Background
Therapeutic efficacy of anti-malarial drugs
Therapeutic efficacy studies: history
Year of protocol publication | ||||
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1965 | 1996 | 2003 | 2009 | |
Classifications for treatment efficacy | Sensitivity: clearance of asexual parasitaemia within 7 days of the first day of treatment, without subsequent recrudescence | Adequate clinical response (ACR): Absence of parasitaemia on Day 14 irrespective of axillary temperature, without previously meeting any of the criteria of ETF or LTF or Axillary temperature < 37.5 °C irrespective of the presence of parasitaemia, without previously meeting any of the criteria of ETF or LTF | Adequate clinical and parasitological response (ACPR): For low to moderate transmission area: Absence of parasitaemia on day 28 irrespective of axillary temperature without previously meeting any of the criteria of early treatment failure or late clinical failure or late parasitological failure For intense transmission area: Absence of parasitaemia on day 14 irrespective of axillary temperature without previously meeting any of the criteria of early treatment failure or late clinical failure or late parasitological failure | Adequate clinical and parasitological response (ACPR): Absence of parasitaemia on day 28 (or day 42), irrespective of axillary temperature, in patients who did not previously meet any of the criteria of ETF, LCF or LPF |
Classifications for treatment failure | Resistance RI: Clearance of asexual parasitaemia as above, but followed by recrudescence before or after day 7 | Early treatment failure (ETF): If the patient develops one of the following during the first 3 day of follow-up: Development of danger signs or severe malaria on day 1, 2 or 3, in the presence of parasitaemia; Axillary Temperature ≥ 37.5 °C on day 2 with parasitaemia higher than the day 0 parasite count; Axillary Temperature ≥ 37.5 °C on day 3 in the presence of parasitaemia; Parasitaemia on day 3 ≥ 25% of the parasite count on day 0 | Early treatment failure (ETF): For all transmission area: development of danger signs or sever malaria on day 1, day 2, day 3 in the presence of parasitaemia; Parasitaemia on day 2 higher than on day 0 irrespective of axillary temperature; Parasitaemia on day 3 with axillary temperature ≥ 37.5 °C; parasitaemia on day ≥ 25% of count on day 0 | Early treatment failure (ETF): Danger signs or severe malaria on day 1, 2 or 3, in the presence of parasitaemia; Parasitaemia on day 2 higher than on day 0, irrespective of axillary temperature; Parasitaemia on day 3 with axillary temperature ≥ 37.5 °C; and parasitaemia on day 3 ≥ 25% of count on day 0 |
Resistance RII: Marked reduction of asexual parasitaemia within the first 7 days of follow-up, but no clearance | Late treatment failure (LTF): Development of danger signs or severe malaria in the presence of parasitaemia on any day from day 4 to 14, without previously meeting any of the criteria of ETF; Axillary temperature ≥ 37.5 °C in the presence of parasitaemia on any day from day 4 to 14, without previously meeting any of the criteria of ETF | Late clinical failure (LCF): For low to moderate transmission area: Development of danger signs or severe malaria after day 3 in the presence of parasitaemia without previously meeting any of the criteria of early treatment failure; presence of parasitaemia and axillary temperature ≥ 37.5 °C (or history of fever) on any day from day 4 to day 28 without previously meeting any of the criteria of early treatment failure For intense transmission area: Development of danger signs or severe malaria after day 3 in the presence of parasitaemia without previously meeting any of the criteria of early treatment failure; presence of parasitaemia and axillary temperature ≥ 37.5 °C (or history of fever) on any day from day 4 to day 14 without previously meeting any of the criteria of early treatment failure | Late clinical failure (LCF): Danger signs or severe malaria in the presence of parasitaemia on any day between day 4 and day 28 (or day 42) in patients who did not previously meet any of the criteria of ETF; and presence of parasitaemia on any day between day 4 and day 28 (or day 42) with axillary temperature ≥ 37.5 °C in patients who did not previously meet any of the criteria of ETF | |
Resistance RIII: No marked reduction of asexual parasitaemia within the first 7 days of follow-up | Late parasitological failure (LPF): For low to moderate transmission area: Presence of parasitaemia on any day from day 7 to day 28 irrespective of axillary temperature and without previously meeting any of the criteria of early treatment failure or late clinical failure For intense transmission area: Presence of parasitaemia on day 14 and axillary temperature < 37.5 °C without previously meeting any of the criteria of early treatment failure or late clinical failure | Late parasitological failure (LPF): Presence of parasitaemia on any day between day 7 and day 28 (or day 42) with axillary temperature < 37.5 °C in patients who did not previously meet any of the criteria of ETF or LCF |
Current protocol
Genotyping to distinguish between recrudescence and reinfection
Pharmacokinetics (PK)/pharmacodynamics (PD)
In vitro and ex vivo phenotypic assay for anti-malarial susceptibility assessment
Principle
WHO microtest | Isotopic | ELISA | Flow cytometry | SYBR green | RSA | |
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Infrastructure | Biosafety laboratory level 2 for parasite culture | Biosafety laboratory level 2 for parasite culture | Biosafety laboratory level 2 for parasite culture | Biosafety laboratory level 2 for parasite culture | Biosafety laboratory level 2 for parasite culture | Biosafety laboratory level 2 for parasite culture |
Equipment | Refrigerator Freezer Microscopy Biosafety cabinet Incubator with gas (CO2, O2 and N2) | Refrigerator Freezer Microscopy Biosafety cabinet Incubator (CO2 and O2) Liquid scintillation counter | Refrigerator Freezer Microscopy Biosafety cabinet Incubator with gas (CO2, O2 and N2) Spectrophotometer | Refrigerator Freezer Microscopy Biosafety cabinet Incubator with gas (CO2, O2 and N2) Flow cytometer | Refrigerator Freezer Microscopy Biosafety cabinet Incubator with gas (CO2, O2 and N2) Fluorimeter | Refrigerator Freezer Microscopy Biosafety cabinet Incubator with gas (CO2, O2 and N2) Flow cytometer) |
Reagents | Reagents for culture | Reagents for culture [3H] hypoxanthine | Reagents for culture HRP2/pLDH monoclonal antibodies Reagents for ELISA | Reagents for culture Fluorochrome | Reagents for culture SYBR Green | Reagents for culture Fluorochrome |
Incubation time (h) | 24–30 | 48 | 72 | 48 | 48 | 48 |
Time to results (12 samples) (h) | 38–42 | 52 | 80 | 54 | 52 | 60 |
Reproducibility | Variable | Good | Variable | Good | Good | Variable to good |
Cost by sample (USD $) | ≥ 5 | ≥ 5 | 1–5 (HRP2) 0.5–2 (pLDH) | ≥ 5 | 0.08 | ≥ 5 |
Advantages | No heavy equipment | Automatic reading | Relatively inexpensive | Highly sensitive | Inexpensive Short Procedure | No heavy equipment (except if using flow cytometry) |
Disadvantages | Labour intensive Requires quality assured microscopy Difficult to standardize Expensive | Use of radioactive reagents Heavy equipment Expensive | High inter-variability between laboratories and users No commercially available Kit for pLDH | Heavy equipment Expensive | Underestimation of parasitemia because of DNA-binding proteins competing with the dye Heavy equipment Interaction between drugs and the dye | Labour intensive (microscopy) Requires quality assured microscopy Difficult to standardize (microscopy) Heavy equipment (if using flow cytometry) Expensive |
Microscopy (WHO microtest)
Isotopic test
ELISA (HRP2 and pLDH)
Fluorescent markers
Flow cytometry
Ring stage survival assay (RSA)
Molecular methods for the detection of genetic polymorphisms associated with anti-malarial drug resistance
Principle
Restriction fragment length polymorphism (RFLP)
Sanger sequencing (capillary electrophoresis)
Next generation sequencing
Real-time-PCR
Ligase detection reaction fluorescent microsphere (LDR-FM) assay
New molecular methods in development
Nucleic acid lateral flow immunoassay (NALFIA)
Q-poc™
Discussion and outlook
Advantages | Disadvantages | |
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In vivo | Relatively easy to standardize No heavy equipment required Provides results directly obtained from patients Provides the evidence required for modifying treatment policies Helps to define the first line and second line treatment for case management Can provide required safety data Confirms association of parasite resistance with in vitro test results (IC50 values) or molecular resistance markers | Logistics constraints (long follow-up with many patients lost to follow up, lack of patients in low transmission settings, expensive) Potential over-estimation of treatment failures because of: inter-individual variation in pharmacokinetics including poor absorption, rapid elimination (diarrhoea, vomiting) and/or insufficient or poor biotransformation of pro-drugs because of human genetic characteristics; extrinsic factors such as poor patient compliance (if the totality of treatment is provided), incorrect dosage, poor drug quality or poor microscopy Potential under-estimation of treatment failures because of host factors such as the immunity or poor microscopy |
In vitro | Provides the intrinsic parasite susceptibility to the drug without confounding factors such as immunity and pharmacology | Difficult to standardize Require a special design (concentration and duration) for certain drugs (i.e. RSA, PSA) Requires good infrastructure and highly trained staff Results not always associated with therapeutic efficacy |
Molecular | Provides direct information on the resistance status of the parasite. When they are validated, their prevalence in a parasite population are often a good indicator of the level of clinical resistance Can provide useful information on the spread of resistance Relatively easy to implement | Requires good infrastructure and highly trained staff Results not always associated with therapeutic efficacy |