6 global update pf hrp2 deletions
TRANSCRIPT
Global Update on P.falciparum HRP2 deletions
XV Reunion Annual de Evaluacion de AMI/RAVREDABogota, Colombia 3‐5 May 2016
Jane Cunningham
What is histidine rich protein 2 (HRP2) ?
• Malaria protein specific to P.falciparum• Found in cytoplasm and surface of Pf infected erythrocytes
• Secreted by the parasite and may be found in plasma and in culture media
• Function not very well understood • May detoxify free haeme by polymerization (acts as haeme polymerase) to inactive haemozoin
Main application of HRP2: RDTs
• Most RDTs that are capable of diagnosing Plasmodium falciparum, target PfHRP2.
• Monoclonal antibodies in RDTs target an epitope abundant in both HRP2 and HRP3 proteins.
• Pf‐HRP2‐detecting RDTs a widely used diagnostic tool for P. falciparum infections.• Generally, PfHRP2‐detecting RDTs have better sensitivity and thermal stability
compared to pLDH‐detecting RDTs• Many more available products meeting WHO procurement criteria
• RDTs detecting P. falciparum alone are suitable for use in most of sub‐Saharan Africa where malaria cases are predominantly caused by P. falciparum.
• 2014: 53% of global RDT sales (166 M) in 2014 were Pf only tests delivered to Africa; in Americas 444,730 Pf only tests; 446,265 combo tests
Reporting of hrp2/hrp3 deletions has a significant potential impact on RDT procurement and case management ‐ accurate reporting and mapping is essential
HRP2 deleted parasites ….old news in South America
January 2010 | Volume 5 | Issue 1 | e8091
o Recommendations against use of HRP2 based RDTs
o Urgent need for investigation of the abundance and geographic distribution of these parasites in Peru and neighboring countries.
o Surveys conducted in: Bolivia, Colombia, Guyana, Peru, Suriname, Brazil, Honduras
41% (61/148) isolates lacked pfhrp2; 21% lacked both pfhrp2 and 3
Rest of world (2010‐2014)HRP2 “fishing expeditions”
Shortcomings in African studies including:
• Use of RDTs of unknown performance/quality or not used at all • Incomplete analyses: flanking genes • Failure to verify the quality of DNA in the sample (amplifying at least 2 single copy genes)
• No published follow up surveys
New survey to be published in 2016• 16 sites in 8 endemic states
Just enough data to raise suspicion
Emphasize accurate reporting of HRP2 deletions
HRP
3
At least 2 eg. msp1, msp2, Glurp
If ELISA not available; use alternative RDT brand
Cheng et al. Malaria Journal 2014, 13:283
2015‐2016 – new reports
Ghana Amoah et al. Malar J (2016) 15:101
Eritrea
India
Two other HBC
China‐MyanmarBorder Acta Tropica 2015; 152, 26‐31
• Use of HRP2 based RDTs in all these countries, but RDT use is not causing HRP2 deletions but it may be sufficient to select for HRP2 deleted parasites, which arose from spontaneous mutations or were imported.
• What other factors could be at play ?
Eritrea
• In late September 2015 National Malaria Control Program/CDC of Eritrea reported to WHO high false negative RDT rates for Pf and Pv with several lots of SD BIOLINE Malaria Ag Pf/Pv, (05FK 80), compared with microscopy in several zones.
1 Maekel2 Anseba3 Gash‐Barka4 Debub5 Northern Red Sea6 Southern Red Sea
Ethiopia
2014‐2015: 28 complaints forms (138 events (95% Pf) from 9 Hospitals in 4 Zobas/zonesRDT retrieved from all affected areas and sent for lot testing at WHO‐FIND lab: Passed
RDT results from Ghindae, Eritrea
SD Bioline Malaria Ag Pf/Pf/Pv (05FK120)T1: HRP2 – negative T2: Pf‐specific p‐LDH ‐ positive T3: Pv‐specific p‐LDH
CareStart™ Malaria HRP2/pLDH (Pf/PAN) COMBO (G0131)T1: HRP2 ‐ negative T2: pan‐pLDH ‐ positive (faint line)
CareStart™ Malaria pLDH (PAN) (G0111)
T1: pan‐pLDH – positive
P.falciparum confirmed by microscopy(120 parasites/µL)
Preliminary results
• Prospective collection 50 Pf microscopy positive samples from two regional hospitals • Site 1: 21/26 (81%) HRP2 Negative • Site 2: 9/25 (36%) HRP2 Negative
• SD Bioline Malaria Ag Pf/Pf/Pv (05FK120) RDT • T1: HRP2; T2: Pf‐specific p‐LDH; T3: Pv‐specific p‐LDH • HRP2 test line negative/pf‐pLDH test line positive = Pf PCR positive + HRP2 PCR negative
• Suggests that this RDT could be useful in screening for the presence of HRP2 deleted parasites
Interim recommendations – Info Note 1. Suspected false‐negative RDT results should be investigated
• Consider range of factors: operator error; degradation of RDTs due to exposure to heat or humidity; low or extremely high parasite density
2. Pf‐hrp2/hrp3 gene deletions should be suspected: • on an individual basis, when a patient sample tests negative
on the HRP2 test line of at least two quality‐assured malaria RDTs and positive on the pan‐ or pf‐pLDH test line of a combination RDT and the sample is confirmed by microscopy to be positive for P. falciparum by two qualified microscopists;
• on a programmatic basis, when a fall in the RDT positivity rate cannot be reasonably explained by other malaria control interventions and/or when the rates of discordance between RDT and microscopy results show systematically higher positivity rates with microscopy (≥ 10–15%) compared to RDTs, where routine quality control is done by cross‐checking or both are performed on the same individuals (e.g. during surveys).
Interim recommendations‐ Info Note
3. Where hrp2/hrp3 gene deletions have been reported, the prevalence should be determined in the affected country and neighbouring countries. This may require specific surveys or adaptation of planned surveys, such as malaria indicator surveys or therapeutic efficacy studies. 4. Analysis of well‐preserved archived specimens may be useful for identifying the existence and geographical location of hrp2/hrp3‐deleted parasite populations. 5. In the absence of confirmed reports of hrp2/hrp3gene deletions, new initiatives to find these gene deletions are not recommended, unless they are prompted by findings for programmes described under 2.
In all cases, investigations should be according to established protocols and with appropriate standards and quality control measures.
Alternatives to HRP2 based RDTs? • Threshold for use of non‐HRP2 based RDTs ? • Consider trade‐offs – pan and pf‐LDH RDTs less sensitive and generate weaker test band
intensities• Overall non‐HRP2 RDT options are very limited
• Surveys – + geographical variation in prevalence – full picture needed to determine best RDT options.
• Surveillance – could use RDTs with HRP2 and pf‐pLDH test lines; HRP2 negative/pf‐pLDH + ‐ collect DBS for PCR and HRP2/HRP3 analysis
Total false
False positive Plasmodium spp.
Infectioni
SD Bioline Malaria Ag P.f (HRP2/pLDH) 05FK90 Standard Diagnostics, Inc. 88 (87 / 52) NA 100 (100 / 97) NA 0.0 0.0CareStart™ Malaria HRP2/pLDH Pf test G0181/G0181‐ET Access Bio, Inc. 91.0 NA 99.0 NA 0.0 0.0
CareStart™ Malaria pLDH 3 Line Test G0121 Access Bio, Inc. 88.9 91.4 100.0 100.0 0.5 0.0CareStart™ Malaria Screen G0231 Access Bio, Inc. 86.9 88.6 100.0 100.0 2.5 0.1
SD Bioline Malaria Ag P.f/P.f/P.v 05FK120 Standard Diagnostics, Inc. 85 (84 / 36) 91.4 100 (100 / 98)f 100.0 0.0 0.0
CareStart™ Malaria pLDH (PAN) G0111 Access Bio, Inc. 84.0 88.6 99.0 97.1 0.0 0.0Advantage Pan Malaria Card IR013025 J. Mitra & Co. Pvt. Ltd. 77.0 100.0 98.0 100.0 0.4 0.0
Pf/pan
Product Product code Manufacturer Panel Detection Score2000 or 5000 parasites/l
Pf samples Pv
samples
Invalid rate (%)l
Clean negative samples
Pf and Pf and Pv
Pan only
Pf only
200 parasites/l
Pf samplesPv
samples
PDS for individual test lines : HRP2/pf‐pLDH
WHO procurement criteria requires minimum PDS @200 p/ul of 75% for Pf and Pv
Access Bio puts both mabs for HRP2 and pf‐pLDH on the same
test line
WHO Response
1. Providing technical support to confirm and map HRP2/HRP3‐deleted parasites where they are suspected on the basis of discordance between the results of HRP2‐RDTs and high‐quality microscopy;
2. Working with relevant groups to adapt planned malaria indicator surveys and demographic and health surveys to include collection of blood samples for molecular testing for malaria, including analysis of pfhrp2/pfhrp3.
3. Working with research groups that hold collections of recently archived samples to screen for the presence of HRP2/HRP3‐deleted parasites;
4. Rigorously reviewing manuscripts submitted for publications and published reports of pfhrp2/pfhrp3 deletions to determine the accuracy of claims and
5. Encouraging test developers and RDT manufacturers to improve the performance of pLDH based tests and identify new target antigens.
6. Identify resources to be able to support 1‐5
Thank you