Diagnostic Practices for Plasmodium vivax Malaria
Infection.
The diagnosis of Plasmodium vivax infection can be broadly categorized into three purposes: identification of clinical cases (passive case detection [PCD]), surveillance (active case detection [ACD]), and clinical trials. Each scenario brings distinct requirements, tools, and pitfalls for diagnosis of the infection.
Passive case detection.
The accurate diagnosis of vivax malaria in an acutely ill patient seeking routine care requires microscopy examination of a Giemsa-stained blood smear (microscopy), or use of an immunochromatographic cassette containing monoclonal antibodies to a P. vivax antigen (rapid diagnostic test [RDT]). Clinical signs and symptoms alone, though frequently used, can neither differentiate malaria infection from other causes of febrile illness, nor distinguish between Plasmodium falciparum and P. vivax or malaria caused by another plasmodia. Competent microscopy is typically more sensitive, specific, and informative (with respect to parasite count and stages present) than RDT. However, the sustainability of microscopy services challenges most health-care systems where endemic malaria occurs.1,2
Microscopy.
Standards for malaria microscopy training, certification, and practice are available from World Health Organization (WHO).3,4 Examination of at least 200 fields of a thick blood film under oil immersion magnification (×1,000) should be undertaken before a negative diagnosis is made. The limit of detection for expert microscopists is considered to be about 10–20 parasites/μL.5,6 Routine competent microscopy in clinical settings is considered unreliable below about 50 parasites/μL.
The density of parasitemia in patients with acute vivax malaria depends upon many factors, including naive versus a state of semi-immunity, age, delay in seeking treatment, self-treatment behavior before presentation, and likely a variety of host and parasite factors.7
The parasite density in P. vivax malaria is typically an order of magnitude lower than P. falciparum in most clinical settings where both these species occur, thus increasing the risk of false negative microscopy diagnosis with acute vivax malaria. Repeated blood film examinations, or increasing the number of microscopy fields to 300 or more in patients suspected of having malaria should be carried out before confidently reporting the patient as negative for malaria parasites.
The primary diagnostic threat in the clinical setting is poor sensitivity. Training of clinical microscopists should be aimed at maximizing parasite detection, even if that is at the cost of a lower level of specificity. In some settings where vivax malaria predominates and transmission of P. falciparum is falling, there may be particularly low sensitivity for P. falciparum.
In addition to relatively intensive training and certification for the microscopist, competent microscopy requires a clean and well-functioning light microscope, clean glass slides, immersion oil with appropriate optical properties, and fresh filtered reagents for Giemsa staining. In many settings of endemic malaria, these essentials represent substantial challenges that cannot be reliably sustained. Where the quality of microscopy services cannot be assured, the use of RDT is recommended.8
Rapid diagnostic tests.
The principle advantage of RDTs is their ease of use and sustainability in resource-challenged settings. RDTs are available from many commercial sources at relatively low cost (usually < US$1/test). Most of these kits are stable at ambient temperature storage for many months. WHO offers standards for training and certification in the use of RDT.9 The sensitivity and specificity of RDT varies greatly among commercial providers, and by species being diagnosed. In general, the kits perform better with P. falciparum infection than that with P. vivax (e.g., 74% versus 37% of test brands scored > 75% “parasite detection score” at a density of 200 parasites/μL, respectively10). However, among the several dozen tests evaluated, a dozen scored ≥ 90% on the parasite detection score for P. vivax and P. falciparum at 200 parasites/μL. These kits may be viewed as suitable for the diagnosis of acute vivax malaria by RDT (see Table 1). Some tests detect P. falciparum histidine-rich protein 2 in addition to a pan-genus antigen (lactate dehydrogenase [pLDH]). Reaction to both indicates presence of P. falciparum, either alone or mixed with any other species, whereas reaction to pLDH alone indicates absence of P. falciparum and presence of any other species, but does not distinguish among these. Other RDTs do offer a P. vivax-specific diagnosis employing aldolase antigen capture with satisfactory performance.11
Top scoring rapid diagnostic test brands for diagnosis of acute vivax malaria*
| Brand | Manufacturer | Product catalog no. |
|---|---|---|
| CareStart kits (5) | AccessBio, Inc. | G0111, GO121, GO131, G0161, GO171 |
| SD Bioline kits (4) | Standard Diagnostics, Inc. | 05FK60, 05FK66, 05FK80, 05FK100 |
| BIONOTE Malaria P.f. and P.v. Ag Rapid Test Kit | Bionote, Inc. | RG19-12 |
| Humasis Malaria P.f/P.v Antigen Test | Humasis Co. Ltd. | AMFV-7025 |
| NanoSign Malaria PF/Pan Ag 3.0 | Bioland Ltd. | RMAP-10 |
Parasite detection score > 90%, false positive rate < 10%, error rate < 10% for both Plasmodium falciparum and Plasmodium vivax at 200 parasites/μL in Round 4 of the World Health Organization evaluation of these devices.10
Active case detection.
ACD is usually conducted through mass blood surveys with the intent of detecting asymptomatic carriers of infection for the purposes of either malaria control or measurement of prevalence of parasitemia in at-risk populations. Asymptomatic carriers typically have much lower parasitemias compared with those seeking medical attention for illness, and RDTs detect only a minority of these infected individuals. The majority of mass blood surveys (historically and today) are performed using microscopy. The growing realization of the importance of detecting low-level parasitemias has spurred development of alternative technologies such as loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR).12–15
Microscopy.
The process of microscopy for the purpose of ACD is similar to that described for PCD. A microscopist has some flexibility in the clinical setting regarding the degree of effort committed to an examination (e.g., > 200 fields and multiple smears examined for a single patient). However, in ACD, only one blood film is usually collected, and thus care must be taken to ensure that the same degree of diligence is applied for all samples collected.
Polymerase chain reaction.
The most widely applied and validated molecular diagnostic is a nested PCR amplification of small subunit ribosomal RNA gene sequences.16 The term nested refers to an initial amplification using primers of genus-wide sequences followed by primers of species-specific character to provide the definitive diagnosis. This technique, and other PCR-based approaches to diagnosis, requires relatively advanced laboratory equipment and technologically skilled execution. Typically, blood blots dried onto filter paper in the field are transported to a laboratory where extraction of DNA and its analysis by PCR is performed. It is also relatively expensive. The advantage of PCR is increased sensitivity to detect parasitemias up to three orders of magnitude lower (depending on blood volume sampled) compared with microscopy or RDT. Its absolute sensitivity in operational use is approximately 1.0 parasite/μL, increasing to 0.02 parasites/μL if > 0.25 mL of venous blood can be collected.
Loop-mediated isothermal amplification.
LAMP of parasite-specific DNA is a more recent technology more suited to ACD in endemic settings. The technique does not require expensive thermocyclers or gel electrophoresis, the readout being a visual color change in a small test tube. Recent evaluation of a commercially available kit (with P. falciparum and pan-genus-specific reagents) showed the technique to be comparable to standard nested PCR technique and superior to expert microscopy.17,18 The procedure can be completed in about 1 hour at the site of collection. A modified LAMP procedure, as well as standard nested PCR, was performed in the diagnosis of P. vivax in large field surveys in China.19
Serology.
Diagnostic techniques employing serological markers have been applied mostly to studies of protective immunity and epidemiology. Most published studies have applied techniques using antigens derived from synthetic peptide vaccine candidates rather than those that may be more informative of active or latent infection. Studies of the vaccine antigen–derived serological assays generally show very poor specificity with regard to diagnosis of active infection. Further work aimed at antibodies specific to acute infection, rather than prolonged protection against such infection, is required and should have the potential to identify populations at greatest risk of parasitemia.
Glucose-6-phosphate dehydrogenase deficiency.
The diagnosis of glucose-6-phosphate dehydrogenase deficiency (G6PDd) is an important diagnostic procedure before initiating the radical cure of P. vivax, which currently requires administration of primaquine (PQ). PQ can cause serious hemolysis in patients with G6PDd, an inherited X-linked highly diverse disorder affecting about 400 million people. G6PDd occurs at a prevalence varying from < 1% to 30% among residents of endemic zones.20 There are many distinct variants of G6PD enzyme each with different degrees of compromise of its key metabolic function—the provision of reducing equivalents via nicotinamide adenine dinucleotide phosphate (NADPH) and reduced glutathione in sustaining redox equilibrium of the cytosol and protecting cell constituents from oxidative damage.21 In most genotypes, the mutant enzyme degrades more rapidly, rendering the older red cells the most deficient. The residual enzyme activity defines a clinically relevant and measurable phenotype for many variants.
Because the G6PDd is X-linked, it is either wholly absent or present (hemizygous) in males. In females, in contrast, it may be absent, homozygous, or heterozygous. Homozygosity in females is relatively rare (the square of the allele frequency), but heterozygosity is common. This is an important clinical and diagnostic problem due to the phenomenon of lyonization of X-linked genetic traits in women. This process results in two distinct populations of red blood cells, that is, those expressing defective or normal G6PD. The relative proportion of cells expressing abnormal enzyme averages 50% but ranges between 0% and 100%. In other words, heterozygous females may be G6PD normal or fully G6PD deficient; however, most have red blood cell populations presenting a mosaic of the two phenotypes. The clinical significance of this in the context of PQ toxicity is unclear, but challenges the widespread deployment of PQ to vulnerable populations.
Clinical diagnosis.
Ascertainment of G6PDd status in a clinical setting can involve the quantitative or qualitative measurement of G6PD activity in hemolysate, cytochemical microscopic examination of whole cells, or inference from genotyping extracted DNA using PCR technology.
Quantitative diagnosis.
Standardized commercially available kits may be used for the spectrophotometric determination of G6PD activity in enzyme units (U) per gram of hemoglobin (gHb). Normal values range approximately from 7 to 10 U/gHb (at 30°C).22 The genetic heterogeneity of phenotypically “normal” G6PD enzyme and varying environmental factors may account for the wide range of activity values.23 Patients presenting values less than 7 U/gHb are classified as G6PD deficient. G6PD activity is usually defined as a percentage of normal activity, as this provides an intuitive measure of likely vulnerability to hemolytic anemia. The “normal” denominator of the estimation is usually defined by the mean G6PD activity of most patients evaluated in any given patient population. Table 2 lists validated quantitative assays and their commercial providers.
G6PDd validated quantitative assays and the commercial providers
| Kit name | Manufacturer | Venipuncture | Test readout | Chemical basis | Cold storage | Laboratory equipment/skills | Cost/test |
|---|---|---|---|---|---|---|---|
| G6PD deficiency | Trinity Biotech, Ireland | Yes | Quantitative | Spectrophotometric | Yes | Yes | |
| G-Six Kinetic | Tulip Group, India | Yes | Quantitative | Spectrophotometric | Yes | Yes | $1.10 |
| R&D G6PD quantitative | N. Dimopoulos S.A., Greece | Optional | Quantitative | Spectrophotometric | Yes | Yes | $0.26–2.50* |
| R&D G6PD qualitative | N. Dimopoulos S.A., Greece | Optional | Qualitative | Ultra-violet fluorescence | Yes | Yes | $0.18 |
| G6PD-WST | Dojindo, Japan | Optional | Qualitative | Dye reduction | Yes | Yes | $2.00 |
| G-SIX | Tulip Group, India | Yes | Qualitative | Met-Hb reduction | No | Yes | $1.12 |
| G6PD deficiency | Trinity Biotech, Ireland | Yes | Qualitative | Ultra-violet fluorescence | Yes | Yes | |
| G6PD deficiency | Trinity Biotech, Ireland | Yes | Qualitative | Dye reduction | Yes | Yes | |
| MBK | Span, India | Yes | Qualitative | Dye reduction | Yes | Yes | |
| BinaxNOW G6PD | Alere/Inverness Medical, United States | Yes | Qualitative | Dye reduction | No | No | $16.00 |
| CareStart G6PD | AccessBio, United States | No | Qualitative | Dye reduction | No | No | $1.50 |
Depends upon numbers of assays run simultaneously, numbers of kits purchased, suppliers of the kits, or use of reagents in lieu of kits.
In the quantitative assay, as in the qualitative assay, diagnosis of G6PDd in patients having recently suffered acute hemolytic anemia is problematic, since some patients may exhibit a normal phenotype as a consequence of the most vulnerable red blood cells having been removed and their replacement by young erythrocytes that have inherently higher G6PD activity levels. The effects of acute malaria on G6PD tests have not been evaluated.
Most G6PD tests require a separate Hb measurement, and this imposes additional complexity and costs. The reason of doing so, however, is the impact of Hb level on the qualitative measurement, for example, below about 8 g/dL Hb, G6PD activity measurements trend sharply higher, probably falsely so.24 G6PD activity measurements from anemic patients should not be considered reliable.
Qualitative.
Standardized commercially available kits allow a visual determination of G6PD phenotype. These all involve the enzymatic conversion of NADP+ to NADPH by G6PD, which may be visualized directly (using fluorescent lighting) or indirectly (using one of several azole dyes that change color in the presence of NADPH). The most commonly used and widely validated qualitative assays are listed in Table 2. An alternative option is the methemoglobin reduction assay.25,26
Cytology.
Cytological techniques can be used to measure the degree of X-inactivation by lyonization in heterozygous females. These methods differentially stain individual red blood cells and permit estimation of the proportion of cells expressing defective G6PD enzyme. A variety of techniques have been described,27 including a recent flow cytometry method of particular promise for clinical and research settings.28
Genetic.
The principal advantage of G6PD genotyping is a diagnosis un-confounded by the physiological variables affecting G6PD activity such as patients suffering acute hemolytic anemia or lyonization in heterozygous females. The primary disadvantage, however, is the uncertainty of a “normal” diagnosis when applying primers to selected known genotypes using standard PCR/restriction fragment length polymorphism (RFLP) techniques. The patient may be deficient but with a genotype not represented in the genetic analysis—a significant hazard with G6PD, which is a complex gene with over 200 known variants documented. New variants of G6PD are routinely found wherever sufficiently detailed investigations can be carried out.29,30 Although whole gene sequencing is certainly possible, it is currently impractical in a routine clinical sense due to its very large size (18.5 kb), complex structure (13 exons), and the occurrence of mutations all along its length. The most commonly applied methodology is PCR/RFLP and requires specific selection of a number of mutation-specific primers limited by practicality.
Point-of-care and survey diagnosis.
Most patients with vivax malaria receive care at home or in the community outside of a clinic. Even when patients attend a clinic or hospital with a laboratory facility, there is often no capacity for G6PDd diagnosis. Similarly, diagnosis of G6PDd for survey purposes also occurs in rural settings and often cannot accommodate relatively sophisticated laboratory techniques.
Point-of-care diagnosis.
There is currently no validated point-of-care (POC) diagnostic device for G6PDd that is practical to apply where most malaria patients live. The obstacles to such a test include cost, complexity, heat sensitivity, and the need for a cold chain to transport and store the kit. A commercial kit in development, however, shows promise in overcoming those issues: the CareStart G6PD™ (AccessBio, Somerset, NJ), which is affordable, easy to use, and is not sensitive to ambient temperature fluctuations. However, although it is capable of reliably detecting G6PD deficiency below 30% of normal activity,31,32 it is currently insensitive to milder deficiencies and most female heterozygotes, a limitation of other qualitative tests. Although promising, CareStart G6PD™ requires more thorough assessment before it may be recommended for routine diagnosis of patients before PQ therapy.33
G6PDd survey.
Surveys to assess the prevalence of G6PDd are often undertaken where laboratory capacity is limited, the test readout being subjective and visually categorized into G6PD deficient, intermediate, or normal. A newer quantitative technique—WST8/1-methoxy-PMS—has been successfully used to survey populations with a quantitative enzyme activity readout. Dried blood blots collected in the field and kept refrigerated are returned to the laboratory for G6PD activity determination in a 96-well spectrophotometric assay format.34,35 The results correlate well with other standard techniques.
Clinical trials.
Evaluation of the safety and efficacy of therapies for acute vivax malaria, which necessarily includes both blood schizontocidal and hypnozoitocidal therapies, employs diagnostics for both the infection and G6PDd. Clinical trials of experimental therapies often demand higher standards of diagnosis than may be applied in routine clinical care, to provide greater assurance of subject safety and the precision of efficacy estimates.
Diagnosis of P. vivax and G6PDd for blood schizontocidal efficacy.
Trials of blood schizontocidal therapies typically only enroll patients with parasitemias above a certain threshold, to increase the likelihood that the associated fever is due to the infection. When parasitemias exceed 500/μL, almost any diagnostic technique will suffice. However, microscopy remains the method of choice for staging the parasite and documenting the parasite clearance.
Screening for G6PDd is required for PQ therapy in trials and experimental blood schizontocide, which do not have a demonstrated safety profile in G6PD-deficient patients.
Diagnosis of P. vivax and G6PDd for hypnozoitocidal efficacy.
The methodology of anti-relapse efficacy trials is challenging, but broadening enrollment criteria may be warranted to include those with subpatent infections. In this context, the use of highly sensitive diagnostic techniques such as LAMP may help to discriminate patient risk groups at enrollment. It is important to identify patients with G6PD variants known to be at high risk of severe hemolysis. Historically, the NADPH spot test (which identifies patients with < 30–40% of normal activity) has been used for this purpose, including a series of trials of PQ as primary prophylaxis in the past decade where subjects received large cumulative doses of this drug over prolonged periods.36 Although valid concerns may be raised regarding insensitivity to milder variants and female heterozygotes, there are no documented cases of acute severe hemolytic anemia following PQ therapy and a classification as normal by the NADPH spot test. At least in the specific instance of PQ, this record of safe use with NADPH spot test screening argues in favor of good safety of this technique or those of similar diagnostic performance. Nonetheless, direct evidence of safety in this practice is lacking.
Diagnosis of recurrent parasitemia.
Clinical efficacy is defined by the clearance and recurrence of parasitemia. In most patients these occur at relatively low levels of patency, and thus expert microscopy is crucial in the assessment of blood films. In contrast to routine clinical microscopy, the microscopist serving clinical trials must undergo rigorous certification to minimize false-positive outcomes, which have potential to significantly underestimate clinical efficacy.37 False negative (intolerable in clinical settings) is less important in the detection of recurrent parasitemia in asymptomatic individuals being routinely followed up. Confirmation of the microscopic diagnosis by PCR techniques is often performed weeks or months later. Though microscopy lacks sensitivity relative to PCR techniques, it is far more unambiguous, whereas false-positive PCR by contamination is a well-known pitfall.
Treatment Practices for P. vivax Malaria
The goals of antimalarial treatment in P. vivax are to reduce the immediate risk to the host, eradicate peripheral asexual parasitemia, prevent the recurrent infection, and interrupt the cycle of transmission.38 The ability of P. vivax to form dormant liver stages (hypnozoites) capable of causing relapsing infections weeks to months after the initial blood-stage infection, provides a major challenge to the complete eradication of parasites from the body. Since no single drug achieves all of these aims, a combination of antimalarials is required targeting a variety of specific key elements of the parasite life cycle.1
Treatment of asexual erythrocytic stages of P. vivax.
Treatment of uncomplicated vivax malaria.
In areas where P. vivax is known to be chloroquine (CQ) sensitive, the WHO recommends 3 days of CQ or an artemisinin combination treatment plus 2 weeks of PQ (provided the affected individual is not G6PD deficient).39 CQ remains a first-line treatment in most parts of the world due to its wide availability, low cost, and long terminal elimination half-life. However, in co-endemic malarious areas, this necessitates a separate treatment approach for P. falciparum and P. vivax.
Most commonly used antimalarial drugs are also active against the asexual stages of P. vivax, the exception being the antifolates, which act slowly,40 and are vulnerable to the rapid development of drug resistance.41,42 Mefloquine,43 atovaquone + proguanil,44 halofantrine,45 piperaquine,46 artesunate,47,48 and pyronaridine,49 all show good efficacy against chloroquine-resistant (CQR) P. vivax in clinical trials.
Artemisinin combination therapies (ACTs) are the treatment of choice for CQR P. vivax.46 WHO-recommended ACTs include artemether–lumefantrine, artesunate–amodiaquine, artesunate–mefloquine, and dihydroartemisinin (DHA)–piperaquine. A fifth ACT, pyronaridine–artesunate, has recently obtained a positive opinion from the European Medicines Agency for the treatment of P. vivax malaria, but it is not yet recommended by the WHO. Artemisinin in combination with effective partner drug have shown excellent cure rates in P. vivax infection.50 ACTs with partner drugs with longer elimination periods provide incidental suppressive prophylaxis against relapse for about a month, but relapse risk thereafter remains relatively high.
The deployment of an ACT-based strategy permits a unified policy for treating both P. falciparum and P. vivax infections, offering a pragmatic approach with operational efficiencies.48 A unified policy also decreases frequent issues of species misdiagnosis in routine practice. The rise and spread of CQR P. vivax has led to a number of countries adopting ACTs as first-line treatment for P. vivax. These include DHA–piperaquine in Indonesia and Cambodia, and artemether-lumefantrine in Papua New Guinea, Solomon Islands, Sudan, Namibia, South Africa, and Vanuatu.51 Unified treatment policy also infers anti-relapse therapy for P. falciparum, where P. vivax is sympatric (see Drug Development for P. vivax section).
Treatment of severe vivax malaria.
Severe and fatal vivax malaria has been reported from Indonesia,52,53 Papua New Guinea,54 India,55 and Brazil.56,57 The main manifestations are anemia and respiratory distress,53,56–62 although series of patients with coma, shock, and renal and hepatic dysfunction associated with vivax malaria have also been described.55–57,61,62 Plasmodium vivax is very sensitive to artemisinin and its derivatives. In the absence of comparative drug trials, physicians have tended to adopt a similar treatment approach for severe vivax malaria as for severe falciparum malaria,1 namely administration of parenteral artesunate, if unavailable, artemether, and if that is also not available then quinine, along with broad-spectrum antibiotic cover and supportive care.
Large-scale multicentered trials in patients in Asia and Africa have demonstrated clear superiority of intravenous artesunate over quinine in reducing case fatality rate in severe falciparum malaria.63,64 Intravenous artesunate also leads to a rapid clinical response in patients with severe vivax malaria,53,61 but there have been no randomized clinical trials in severe P. vivax malaria.
Specific antimalarial treatment recommended in severe vivax malaria includes the following in order of preference:
-
Artesunate: 2.4 mg/kg body weight, intravenously or intramuscularly given on admission (time = 0), then at 12 and 24 hours, and then once a day. This is the treatment of choice.
-
Artemether: 3.2 mg/kg body weight, intramuscularly given on admission, then 1.6 mg/kg body weight per day.
-
Quinine: 20 mg quinine salt/kg body weight on admission (intravenous infusion in 5% dextrose/dextrose saline over a period of 4 hours) followed by maintenance dose of 10 mg/kg body weight 8 hourly (maximum infusion rate 5 mg salt/kg/hour).
Parenteral antimalarials should be administered for at least 24 hours. Once the patient can accept oral therapy, full course of oral ACT should be given to the patients. Full details are available in the latest Malaria Treatment Guidelines.1
CQR P. vivax.
The first reports of CQR P. vivax were published in 1989 from Australian travelers to Papua New Guinea,65 and in 1991 as an endemic problem in Indonesia,66 30 years after the documentation of CQR P. falciparum. Although intrinsic differences in the transmission dynamics between these two species may account for much of the time lag, it is likely that this also reflects the inherent complexity of defining antimalarial treatment efficacy of P. vivax.67 High-grade CQR P. vivax has been documented on the island of New Guinea, where patients treated with CQ have been observed to have early clinical deterioration requiring hospitalization, delayed parasite clearance, and early recurrent parasitaemia.46,68,69 Evidence for declining CQ efficacy against P. vivax, albeit to a lesser degree, has been reported from across the vivax-endemic world (Figures 1–3).70–72
Clinical reports of chloroquine-resistant Plasmodium vivax. Red icons highlight areas of chloroquine-resistant parasites defined by greater than 10% recurrence (and lower 95% confidence interval [CI] > 5%) by day 28 with or without measurement of chloroquine drug concentration; dark orange diamonds locate area suggestive of resistance as defined by 5–10% recurrence by day 28, confirmed with adequate chloroquine drug concentrations; light orange icons locate sites of possible resistance as defined by less than 5–10% recurrence by day 28 but with lower 95% CI < 5% by day 28, without drug concentrations. Yellow icons represent case reports. Details of clinical trials are provided in Supplemental Annex A.
Citation: The American Society of Tropical Medicine and Hygiene 95, 6_Suppl; 10.4269/ajtmh.16-0171
Clinical reports of chloroquine-sensitive (CQS) Plasmodium vivax. CQS was defined as < 5% recurrence by day 28, no early administration of primaquine, and all patients from symptomatic clinical presentation. Yellow icons represent studies before 2007, orange icons studies between 2007 and 2012. Details of clinical trials are provided in Supplemental Annex A.
Citation: The American Society of Tropical Medicine and Hygiene 95, 6_Suppl; 10.4269/ajtmh.16-0171
Risk of recurrence at the end of the study following very low-dose primaquine (PQ) (total dose ≤ 2.5 mg/kg), low-dose PQ (total dose > 2.5 mg/kg to < 5.0 mg/kg), high-dose PQ (total dose > 5.0 mg/kg). Indonesia and Papua New Guinea [closed circles]; Thailand and Vietnam (open circles); South and Central America (open squares); Indian subcontinent, Middle East, and Horn of Africa (open diamonds); and Korea and China (closed diamonds). The U.S. studies of induced malaria and returning soldiers are categorized according to origin of infecting strain.
Citation: The American Society of Tropical Medicine and Hygiene 95, 6_Suppl; 10.4269/ajtmh.16-0171
In vivo efficacy.
The WHO's protocols for the evaluation of antimalarial efficacy focus primarily on the treatment of P. falciparum. These guidelines have undergone extensive revision over the last 20 years, the more recent versions extending their scope to investigate the therapeutic efficacy against P. vivax infections. Current guidelines for assessing CQ recommend supervised treatment and follow-up for a minimum of 28 days, accompanied by measuring whole blood CQ and desethylchloroquine level at the day of failure. Recurrent infections during this period presenting with whole blood CQ plus desethylchloroquine concentration exceeding 100 ng/mL are considered as resistant irrespective of whether they are relapse, recrudescence, or reinfection.
A major confounding factor in interpreting clinical drug efficacy against P. vivax is an inability to distinguish reliably between relapse, recrudescence, or reinfection. A variety of methodologies have been developed for P. vivax with as few as three polymorphic markers proving to be sufficient to discriminate homologous from heterologous infections.73–76 However, recurrence of P. vivax genetically identical to the pretreatment isolate can occur from either a true recrudescence of the initial infection or a relapse from hypnozoites generated from the prior blood-stage infection74,77; unsurprisingly molecular methods are unable to distinguish between these alternatives. Relapses are also commonly genetically heterologous. The confounding effect of relapsing infections varies considerably between geographical locations, both for the absolute risk of relapse and the timing at which these occur. In equatorial regions, 50–80% of patients can have a relapse starting within 3 weeks of the initial infection (if a rapidly eliminated drug is used for treatment), whereas in patients infected by temperate strains, the risk of relapse may fall to 5–20%, recurrences occurring many months after the initial infection.78 Major impediments to defining CQ resistance and common causes of misdiagnosis of CQ resistance and susceptibility come from multiple aspects (Table 3).
Common sources for misdiagnosis of CQR and CQS Plasmodium vivax
| Explanation | Recommendation | |
|---|---|---|
| Incorrect diagnosis of CQS | ||
| Enrollment of patients without clinical disease | Host immunity in asymptomatic patients enrolled from cross-sectional surveys, may facilitate clearance of parasitaemia even following partially effective drug treatment | Restrict efficacy trials to patients presenting with clinical disease |
| Coadministration of early PQ | Early PQ has schizontocidal activity that can increase parasite clearance and prevent recrudescent infections | Primaquine treatment should be delayed until the end of the follow-up period |
| Short duration of follow-up | Early evidence of resistance is manifest by late recrudescence | Patients should be followed up for a minimum of 28 days |
| Incorrect diagnosis of CQR | ||
| Incomplete treatment course | From poor patient adherence | Supervision of drug treatment |
| Dose of chloroquine administered too low | Prescription of inadequate mg/kg dose | Documentation of exact dose of drug administered |
| Poor absorption of drug | Either from poor quality drug, reduced gastrointestinal absorption | Measurement of drug blood concentrations on day 7 and the day of parasite recurrence |
| Poor drug quality | Faulty product | Confirmation of adequate drug levels, pharmacologic evaluation of study drugs, and purchase only from certified, trusted producers |
| Inadequate sample size | Leading to wide confidence intervals of high failure rates derived from very few cases | Recruitment of an adequate sample (> 70 patients) |
CQR = chloroquine resistant; CQS = chloroquine sensitive; PQ = primaquine.
A recent review of the literature identified 135 prospective clinical trials of antimalarial efficacy against the erythrocytic stages of P. vivax monoinfection published between 1980 and 2013, of which full manuscripts were available for 124 studies. There have also been 28 case reports of CQ resistance. A complete list of these extracted data is available in Supplemental Annex A. In total there have been 121 treatment arms documenting the efficacy of CQ (enrolling 13,878 patients) and 21 treatment arms assessing the clinical efficacy of ACTs (artemether–lumefantrine, eight; DHA–piperaquine, seven; sulfadoxine/pyrimethamine, three; and one each for amodiaquine–artesunate, pyronaridine–artesunate, and artemisinin–naphthoquine). CQ efficacy has been quantified at 97 geographical locations, of which 47 (48%) revealed reduced potential susceptibility (Figure 1 and Supplemental Annex A). Numerous studies demonstrate the great therapeutic benefit of primaquine therapy at low (Figure 4) or high (Figure 5) dosing when combined with chloroquine versus chloroquine alone, i.e., odds of recurrence of 0.14 (0.06–0.35) or 0.03 (0.01–0.13), respectively.
Forest plot of the effectiveness of low-dose primaquine in studies with a control arm. * Indian subcontinent, ^ United States (Korea), ^^ United States (Chesson), # Thailand, ## Indonesia, + Ethiopia.
Citation: The American Society of Tropical Medicine and Hygiene 95, 6_Suppl; 10.4269/ajtmh.16-0171
Forest plot of the effectiveness of high-dose primaquine in studies with a control arm. * Indian subcontinent, ## Indonesia.
Citation: The American Society of Tropical Medicine and Hygiene 95, 6_Suppl; 10.4269/ajtmh.16-0171
Ex vivo and molecular assays.
The in vitro assessment of parasite drug susceptibility has proven to be very useful in the investigation and mapping of drug-resistant P. falciparum; however, the development of similar tests in P. vivax is more challenging. Unlike P. falciparum, the parasite preferentially invades young red blood cells, limiting its reproductive capacity and ability to adapt in continuous in vitro culture.79,80 Without culture adaptation, the ex vivo assessment of drug susceptibility in P. vivax field isolates has been limited to clinical samples derived directly from the human host and subjected to short-term culture and drug exposure as exemplified by the schizont maturation test.79–82 The inability to sustain in vitro growth restricts analysis of field isolates to a single time point making an assessment of reproducibility difficult. Despite its limitations, the current schizont maturation assay has demonstrated utility in discriminating parasite populations with different degrees of CQR,83–85 characterizing drug susceptibility profiles of P. vivax to commonly used antimalarial drugs,86,87 and screening susceptibility to novel therapeutic agents.88–93 Development of methods capable of sustaining P. vivax in continuous in vitro culture will transform the current ex vivo assay, accommodating cryopreservation of field isolates to reduce the reliance on the analysis of fresh isolates.
The identification of a molecular marker of CQ resistance in P. vivax remains elusive. Early studies failed to show a strong correlation between pvcrt-o and the CQR phenotype,84,94,95 although more recently interest has focused on the transcription level of pvcrt-o and its overexpression.96 A sequence polymorphism in pvmdr1 conferring Y976F has been reported in a number of studies and may correlate with CQ resistance84,97; however, since CQ resistance can occur in isolates with wild type pvmdr1, pvmdr1 mutations are likely to be at best minor determinants of CQ susceptibility.84,98,99
Treatment of liver stages of P. vivax.
Determinants of efficacy.
For over 60 years, clinicians, policy makers, and patients have relied on PQ, an 8-aminoquinoline, for the radical cure of P. vivax. Primaquine is the only licensed antimalarial with proven hypnozoitocidal activity, but can result in significant hemolysis particularly in those with G6PDd.100,101 In view of the risk of adverse reactions, PQ dosing strategies are influenced more by concerns over toxicity than by their absolute efficacy. These concerns are particularly important in poorly resourced settings where routine G6PDd testing is often unavailable.
The predominant determinant of therapeutic efficacy appears to be the total dose of PQ administered rather than the daily dosage or duration of therapy.102 In an attempt to reduce potential toxicity, the WHO guidelines for the radical cure of vivax malaria currently recommends the use of a daily dose of 0.25 mg/kg/day (3.5 mg/kg total dose) PQ taken with food once daily for 14 days, coadministered with CQ or ACT depending on CQ sensitivity in the region.103 In southeast Asia and Oceania, the same guidelines recommend a higher daily dose of 0.5 mg/kg (7.0 mg/kg total dose) in view of the high risk of relapses.
A recent review of the published literature identified 87 clinical trials presenting data on 59,735 patients enrolled in 156 treatment arms104; the extracted data from these studies are presented in Supplem