1. Multi-Drug Resistant Malaria:
Plasmodium falciparum at our door step
Vani Vannappagari, MBBS, MPH, PhD.
WorldWide Epidemiology , GlaxoSmithKline
Presented at the 41st Annual Symposium
“Global Movement of Infectious Pathogens and Improved Laboratory
Detection”
Eastern PA Branch-American Society for Microbiology
November 17, 2011
Thomas Jefferson University, Philadelphia

2. Malaria: Morbidity and Mortality
There are ~250 million malaria cases annually
~A million malaria related deaths annually
85% of deaths occur among the <5 year age group
– By some calculations a child dies of malaria, every 30
seconds
Over 3.2 Billion people are at risk for malaria and over 1.2
billion people are at high risk for malaria.

3. Protozoa from the Plasmodium genus
Five species causing human disease
– Plasmodium falciparum
– Plasmodium vivax
– Plasmodium ovale
– Plasmodium malariae
– Plasmodium knowlesi
P. falciparum, the most virulent, accounts for >75% of
malaria cases in SSA where ~80% of malaria cases and
~90% of malaria deaths occur
P. vivax accounts for 10-20% of malaria cases in SSA;
outside of SSA, it accounts for >50% of all malaria cases

4. Malaria Cases Due to Plasmodium falciparum
World Malaria Report, 2008

5. Malaria Cases Due to Plasmodium falciparum
and Plasmodium vivax
Feachem, Lancet. 2010 November 6; 376(9752): 1566–1578

6. Clinical Presentation of Malaria
Source: CDC

7. Diagnosis of Malaria
Prompt and accurate diagnosis
Ideal diagnostic tests need to be rapid, affordable,
low-maintenance, minimal training, able to detect
low density parasitemia and distinguish species
There are several methods of diagnosing malaria
including:
– Clinical
– Microscopy
– Rapid Diagnostic Tests
– Polymerase Chain Reaction (PCR)
– Other tests

8. Clinical Symptoms as a Guide to Diagnosis
WHO recommendations for laboratory-confirmed diagnosis of malaria
infections prior to treatment in all cases
Clinical features cannot reliably distinguish severe malaria from other
severe viral and bacterial infections in children
Specificity of clinical diagnosis (i.e. declared fever) is only 20-60%
compared with microscopy
127 (6%) of 2048 children admitted to hospital in Kenya and
Mozambique with severe falciparum malaria had concurrent positive
blood cultures (Bassat, 2009)
At autopsy, seven of 31 (22.6%) Malawian children with a clinical
diagnosis of cerebral malaria were found to have died from other causes.
(Taylor, 2004)

9. Microscopy
Operational Gold Standard for malaria diagnosis
Under optimum conditions, microscopy can detect 20-50
parasites per μL blood
Advantages of using microscopy include parasite
quantification and species identification
Achieving high sensitivity requires training and quality
control of microscopists, adequate equipment, and
maintenance

10. Mode of Action of Rapid Diagnostic Tests

11. P. falciparum panel detection score of malaria RDTs at low (200) and high (2000
or 5000) parasite density (parasites/μl) according to target antigen type (HRP2
or pLDH)

12. Polymerase Chain Reaction (PCR) Tests –Gold
Standard
Numerous PCR assays have been developed for the laboratory diagnosis
of malaria, including nested and real-time PCR techniques
PCR-based assays are highly specific and sensitive, capable of detecting
as few as 5 parasites per μL of blood
PCR can readily detect mixed-species infections and may be automated
to enable the processing of large numbers of samples.
PCR tests are mostly used in research settings for diagnostic
confirmation and identification of molecular markers of resistance
In areas with high transmission, detection of low-level parasitaemia may
not be clinically relevant

13. Nucleic Acid Lateral Flow Immunoassay (NALFIA)
NALFIA is a simple readout system for nucleic acids and has been
successfully applied for the detection of food-borne pathogens
such as Bacillus cereus and Salmonella
Rapid immunochromatographic test to detect labeled amplicon
products on a nitrocellulose stick coated with specific antibodies
Lower detection limit is 0.3 to 3 parasites/μL, 10-fold more
sensitive than gel electrophoresis analysis
More sensitive than microscopy and a good alternative to detect
PCR products while circumventing using electricity or expensive
equipment
 First step toward molecular field diagnosis

14. Loop-Mediated Isothermal Amplification (LAMP)
LAMP (Loop-mediated isothermal amplification), allows
rapid amplification and detection of a target genetic
sequence with minimal instrumentation and simplified
sample processing
Can detect 1-6 parasites / l with minimal sample
processing that requires no sophisticated equipment
and the results can be read with the naked eye
Early results have proved the utility of this approach for (-) (+)
identifying parasite-positive individuals in population
surveys, even at low transmission intensities
Currently prototype still under study

15. Other Diagnostic Methods
Fluorescence microscopy
Flow cytometry
Life lens smart phone application
– Smartphone application.
– A user takes a photo of a blood sample using a phone and
the high resolution imaging sensor determines whether
the blood is infected with malarial parasites

16. Frequently Used Diagnostic Methods
Clinical diagnosis - least expensive and most commonly used
method in most malaria endemic areas
Globally, ~73% of reported suspected malaria cases in 2009
were parasitologically-confirmed before treatment
 In SSA, only ~35% confirmed before treatment
 Fosters drug resistance
Microscopy - most popular parasitological method of
detecting malaria infection
 Only available in better-equipped clinics
Use of RDTs also on the rise, but still remain out of reach to
many due to cost

17. Available Treatments for Malaria

18. Recommended Treatments for Uncomplicated
P. falciparum Malaria
First-line treatments
 Artemisinin-based combination treatments (ACTs)
 Recommended ACTs include: artemether plus
lumefantrine, artesunate plus amodiaquine,
artesunate plus mefloquine, and artesunate plus
sulfadoxine-pyrimethamine
Second-line treatments
 Alternative ACT known to be effective in that particular
region
 Artesunate plus tetracycline or doxycycline or
clindamycin
 Quinine plus tetracycline or doxycycline or clindamycin

19. Recommended Treatments for Severe P. falciparum
Malaria
First-line treatment
Parenteral artesunate
Second-line treatment
Artemether or quinine if parenteral artesunate is
not available

20. P. falciparum Drug Resistance
P. falciparum has become variably resistant to all drug
classes except artemisinin derivatives
 Particularly resistant to former first-line drugs
chloroquine (median treatment failure rate up to 100%)
and sulfadoxine-pyrimethamine (median treatment
failure rate as high as 52.8% in some areas)
Chloroquine remains effective only in Central America

21. P. Falciparum Resistance to Former First-line Treatments

22. Multi-Drug Resistance
Multi-drug resistance has been established in southeast Asia,
South America, and SSA
There is high and increasing global resistance of P.
falciparum to amodiaquine, mefloquine and lumefantrine

23. Factors Contributing to Development of
Resistance
Uncontrolled use of combination therapies
Monotherapy
Sub-therapeutic levels, substandard and counterfeit drugs.

24. Delaying the Development and Spread of Resistance
Early diagnosis and appropriate treatment of malaria
– Treatment: prompt provision of antimalarial drugs (ACTs
for P. falciparum and chloroquine and primaquine for P.
vivax).
Optimizing insect vector control (Indoor residual spraying of
insecticide, insecticide treated bed nets)
Strengthening disease management and surveillance
systems and monitoring for drug resistance

25. Vaccine Targets based on the life cycle of the
parasite
Pre-erythrocytic
vaccines
Blood-stage vaccines
Transmission-blocking
vaccines

26. Challenges in Developing a Malaria Vaccine
Parasite has developed immune evasion strategies
Each infection presents thousands of antigens
Different antigens at different stages of the life cycle
Multiple infections (different strains and/or different
species)
Inadequate data on the immune response to infection

27. Most Advanced Vaccine Candidate - RTS,S Malaria
RTS,S is a fusion protein of a portion of the
circumsporozoite protein (CSP) with the hepatitis B
surface antigen produced as a recombinant particle and
combined with a proprietary adjuvant AS02
(GlaxoSmithKline)
Phase II trials showed reduction of clinical malaria by 35%
-55% and severe malaria by 49%
Phase 3 trials begun in 2009 to recruit up to 16,000
children in 7 African countries
Results expected in 2012

28. Questions?

29. Back-up slides

30. Population at risk for malaria in 2009 according to
World Health Organization Regions
Region Population* Population at risk Population at risk
for malaria* (low & for malaria* (high
high) (%) only) (%)
Africa 821.8 696.3 (84.7) 567.0 (69.0)
Americas 543.3 158.5 (29.2) 42.1 (7.7)
East Mediterranean 555.2 307.3 (55.3) 115.9 (20.9)
Europe 276.9 2.6 (0.9) 0.2 (0.1)
South-East Asia 1,783.3 1,249.2 (70.0) 449.5 (25.2)
Western Pacific 1,637.8 866.4 (52.9) 68.5 (4.2)
Total 5,618.2 3,280.3 (58.4) 1,243.2 (22.1)
*-Millions
Source: World Malaria Report 2010

31. Estimated numbers of malaria cases (in millions)
and deaths (in thousands) by WHO Region, 2008
Region No. of malaria No. of deaths * % of deaths
cases * under 5 years
Africa 208 (155-276) 767 (621-902) 88
The Americas 1 (1-1) 1 (1-2) 30
East Mediterranean 9 (7-11) 52 (32-73) 77
Europe 0 0 3
South-East Asia 24 (20-29) 40 (27-55) 34
Western Pacific 2 (1-2) 3 (2-5) 41
Total 243 (190-311) 863 (708-1003) 85
*-(5th-95th Percentile)
Source: World Malaria Report 2009

32. The prevalence of mixed Plasmodium infections detected
at admission by microscopy and PCR
Countries Mixed infections; Mixed infections; Cryptic species
microscopy (%) PCR (%)
Australia 0/23 (0) 4/23 (17) Pv
Thailand 1/137 (0.5) 9/173 (5) Pv
Thailand 1/196 (0) 25/196(13) Pv; Pm
Thailand 0/48 (0) 6/48(12.5) Pf; Pv
Thailand 18/475 (4) 356/548 (65) Pf; Pv; Pm; Po
Venezuela 0/100 (0) 17/100(17) Pf; Pv
Equatorial Guinea 11/159 (7) 44/159 (28) Pf; Pm; Po
Spain 2/192 (1) 9/192 (5) Pv; Pm; Po
Papua New 80/1470 (0.01) 113/173 (65) Pf; Pv; Pm; Po
Guinea
Laos 2/58 (3.4) 27/117 (23.1) Pf; Pv; Pm; Po
Thailand 7/300 (2.3) 26/151 (17) Pf; Pv; Pm; Po
Source: Maymax et al. Trends in Parasitology 2004

33. Countries with the highest number of confirmed P.
falciparum cases in 2009
Microscopy/RDTs Microscopy/RDTs P. falciparum
Country Population taken positive cases*
Uganda 32,709,864 3,6112,418 1,301,337 1,275,310
India 1,198,003,273 103,395,721 1,563,344 837,130
Ghana 23,837,261 2,899,497 1,104,370 924,095
Ethiopia 82,824,732 1,328,114 1,036,316 594,751
Benin 8,934,986 --- 889,597 534,590
Liberia 3,954,977 1,003,961 839,581 212,657
Indonesia 229,964,721 2,461,428 544,470 212,501
Togo 6,618,613 734,303 391,338 191,357
Myanmar 50,019,774 940,050 436,068 121,636
Congo 3,683,181 203,160 92,855 92,855
Note: Microscopy slides/RDTs taken may be lower than microscopy slides/RDTs positive if a country did not
report number of RDTs examined (taken), * Proportion of P. falciparum out of total confirmed malaria cases
Source: World Malaria Report 2010

34. Populations at high risk for malaria infection
High transmission areas
– Children under five years
– Pregnant women
– HIV infected individuals
– Travellers from non-endemic areas
Low transmission areas
– All age groups
– Travellers from non-endemic areas

35. Glucose-6-phosphate Dehydrogenase (G6PD) Deficiency
G6PD is required by all cells for protection from damage by oxidation.
For the red cell, this is the sole source of protection against oxidant
damage in the form of free radicals generated by the conversion of
oxy- to deoxyhemoglobin and by peroxides generated by
phagocytosing granulocytes.
Approximately 330 million people worldwide affected with highest
prevalence in individuals of African, Mediterranean and Asian
heritage.
Since this is an X-linked gene, prevalence among females is higher
but they are generally asymptomatic. Many variants of G6PD alleles
have been identified

36. Global distribution of G6PD deficiency
Nkhoma et al, 2009

37. G6PD variants
• Class I: severely deficient, associated with chronic
nonspherocytic hemolytic anemia
–
• Class II: severely deficient (1%-10% residual activity),
associated with acute intermittent hemolytic anemia
(G6PD Mediterranean)
–
• Class III: moderately deficient (10%-60% residual
activity) - intermittent hemolysis usually associated with
infection or drugs
• Class IV: normal activity (60%-100%)
• Class V: increased activity (>100%-150%)

38. Treatment of Malaria
Objectives of treatment include:
– Prevent progress to severe disease and complications
– Prevent development of antimalarial drug resistance
– Reduce transmission

39. Treatment guidelines for uncomplicated falciparum malaria
Artemisinin-based combination therapies (ACT) are the recommended
treatments for uncomplicated falciparum malaria.
ACTs should include at least 3 days of treatment with an artemisinin
derivative
The following ACTs options are recommended:
– Artemether + lumefantrine; artesunate + amodiaquine; artesunate +
mefloquine; artesunate + sulfadoxine-pyrimethamine; and
dihydroartemisinin + piperaquine .
Second-line antimalarial treatment:
– Alternative ACT known to be effective in the region;
– Artesunate plus tetracycline or doxycycline or clindamycin.
– Quinine plus tetracycline or doxycycline or clindamycin.
Source: WHO Guidelines for the Treatment of Malaria, 2010

40. Treatment guidelines for severe falciparum malaria
For adults, artesunate i.v. or i.m
– Quinine remains an acceptable alternative
For children (especially in the malaria endemic areas of Africa) the
following options are recommended as there is insufficient evidence to
recommend any of these antimalarial medicines over another:
– artesunate i.v. or i.m.; quinine (i.v. infusion or divided i.m. injection);
artemether i.m.
Give parenteral antimalarials for a minimum of 24hrs once started
(irrespective of the patient's ability to tolerate oral medication earlier),
and, thereafter, complete treatment by giving a complete course of:
– an ACT; artesunate + clindamycin or doxycycline; quinine +
clindamycin or doxycycline
Source: WHO Guidelines for the Treatment of Malaria, 2010

41. Treatment guidelines for special populations –pregnant
women
First trimester:
– Quinine + clindamycin
– An ACT is indicated only if this is the only treatment immediately
available, or if treatment with quinine + clindamycin fails or
compliance issues with a 7-day treatment.
Second and third trimesters:
– ACT known to be effective in the country/region or artesunate +
clindamycin or quinine + clindamycin
Source: WHO Guidelines for the Treatment of Malaria, 2010

42. Treatment guidelines for other special populations
Lactating women
– Lactating women should receive standard antimalarial treatment
(including ACTs) except for dapsone, primaquine and tetracyclines.
Infants and young children
– ACTs with attention to accurate dosing and ensuring
Travellers returning to non-endemic countries:
– atovaquone-proguanil
– Artemether +lumefantrine
– dihydroartemisinin + piperaquine
– quinine + doxycycline or clindamycin.
Source: WHO Guidelines for the Treatment of Malaria, 2010