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

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.

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

Malaria Cases Due to Plasmodium falciparum

World Malaria Report, 2008

Malaria Cases Due to Plasmodium falciparum and Plasmodium vivax

Feachem, Lancet. 2010 November 6; 376(9752): 1566–1578

Clinical Presentation of Malaria

Source: CDC

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

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)

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

Mode of Action of Rapid Diagnostic Tests

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)

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

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

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

(+) (-)

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

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

Available Treatments for Malaria

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

Recommended Treatments for Severe P. falciparum Malaria

First-line treatment

Parenteral artesunate

Second-line treatment

Artemether or quinine if parenteral artesunate is not available

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

P. Falciparum Resistance to Former First-line Treatments

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

Factors Contributing to Development of Resistance

Uncontrolled use of combination therapies

Monotherapy

Sub-therapeutic levels, substandard and counterfeit drugs.

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

Vaccine Targets based on the life cycle of the parasite

Pre-erythrocytic

vaccines

Blood-stage vaccines

Transmission-blocking

vaccines

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

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

Questions?

Back-up slides

Population at risk for malaria in 2009 according to World Health Organization Regions

Region Population* Population at risk

for malaria* (low &

high) (%)

Population at risk

for malaria* (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

Estimated numbers of malaria cases (in millions) and deaths (in thousands) by WHO Region, 2008

Region No. of malaria

cases *

No. of deaths * % of deaths

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

Source: World Malaria Report 2009

*-(5th-95th Percentile)

The prevalence of mixed Plasmodium infections detected at admission by microscopy and PCR

Countries Mixed infections;

microscopy (%)

Mixed infections;

PCR (%)

Cryptic species

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

Guinea

80/1470 (0.01) 113/173 (65) Pf; Pv; Pm; Po

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

Countries with the highest number of confirmed P. falciparum cases in 2009

Country Population Microscopy/RDTs taken

Microscopy/RDTs positive

P. falciparum 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

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

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

Global distribution of G6PD deficiency

Nkhoma et al, 2009

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%)

Treatment of Malaria

Objectives of treatment include: – Prevent progress to severe disease and complications

– Prevent development of antimalarial drug resistance

– Reduce transmission

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

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

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

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