Therapeutic efficacy studies: history

The first major protocol revision was made in 1996, extending the follow up period to 14 days, with blood samples taken only on day 0, day 3, day 7, and day 14 of follow-up. Parasitological and clinical responses were taken into account for the evaluation of therapeutic efficacy [46]. The responses to the treatment were classified into ‘adequate clinical response’ (ACR), ‘early treatment failure’ (ETF) and ‘late treatment failure’ (LTF) (Table 1). However, the protocol remained more appropriate for high transmission regions.

A second major revision was carried out in 2003, with recommendations for the evaluation of drugs in low and moderate transmission settings, to fill the gap of the previous standardized protocol version from 1996 [47]. The protocol included a 28-day follow-up period for low and moderate transmission settings, and a 14-day follow-up period for high transmission settings. A 28-day follow-up period was also recommended for the evaluation of drugs with long half-life. The protocol also suggested the use of genotyping by polymerase chain reaction (PCR) to differentiate recrudescence from re-infections during the follow-up period, especially for studies in high transmission areas [48]. The treatment responses were re-classified into four categories: ‘adequate clinical and parasitological response’ (ACPR), ‘early treatment failure’ (ETF), ‘late clinical failure’ (LCF) and ‘late parasitological failure’ (LPF) (Table 1). Even when this new protocol responded to the needs of different transmission settings, some confusion arose due to the use of the same classification of treatment failures, but with different definitions according to transmission intensity.

Current protocol

The current protocol was developed in 2009, and incorporates recommendations for all endemic regions in a single procedure (Table 1). For all endemic areas, both clinical and parasitological observations are taken into account for the interpretation of treatment responses [44]. The protocol also includes recommendations for an emergency treatment in case of study exclusions. Patients excluded from the study are either censored or removed from the analysis, depending on the type of analysis that is required, even though, Kaplan–Meier analysis is the preferred to the per protocol analysis [44]. At least 28 days (or 42–63 days for drugs with longer half-lives) are required for follow-up. This protocol includes a recommendation as well to take a blood sample to measure the concentration of the drug at day 7, which is a good predictor of drug absorption and its correlation with treatment failure [49, 50].

Genotyping to distinguish between recrudescence and reinfection

In the current therapeutic efficacy protocol, systematic genotyping is recommended in cases of clinical or parasitological failure, to distinguish recrudescence from re-infections, using three highly polymorphic genes: merozoite surface protein 1 (msp1), merozoite surface protein 2 (msp2), and glutamate rich protein (glurp) [44]. Genotypic profiles obtained from samples collected on day 0 (i.e., from the initial parasite infection) and on day X (i.e., the day of follow-up observation) are compared. Identical profiles confirm recrudescent cases, while different profiles are indicative of a re-infection. Notably, these genes encode antigens under immune selective pressure, and this might bias the interpretation of dissimilar parasites in paired blood samples [51]. Moreover, despite the WHO recommendation, some studies only use 1 or 2 of the three genes, or use them sequentially and only test some of the genes, and other use more than three genes, which limits the comparison between different studies and could compromise the interpretation of the results [52]. The results of the genotyping are often detected by agarose gel, and there can be high inter-individual variation in results interpretation depending on the quality of the gel and the experience of the laboratory technician [53]. This is due to the lack of resolution of the current genotyping methods that do not allow to correctly distinguish polymorphisms on agarose gel for digested PCR products with long fragments such as those from glurp [54]. Moreover, genotyping could not give accurate estimates, especially in area of high transmission intensity due to the high number of infecting parasite strains (multiplicity of infection) [52].

Other methods have been developed to distinguish recrudescent and new infections, using microsatellite markers [55] and capillary electrophoresis [56, 57]. Microsatellites are simple sequence repeats in the Plasmodium genome, generally not more than three nucleotides, and hundreds have been described [58]. They are generally not under immune selection, and the sizes of alleles fall at predictable, discrete lengths that may enable easy comparison across multiple samples and laboratories [59]. By measuring the size of microsatellites using capillary electrophoresis, which has a resolution of one nucleotide and is highly reproducible, the full diversity of length polymorphisms present in a population can be evaluated [55]. Many procedures for genotyping by microsatellites have been discussed [60], and some studies have shown that it is difficult to conclude with certainty the distinction between a recrudescence and a reinfection. The use of microsatellite markers in combination with msp1, msp2, and glurp genes may improve the sensitivity and standardization methods of P. falciparum genotyping [55, 61, 62].

Pharmacokinetics (PK)/pharmacodynamics (PD)

It has been shown that host factors, such as bioavailability and immune response, can influence the therapeutic response [63, 64]. Apparent drug failures may in fact reflect issues with the metabolism of the drug rather than innate parasite resistance. The impact of drug metabolism on the treatment outcome can be assessed by PK (dynamics of the drug concentration resulting from administration of a certain drug dose) and PD (impact of a certain drug concentration on the parasite density) [65]. Bioavailability is one of the most important factor, referring to the proportion of the absorbed drug that is transformed into the active metabolite, enters the blood circulation, and has an anti-malarial effect [49, 65]. Only a few years ago, the WHO started promoting pharmacology assessment as part of anti-malarial efficacy studies, by publishing specific methods and procedures [50]. The WHO’s protocol recommends taking a blood sample (venous or capillary blood) from the patient, several times per day, to assess drug concentration dynamics over the course of the treatment. These data are analysed using different PK models appropriate for each drug [66]. The results show if the drug has reached the required therapeutic concentration that correlates with elimination of the parasite, or if the concentration has been sub-optimal, in which case a treatment failure should not be interpreted as parasite resistance [66, 67]. Studies with different anti-malarial drugs have shown that in some patients, especially young children, who are frequently those involved in therapeutic efficacy studies in high transmission settings, tend to receive a lower dose of the drug leading to suboptimal blood concentrations [68, 69]. This may lead to patient outcomes misclassifications as treatment failure due to drug resistance whereas the patient didn’t reach the optimal drug concentration to clear the parasites.

Principle

The assessment of P. falciparum parasites susceptibility to anti-malarial drugs can be performed phenotypically, using parasite strains collected from patients (ex vivo) or with culture-adapted isolates (in vitro) [70]. The assessment can be done by culturing parasites in the presence of anti-malarial drugs at varying concentrations to determine the growth inhibitory effect of the drugs (Fig. 1), or by exposing parasites to a specific high concentration for a relatively short period [71]. The parasite culture is done in laboratory flasks with liquid medium and red blood cells, supplemented with amino acids, human serum, or bovine serum albumin (BSA). Antibiotics can also be added to the culture medium to avoid bacterial contamination. The susceptibility assays are usually done in assay plates (96-, 24-, or 12-well) which have been previously coated with varying concentrations of the anti-malarial drugs. The parasite growth is then measured using various techniques, and results used to determine either the concentration that inhibits parasite growth by 50% (50% inhibitory concentration; IC₅₀) [73] or the survival rate [71]. Currently, in vitro/ex vivo drug sensitivity assays use one of several different methods to measure parasite growth [74], as described below and in Table 2.

	WHO microtest	Isotopic	ELISA	Flow cytometry	SYBR green	RSA
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)

The principle of the assay is based on counting parasite growth by microscopy. Parasites are cultured with different concentrations of drugs for 24–30 h. The baseline parasitemia differs between the different techniques, and varies from 0.1 to 1% of red blood cells being infected. Parasite growth in the different plate wells with different drug concentrations is determined by counting the parasites by microscopy, on a thin film, after Giemsa staining [75, 76]. One must take into account the number of parasites at different developmental stages (ring stage or schizont) for the interpretation of the results.

Isotopic test

The principle of the assay is based on measuring parasite growth by adding radioactive dye in the culture that is incorporate into parasite DNA. Parasites are cultured with different concentrations of a drug for 48 h, and a radioactive marker, i.e., tritium labelled hypoxanthine, is added to the culture medium [77, 78]. Hypoxanthine is a DNA precursor, and the tritium-labelled hypoxanthine is incorporated into the parasite DNA during the culture phase. After the 48 h incubation period, the culture is filtered through filter paper and the paper dried. A scintillating liquid is added to the paper, and in a beta-counter machine, the parasite DNA is measured as counts per minute (CPM), and resulting values are used to calculate the IC₅₀.

ELISA (HRP2 and pLDH)

The principle of the assay is based on assessing parasite growth by measuring the concentration of proteins produced by the cultured parasites [79]. Parasites are cultured with different concentrations of a drug for 72 h. After 72 h, the culture plates are frozen at − 20 °C and thawed (several times if needed) to ensure cell lysis. In parallel, ELISA plates are coated with monoclonal antibodies directed against the Plasmodium spp. LDH (lactate dehydrogenase), or the P. falciparum-specific HRP2 (histidine-rich protein 2) at 4 °C overnight [80, 81]. The wells of the antibody coated plates are then incubated with parasite culture mixes transferred from the thawed parasite culture plates. After an incubation period of 2 h, biotinylated antibodies and colorimetric detection reagents are added. Finally, the plates are read on a spectrophotometer at 450 nm, and resulting absorbance values are used to calculate the IC₅₀ and determine the parasite susceptibility to anti-malarial drugs [82, 83].

Fluorescent markers

The principle of the assay is based on measuring parasite growth using a fluorescent marker that will react with DNA and RNA [84]. Parasites are cultured with different concentrations of a drug for 48 h or 72 h. After 48/72 h of incubation, the parasite culture plate is frozen at − 80 °C until the SYBR Green I assay is performed. The plate is thawed for 2 h at room temperature on the day of analysis, and the contents are shaken briefly, before transferring to a new plate, where SYBR Green is added and incubated at room temperature for 1 h. Finally, the plate is read on a fluorimeter, the intensity of fluorescence corresponding to the quantity of the DNA in the culture, and the IC₅₀ is determined from the obtained values.

Flow cytometry

The principle of the assay is based on measuring parasite growth by counting the number of infected red blood cells. Parasites are cultured for 48 h with different concentrations of a drug [85]. The method is based on the detection of infected red blood cells by marking intra-erythrocytic parasite DNA with a fluorescent dye. Various types of permeable markers can be used to mark the DNA [85]. The mixture of infected and non-infected red blood cells is then analysed in a cytometer, and the results analysed to determine the amount of infected cells, hence the number of parasites having grown in absence or in presence of the anti-malarial drug, to determine the IC₅₀ [86, 87].

Ring stage survival assay (RSA)

This method was developed specifically to assess resistance of P. falciparum parasites to artemisinin derivatives that cannot be well detected by the classical in vitro/ex vivo assays [71, 88]. This is due to the fact that the decreased susceptibility to artemisinin affects the ring stages only [89], hence a resistance phenomenon cannot be well detected in a culture that goes through all the parasites stages. For in vitro assays, parasites are cultured without any drug to reach a high parasite density (≥ 0.2%), then a tight synchronization step (ring-stages aged from 0 to 3 h) is performed to eliminate the schizont stages, and the ring stage parasites are placed into culture in the presence of 700 nM of dihydroartemisinin (DHA). This concentration represents the typical therapeutic concentration found in patients treated with artemisinin derivatives. DHA is removed after 6 h (a physiologically relevant duration), and the parasites are placed into a fresh culture mix for another 66 h. For ex vivo assays, samples collected from patients are processed within 24 h. Plasma is removed and the blood washed three times in RPMI-1640. If the parasitemia is greater than 1%, it is adjusted to 1% by adding uninfected erythrocytes, but parasites are not experimentally synchronized, they are directly exposed to 700 nM of dihydroartemisinin (DHA) for 6 h, and then placed into a fresh culture mix for another 66 h. For both in vitro and ex vivo, survival rates are assessed microscopically or by flow cytometry by counting the proportion of viable parasites that developed into second-generation rings or trophozoites with normal morphology at 66 after drug removal [90].

Principle

Molecular methods have allowed for a better understanding of the emergence and spread of anti-malarial drug resistance. In the last two decades, the mechanisms of resistance to the most widely used anti-malarial drugs have been revealed in part using molecular techniques, and anti-malarial resistance is often associated with single nucleotide polymorphisms (SNPs) or amplifications of the genes coding for drug target proteins or transporters (CNVs) [91]. Various methods have been developed for the assessment of these known resistance markers. Resistance to chloroquine is associated with point mutations in two different genes: P. falciparum chloroquine-resistance transporter (Pfcrt) and P. falciparum multidrug resistance gene 1 (Pfmdr1) [92–94]. Resistances to sulfadoxine/pyrimethamine are associated with point mutations in P. falciparum genes coding for dihydrofolate reductase (Pfdhfr) [95, 96] and dihydropteroate synthetase (Pfdhps) [97], while resistance to mefloquine has been associated with gene amplification in Pfmdr1gene [98, 99]. Mutations in the parasite cytochrome b are associated with atovaquone resistance [100] and resistance to piperaquine has been associated with gene amplification in plasmepsin 2 and 3 genes [101, 102]. More recently, decreased susceptibility to artemisinin derivatives has been associated with point mutations in the propeller domain of a Kelch gene located on the chromosome 13 (K13) [33].

The basic principle of most of methodologies assessing genomic markers associated with drug resistance is based, after DNA extraction, on the amplification of the gene or loci of interest, using the polymerase chain reaction (PCR). To increase the detection sensitivity, a second amplification can be used to amplify the PCR product of the primary PCR (nested PCR). In resource-limited settings, blood samples are often collected and dried onto filter papers and then stored and transported appropriately to the laboratory. Parasite DNA obtained from dried blood spots can be stable when adequately stored until analysis (several years at room temperature). The transport of these biological samples therefore doesn’t require any specialized equipment for storage; however it is important that the blood spots are well dried to avoid growth of fungi and deterioration of the DNA. It is also critical to avoid cross contamination between different samples when storing and cutting the blood spots. The DNA extraction can be performed using a variety of methods, ranging from simple Chelex methods to commercial kits [103]. The amplification by PCR only requires a small amount of extracted DNA.

Restriction fragment length polymorphism (RFLP)

The PCR products are digested with specific restriction enzymes, to determine if the sequence of the codons of interest is present [104]. These restriction enzymes only cut the nucleotide sequence at specific sites which results in PCR product fragments of specific sizes. After enzymatic digestion the digested amplification products are then analysed by agarose gel electrophoresis to determine the length of the digested DNA products. The result can then be interpreted as ‘mutant’ (i.e., mutation associated with drug resistance), ‘wild-type’ (i.e., absence of mutation associated with drug sensitivity) or mixed (presence of both mutant and wild-type alleles, i.e. a mixture of parasite strains) [105].

Sanger sequencing (capillary electrophoresis)

Sequencing can be performed on the gene or loci of interest. Sanger sequencing (chain termination sequencing) is a method of DNA sequencing based upon the selective incorporation of chain terminating dideoxynucleotides (ddNTPs) during in vitro DNA replication [106]. There are two classical methods of Sanger sequencing which utilize fluorescently labelled primer or labelled dNTPs. In most cases both strands of the targeted genomic DNA are sequenced. The sequences are then reassembled using dedicated software and compared to a sequence from reference strain (often 3D7) to look for new point mutations. The length of the DNA target which can be sequenced with high confidence is based upon the technique and the method utilized and how well the method has been optimized. In general 400–700 bases is an average read length for Sanger sequencing.

Next generation sequencing

Next generation sequencing (NGS) can be performed on the gene or loci of interest or the whole genome to look for potential new mutations or mechanisms of resistance. NGS can be used as well to track the origin and spread of resistant parasites using microsatellites data analysis [36, 37, 107]. Different samples are pooled either after or before DNA extraction and are sequenced together. A DNA library is then prepared consisting of small fragments of the DNA, not all of the same size, but on average 100 base pairs. NGS systems are able to sequence millions of small base pairs in parallel based on the “shotgun” approach in which millions of short nucleotides are sequenced in parallel for only one strand of the DNA. Deep sequencing refers to sequencing a genomic region multiple times, sometimes hundreds or even thousands of times. This NGS approach allows detection of rare clonal types compared to the classic sequencing by capillary electrophoresis [108]. The sequencing data are analysed by bioinformatics tools to reconstruct the gene of interest or the whole genome and compare it to a reference strain (usually 3D7) [109].

Real-time-PCR

Real-time PCR can be used to detect both SNPs and gene copy number. Real-time PCR is a PCR conducted on a specific piece of equipment, which allows for the real time observation of the DNA amplification using nucleic acid stain dyes (i.e. SYBR green), fluorescence-labelled amplification primers and/or probes. When the technique is used for quantitative assay to detect changes in gene copy number, fluorescent or intercalating dyes are added to the PCR mix to detect PCR product as it accumulates in real time during PCR cycles. The measured fluorescence is proportional to the total amount of amplicon; the change in fluorescence over time is used to calculate the amount of amplicon produced in each cycle. The cycle number at which the fluorescent signal emitted during the amplification first cross a threshold (Ct) corresponds the amount of target that was present in the reaction at cycle 1 of the reaction (starting concentration) [110]. In order to perform SNP genotyping, two specific probes labelled with different dyes are used, the first for the wild type allele and the second for the mutant allele. Those oligonucleotides are constructed with a fluorescent dye attached at the 5′ end and a quencher at the 3′ end are used to detect SNPs. When the specific target sequence is present, the oligonucleotide anneals and during the extension phase of PCR, the probe is cleaved. The cleavage of the probe will remove the probe from the target DNA strand and separates the reporter dye from the quencher, allowing the increase of the fluorescence signal which can be detected. This process is repeated each cycle, resulting in an increase in fluorescence intensity proportional to the amount of the PCR product. The mutant, wild-type, or mixed genotype can therefore be determined by observing the real-time increase of the fluorescence of the given fluorophore(s) [111].

Ligase detection reaction fluorescent microsphere (LDR-FM) assay

PCR products are amplified in a multiplex ligase detection reaction containing specific primers for each mutation and common primers. The specific primers are designed in a way that they contain a tag at the 5′ end complementing a sequence attached to a Luminex-Tag bead and 3′ end specific for the mutation of interest. After this second amplification, products are hybridized to Luminex-Tag beads. To quantify the abundance of different alleles, labelled products are then run on a Luminex instrument, and results are read as fluorescence intensity for each reaction in a 96-well format [112].

New molecular methods in development

In addition to the most common methods described above, new molecular methods have been developed over the last years or are being developed. The first step of these methods is the same as for the ones above, consisting of a PCR amplification. However there are different approaches for the subsequent detection of the mutations.

Nucleic acid lateral flow immunoassay (NALFIA)

For this technique, there is no DNA extraction step needed. The blood is directly added to the PCR reaction, and the target gene or part of gene is amplified for 1 h. Thereafter, the product can be visualized with NALFIA, which is a rapid immunochromatographic test to detect labelled amplicon products on a nitrocellulose stick coated with specific antibodies. The amplicons are labelled via specific primers that contain a biotin molecule and a hapten. This complex is detected by direct interaction with a colloidal, neutravidin-labelled carbon particle [113]. Like a malaria rapid diagnostic test, the NALFIA has a positive control, which is the human housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). This technique has already been used to detect molecular markers associated with anti-malarial drug resistance and showed good correlation with standard sequencing and real-time PCR methods [113].

Q-poc™

This method has been developed by a company called Quantum DX in collaboration with academic institutions [114]. The Q-POC™ is a simple handheld molecular device that could provide results in less than 20 min. The device does include a cassette to both collect the sample and perform sample preparation, DNA amplification and detection by microarray. The device can differentiate species of Plasmodium parasites, and can also detect the different molecular markers associated with anti-malarial drug resistance. The company in collaboration with St George’s, University of London, is developing a malaria specific assay for molecular markers of anti-malarial resistance under the EU funded project NanoMal [114].