studies Test Validation N Locations Immy CrAg LFA
Multi-site, Multiple studies
1000s
Multiple
Biosynex CryptoPS
Single Site
186 serum, plasma;
23 CSF
Cameroon
StrongStep, Liming Bio
Two-site, one study
143 CSF;
167 plasma
Uganda
FungiXpert® Genobio Pharmaceutical
One study
40 CSF
Mozambique

7. CrAg LFA Use to Data Test N CrAg+ N CrAg- Immy CrAg LFA 10,000’s
Biosynex CryptoPS
14 blood
4 CSF
172 blood
17 CSF
StrongStep, Liming Bio
101 CSF
55 plasma
42 CSF
112 plasma
FungiXpert® Genobio Pharmaceutical
14 CSF
26 CSF

8. Positive Predictive Value Negative Predictive Value
Immy CrAg LFA CSF Validation Study (Data on First prospective study, Multiple others)
Diagnostic Test n
Sensitivity
Specificity
Positive Predictive Value
Negative Predictive Value
CRAG LFA
666
99.3%
(435/438)
99.1%
(226/228)*
99.5%
(435/437)
98.7%
(226/229)
CSF Culture †
806
90.0%
(459/510)
100%
(296/296)
(459/459)
85.3%
(296/347)
100 mL CSF volume
524
94.2%
(309/328)
(196/196)
(309/309)
91.2%
(196/215)
10 mL CSF volume
282
82.4%
(150/182)
(100/100)
(150/150)
75.8%
(100/132)
India Ink
805
86.1%
(438/509)
97.3%
(288/296)
98.2%
(438/446)
80.2%
(288/359)
CRAG-latex (Meridian)
279
97.8%
(176/180)
85.9%
(85/99)
92.6%
(176/190)
95.5%
(85/89)
CRAG-latex (Immy)
749
97.0%
(452/466)
(283/283)
(452/452)
95.3%
(283/297)
* All CSF CRAG lateral flow assay (LFA) “false positives” did not have any other pathogen identified, but did not have a serum or plasma specimen available for testing of cryptococcal antigenemia to determine if this is enhanced detection.
† Different quantitative CSF culture methods were used in with 10 mL input volume vs with 100 mL CSF volume
Boulware DR. Rolfes MA, Rajasingham R, von Hohenberg M, Qin Z, Taseera K, Schutz C, Kwizera R, Butler EK, Meintjes G, Muzoora C, Bischof JC, Meya DB. Multisite validation of cryptococcal antigen lateral flow assay and quantification by laser thermal contrast. Emerg Infect Dis. 2014; 20;

9. Biosynex vs. Immy CrAg Serum Immy CrAg+ Immy CrAg- BioSynex CrAg+ 11
Biosynex Crag- 3
172
78%
Plasma
Immy CrAg+
Immy CrAg-
BioSynex CrAg+ 11
Biosynex Crag- 1
174
92%
CSF
Immy CrAg+
Immy CrAg-
BioSynex CrAg+ 4
Biosynex Crag- 19
Temfack E, Kouanfack C, Mossiang L, Loyse A, Fonkoua MC, Molloy SF, Koulla-Shiro S, Delaporte E, Dromer F, Harrison T, Lortholary O. Cryptococcal Antigen Screening in Asymptomatic HIV-Infected Antiretroviral Naïve Patients in Cameroon and Evaluation of the New Semi-Quantitative Biosynex CryptoPS Test. Front Microbiol. 2018; 9: 409.

10. Biosynex or Immy vs. Meridian EIA
Serum
EIA+
EIA Negative
BioSynex CrAg+ 8 3
Biosynex Crag-
175
Serum
EIA+
EIA Negative
Immy CrAg+ 8 6
Immy Crag-
172
Conclusion: EIA is a poor reference standard
Temfack E, Kouanfack C, Mossiang L, Loyse A, Fonkoua MC, Molloy SF, Koulla-Shiro S, Delaporte E, Dromer F, Harrison T, Lortholary O. Cryptococcal Antigen Screening in Asymptomatic HIV-Infected Antiretroviral Naïve Patients in Cameroon and Evaluation of the New Semi-Quantitative Biosynex CryptoPS Test. Front Microbiol. 2018; 9: 409.

11. Biosynex Conclusions Excellent Specificity
78% (11/14) Sensitivity of Serum (95%CI: 49% to 95%)
92% (11/12) Sensitivity of Plasma (95%CI: 61% to 99%)
4 of 4 CSF samples CrAg positive.
Of serum specimens with Immy titers of <1:100, only 2 of 5 were positive by Biosynex
Missed titers of 1:10 and 1:20
Detected one at 1:20 and one at 1:80.
Needs more validation on CSF and low blood titers
Temfack E, Kouanfack C, Mossiang L, Loyse A, Fonkoua MC, Molloy SF, Koulla-Shiro S, Delaporte E, Dromer F, Harrison T, Lortholary O. Cryptococcal Antigen Screening in Asymptomatic HIV-Infected Antiretroviral Naïve Patients in Cameroon and Evaluation of the New Semi-Quantitative Biosynex CryptoPS Test. Front Microbiol. 2018; 9: 409.

12. StrongStep CrAg Validation
Retrospective and Prospective components
Kampala and Mbarara, Uganda
Plasma
Immy CrAg+
Immy CrAg-
StrongStep CrAg+ 54 12
StrongStep Crag- 1
101
CSF
Immy CrAg+
Immy CrAg-
StrongStep CrAg+
101 1
StrongStep Crag- 41
Mpoza E, Mukaremera L, Kundura DA, Akampurira A, Luggya T, Tadeo KK, Pastick KA, Bridge SC, Tugume L, Kiggundu R, Musubire AK, Williams DA, Muzoora C, Nalintya E, Rajasingham R, Rhein J, Boulware DR, Meya DB, Abassi M. PLoS One. 2018; 13(1):e doi: /journal.pone

13. Strongstep CrAg LFA performance
Sample N
Sensitivity
Specificity
Adjusted Positive Predictive Value
Adjusted Negative Predictive Value
P-value
CSF
143
100%
(101/101)
98%
(41/42)
96%
0.99
Plasma
167
(54/55)
90%
(101/112)
50%
99.8%
0.009
The adjusted positive and negative predictive values are calculated for a CrAg antigenemia prevalence of 9% in Kampala, Uganda and cryptococcal meningitis prevalence of 37% in Sub-Saharan Africa
Mpoza E, Mukaremera L, Kundura DA, Akampurira A, Luggya T, Tadeo KK, Pastick KA, Bridge SC, Tugume L, Kiggundu R, Musubire AK, Williams DA, Muzoora C, Nalintya E, Rajasingham R, Rhein J, Boulware DR, Meya DB, Abassi M. PLoS One. 2018; 13(1):e doi: /journal.pone

14. StrongStep Discordant Specimens
StrongStep CrAg LFA
Immy CrAg Plasma
Immy CrAg CSF
CSF culture
Clinical Diagnosis
Classification
CSF 1
Positive
N/A
Negative
No Meningitis
False Positive
Plasma
39,000 CFU/mL
Cryptococcal Meningitis
False Negative 2
Viral Meningitis 9
No CrAg Antigenemia
Conclusions: A Validation study is more than 2 x 2 table.
One needs to define cases and controls based on their true disease state.
This is more difficult, requires collection of clinical outcome data – not just samples
Mpoza E, Mukaremera L, Kundura DA, Akampurira A, Luggya T, Tadeo KK, Pastick KA, Bridge SC, Tugume L, Kiggundu R, Musubire AK, Williams DA, Muzoora C, Nalintya E, Rajasingham R, Rhein J, Boulware DR, Meya DB, Abassi M. PLoS One. 2018; 13(1):e doi: /journal.pone

15. Dynamiker CrAg LFA Data Courtesy of: Jahit Sacarlal
Retrospective pilot, n=10 CSF
5 CrAg+ Immy = Dynamiker CrAg+
5 CrAg- Immy = Dynamiker CrAg-
Prospective Two lab Study in Maputo, Mozambique
One clinical site
Two blinded, independent labs
CSF Culture
Lab 1 Culture+
Lab 1 Culture-
Dynamiker CrAg+ 8 1
Dynamiker CrAg- 21
Sacarlal J, Lucas G, Simbine S, Relvas V, Quiboane ZC, Mogodiri T, Sun K. Evaluation of the Dynamiker Cryptococcal Antigen Lateral Flow Assay (LFA) in HIV patient admitted in Mozambique. Unpublished

16. Inter-lab Agreement CSF Culture Lab 1 Culture+ Lab 1 Culture-6 2
Lab 2 Culture- 22
CSF
Lab 1 India Ink+
Lab 1 India Ink-
Lab 2 India Ink+ 5 3
Lab 2 India Ink- 22
Sacarlal J, Lucas G, Simbine S, Relvas V, Quiboane ZC, Mogodiri T, Sun K. Evaluation of the Dynamiker Cryptococcal Antigen Lateral Flow Assay (LFA) in HIV patient admitted in Mozambique. Unpublished

17. Dynamiker Manufacturer Data
In Vitro
Control Reagent+
Control Reagent-
Dynamiker CrAg+
122 1
Dynamiker CrAg- 5 19
PLATELIA™ commercial reagent
Sensitivity: 96% Specificity 95%
CSF
Case Adjudication+
Case Adjudication-
Dynamiker CrAg+
122 1
Dynamiker CrAg- 24
Sensitivity: 100% Specificity 95%
Manufacturer Study: Methods very unclear.
Use latex agglutination (?Brand), culture, and case adjudication as ref standard.

18. References StrongStep
Mpoza E, Mukaremera L, Kundura DA, Akampurira A, Luggya T, Tadeo KK, Pastick KA, Bridge SC, Tugume L, Kiggundu R, Musubire AK, Williams DA, Muzoora C, Nalintya E, Rajasingham R, Rhein J, Boulware DR, Meya DB, Abassi M. PLoS One ; 13(1):e doi: /journal.pone
Biosynex
Temfack E, Kouanfack C, Mossiang L, Loyse A, Fonkoua MC, Molloy SF, Koulla- Shiro S, Delaporte E, Dromer F, Harrison T, Lortholary O. Cryptococcal Antigen Screening in Asymptomatic HIV-Infected Antiretroviral Naïve Patients in Cameroon and Evaluation of the New Semi-Quantitative Biosynex CryptoPS Test. Front Microbiol Mar 13;9:409. doi: /fmicb
Dynamiker
Sacarlal J, Lucas G, Simbine S, Relvas V, Quiboane ZC, Mogodiri T, Sun K. Evaluation of the Dynamiker Cryptococcal Antigen Lateral Flow Assay (LFA) in HIV patient admitted in Mozambique. Unpublished

19. What Specimen to Test: Distribution of CrAg positivity in Clinical Meningitis in Uganda 2013-17
CrAg test requires performance on CSF and blood (serum, plasma, or whole blood)

20. Discussion General Discussion
Information to be included with an application for inclusion in the WHO List of Essential In Vitro Diagnostics Device (IVD)s.

21. Box 1. Info to be included with an application for inclusion in the WHO List of IVDs
1. Summary statement of the proposal for inclusion:
Cryptococcal meningitis as the most common cause of meningitis in Africa requires diagnostics. Microscopy alone is insufficient as for example, the most common cause of meningitis with negative CSF India Ink is still cryptococcal meningitis.
2. Name of the focal point in WHO submitting the application ?
3. Name of the organization(s) consulted and/or supporting the application
Essential Diagnostics for Advanced HIV Disease Meeting, April 2018
4. Generic name (and brand name when only one IVD is suitable/available) of the IVD as appropriate:
Cryptococcal Antigen lateral flow assay
5. Whether listing is requested as a specific IVD from a single manufacturer or as a type of IVD technology.
IVD Technology
6. Information supporting the public health relevance (epidemiological information on disease burden, assessment of current use, target population)
Rajasingham, R et al. Lancet Infectious Diseases 2017
Target Population: HIV-infected persons with advanced disease or CNS symptoms

22. Box 1. Info to be included with an application for inclusion in the WHO List of IVDs
7. Characteristics of the IVD:
intended use: both diagnosis and screening
product presentation: Lateral flow assay + cassette
infrastructure level requirement: none/minimal
target user: all levels of healthcare system
sample type and volume: CSF, serum, plasma ~40mcL
sample handling: minimal, + Blood processing
Performance: >98% sensitivity and 99% specificity
Specificity important on blood. Sensitivity with a range of titers, detectable to 1:5 of Immy titer
time to results: <20 minutes
storage conditions: room temperature
operation conditions:
shipping requirements: Room Temp
training requirements:
associated equipment: Pippette
Throughput: variable
need for maintenance: None Connectivity: N/A

23. Box 1. Info to be included with an application for inclusion in the WHO List of IVDs
8. Use of the IVD details (as part of a diagnostic testing algorithm; reference to existing WHO and other clinical guidelines; need for special diagnostic or treatment facilities and skills)
Pre-ART Screening in CD4<200 as per 2017 WHO Guidelines for Advanced HIV Disease
Suspected Meningitis (Blood and CSF)
9. Summary of laboratory evaluation studies
Previously Summarized (Expand for prior published Immy studies)
10. Summary of validation studies in a variety of clinical settings:
Identification of clinical evidence (search strategy, systematic reviews identified, reasons for selection/exclusion of particular data)
Summary of available data (appraisal of quality, performance, ease of use, summary of results)

24. Box 1. Info to be included with an application for inclusion in the WHO List of IVDs
11. Summary of evidence on safety. N/A
12. Summary of data on comparative cost and cost-effectiveness:
Screening: Rajasingham JAIDS 2012; Jarvis PLoS One 2013
Meningitis Dx: Durski K. JAIDS 2013
13. Summary of regulatory status of the IVD:
FDA Approval: Immy
CE Mark (EU): Immy, Biosynex
WHO PQ: Not available from WHO for most common cause of meningitis.
14. Proposed text for the WHO List of Essential IVDs.
Text: