This document summarizes a study on submicroscopic and asymptomatic Plasmodium infections in western Thailand. The study found high rates of submicroscopic infections (11.4%) in asymptomatic villagers using qPCR screening. Among symptomatic patients, qPCR also detected frequent cryptic mixed infections (26%) that were missed by microscopy. Serological analysis found evidence of prior exposure in all individuals and correlated with qPCR results. The findings have implications for malaria control and elimination efforts, suggesting submicroscopic infections are more common than previously thought. Improved diagnostics beyond microscopy may be needed to accurately detect ongoing transmission.

1. Submicroscopic and asymptomatic
Plasmodium falciparum and P. vivax
infections are common in western Thailand
- the molecular and serological evidence -
Elisabeth Baum,
Ph.D.
Department of
Medicine
University of
California, Irvine

2. ! Mesoendemic for malaria
! P. vivax (60%), P. falciparum (40%);
P. malariae and P. ovale
! Control phase, entering pre-elimination
! On track for >75% decrease in incidence in
2000-2015 (WHO World Malaria Report 2013)
Malaria in Thailand

3. Source: WHO South East Asia Regional Office – Malaria Statistics for Thailand 2013
0
1
2
3
4
5
6
7
1990
1991
1992
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
Annual Parasite Incidence
(malaria positives in 1,000 population at risk)
Successes Challenges
! Drug resistance in P. falciparum
(artemisinin) and P. vivax (chloroquine)
! Influx of refugees, migrant workers
and displaced persons camps along
borders
Malaria in Thailand

4. Tak Province
! One of the highest Plasmodium
prevalences in the country
Source: WHO Thailand National Strategic Plan for Malaria Control and Elimination (2011)
Tak
Parasite prevalence is highest on border regions
Tak – Myanmar river border Refugees from Myanmar entering Tak
API per 1,000
Myanmar
Laos
Cambodia
Malaysia

5. Active Case Detection (ACD)
! Weekly household visits
Mass Blood Surveys (MBS)
! Quarterly cross-sectional
Malaria Surveillance in Tak Province
Passive Case Detection (PCD)
! Hospital (1)
! Malaria clinics (5)4 villages

6. Malaria at a Glance in Tak, Thailand
0
2
4
6
8
10
12
14
16
18
20
0
1000
2000
3000
4000
5000
6000
2011 2012 2013
%Smearpositiverate
Bloodsmearsexamined
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0
500
1000
1500
2000
2500
3000
3500
4000
MBS1 MBS2 MBS3 MBS4 MBS5
%Smearpositiverate
Bloodsmearsexamined
Mass Blood Surveys
(4 sentinel villages)
Passive Case Detection
(1 hospital, 5 clinics)
2011 2012 2013

7. Low incidence of clinical cases and low parasite prevalence in the population…
…is Plasmodium really disappearing from Tak, or are we missing something?

8. ! qPCR screening and serology using a
protein microarray
! Whole blood from finger-prick
! Peak malaria season 2012 (May)
! Samples from:
! MBS from one village
! PCD from hospital
! Matched expert microscopy
Methods

9. Sample Sources
Community MBS
Mae Salid Noi
n=379
Samples collected
qPCR screening
Microarray serology
n=219
n=93
>11 y
>15 y
! No symptoms during ±2 weeks from MBS
! 2 had fever on MBS (0.91%)
n=379
>0 y

10. Sample Sources
Community MBS
Mae Salid Noi
n=379
Samples collected
qPCR screening
Microarray serology
n=219
n=93
>11 y
>15 y
n=97
n=61
n=60
>15 y
>15 y
Hospital PCD
Tha Song Yang
>1 y>0 y

11. qPCR Screening
• 100-150µL of RBC pellet
• Total DNA extraction and purification
• Species-specific 18S rRNA gene
o P. falciparum
o P. vivax
• Parasite detection limit 50 parasite/mL

12. Infectious Status of Samples from Community MBS
qPCR Microscopy
0
5
10
15
20
190
200
210
220
Resultbymethod(n=219)
• 11.4% infections
• 92% asymptomatic
o (1 Pf, 1 Pv with fever)
• 100% submicroscopic
Negative Pv Pf

13. Infectious Status of Samples from Hospital PCD
Microscopy
Pf
Pv
Pf + Pv
Neg
qPCR
Pf
5
0
0
2
Pv
0
8
0
5
Pf + Pv
2
5
0
0
Neg
0
0
0
34
0
10
20
30
40
qPCR Microscopy
Resultbymethod(n=61)
Negative Pv Pf Pf + Pv
• 44.3% Plasmodium infections
• 26% mixed-species
• 100% submicroscopic 2° species

14. Frequent submicroscopic asymptomatic infections amongst villagers,
and frequent cryptic mixed infections amongst malaria patients
Community (MBS) Hospital (PCD)
• 44.3% infections (32.8% by LM)
• 26% mixed-species (0% by LM)
• 100% submicroscopic 2° species
• 11.4% infections (0% by LM)
• 92% asymptomatic
• 100% submicroscopic

15. Protein Microarray Serology
• Serology provides clues of past and current exposure
• How serology correlates with qPCR results?
• Protein microarray displaying 500 P. falciparum and
500 P. vivax proteins
Source of Samples
! Community MBS (n=93, adults)
Lack of fever for 5 months (ACD records)
! Hospital PCD (n=60, adults)
Limited information regarding symptoms and malaria history
(some febrile, some not)

16. Community (Asymptomatic) Malaria Clinic (Symptomatic)
Negative PF+ PV+ Negative PF+ PFPV+ PV+
P.falciparumproteins(n=281)P.vivaxproteins(n=177)
0 2 >5
Intensity of antibody binding to P. falciparum and P. vivax proteins, as measured by Z-scores of signal intensity
! 1,000 proteins
! 458 sero-reactive
Community (asymptomatic) Hospital (symptomatic)
281 P. falciparum
antigens
177 P. vivax
antigens

17. 0
50
100
150
200
250
300
350
400
450
500
Countofsero-reactiveproteins(n=458)
Community MBS n=93 Hospital PCD n=60
(Healthy) (Symptomatic)
Number of antigens recognized by individual serum samples

18. Intensity of Antibody Binding
Community MBS Hospital PCD Community MBS Hospital PCD
P. falciparum antigens P. vivax antigens
Negative
PF +
PV +
Mixed sp.
Meansignalintensityand95%CI

19. Conclusions
! Serological evidence of exposure in all individuals tested (100% sero-positivity)
! Molecular screening detected an unexpected number of sub-microscopic
infections amongst asymptomatic villagers
! Molecular screening detected cryptic mixed-species infections amongst malaria
patients, missed by microscopy
! Implications for control/elimination and correct treatment?

20. Implications
Source: The malERA Consultative Group on Diagnoses and Diagnostics (2011) A Research Agenda for Malaria Eradication: Diagnoses and Diagnostics. PLoS Med 8(1)
Malaria in Tak is here?
The way forward?
Light microscopy
RDTs
Molecular (DNA)
Serology
Clinical diagnosis
High prevalence Low prevalence Pre-elimination Eliminated
Parasite density

21. Elisabeth Baum,
Ph.D.
Department of
Medicine
University of
California, Irvine
Funding
NIH Centers for Excellence in Malaria Research (ICEMR), Southeast Asia. U19
AI089672
UC Irvine Huw Davies, Aarti Jain, Eugenia Lo, Ming-Chieh Lee, Philip Felgner,
Guiyun Yan
Antigen Discovery Inc. Arlo Z. Randall, Douglas M. Molina, Xiaowu Liang
Mahidol Vivax Research Center Jetsumon Sattabongkot, Kirakorn Kialtibutr,
and field crew
Vector Borne Disease Training Center Jeeraphat Sirichaisinthop, and field crew
Pennsylvania State University Liwang Cui
*This work was submitted to Malaria Journal
Thank you!