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Real-Time Polymerase Chain Reaction Assay for the Rapid Detection and Characterization of Chloroquine-Resistant Plasmodium falciparum Malaria in Returned Travelers

  1. Gabriella A. Farcas1,2,3,
  2. Rainer Soeller4,
  3. Kathleen Zhong1,
  4. Alireza Zahirieh1, and
  5. Kevin C. Kain1,2,3
  1. 1Tropical Disease Unit, University Health Network—Toronto General Hospital, Toronto, Canada
  2. 2Institute of Medical Science, Faculty of Medicine, Toronto, Canada
  3. 3McLaughlin-Rotman Centre, McLaughlin Center for Molecular Medicine, University of Toronto, Toronto, Canada
  4. 4Artus GmbH, Hamburg, Germany
  1. Reprints or correspondence: Dr. Kevin Kain, Tropical Disease Unit, Eaton South 9-412, Toronto General Hospital, 200 Elizabeth St., Toronto, Ontario M5G 2C4, Canada (kevin.kain{at}uhn.on.ca).
 
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Abstract

Background. Imported drug-resistant malaria is a growing problem in industrialized countries. Rapid and accurate diagnosis is essential to prevent malaria-associated mortality in returned travelers. However, outside of a limited number of specialized centers, the microscopic diagnosis of malaria is slow, unreliable, and provides little information about drug resistance. Molecular diagnostics have the potential to overcome these limitations.

Objective. We developed and evaluated a rapid, real-time polymerase chain reaction (PCR) assay to detect Plasmodium falciparum malaria and chloroquine (CQ)—resistance determinants in returned travelers who are febrile.

Methods. A real-time PCR assay based on detection of the K76T mutation in PfCRT (K76T) of P. falciparum was developed on a LightCycler platform (Roche). The performance characteristics of the real-time assay were compared with those of the nested PCR—restriction fragment-length polymorphism (RFLP) and the sequence analyses of samples obtained from 200 febrile returned travelers, who included 125 infected with P. falciparum (48 of whom were infected CQ-susceptible [K76] and 77 of whom were CQ-resistant [T76] P. falciparum), 22 infected with Plasmodium vivax, 10 infected with Plasmodium ovale, 3 infected with Plasmodium malariae malaria, and 40 infected with other febrile syndromes. All patient samples were coded, and all analyses were performed blindly.

Results. The real-time PCR assay detected multiple pfcrt haplotypes associated with CQ resistance in geographically diverse malaria isolates acquired by travelers. Compared with nested-PCR RFLP (the reference standard), the real-time assay was 100% sensitive and 96.2% specific for detection of the P. falciparum K76T mutation.

Conclusion. This assay is rapid, sensitive, and specific for the detection and characterization of CQ-resistant P. falciparum malaria in returned travelers. This assay is automated, standardized, and suitable for routine use in clinical diagnostic laboratories.

Rapid and accurate diagnosis is essential for effective treatment of malaria. Delays in diagnosis have been associated with progression to severe and fatal malaria in areas where the disease is endemic and nonendemic [1–3]. The century-old technique of microscopic examination of stained blood smears is still considered to be the gold standard for malaria diagnosis. However, even in expert hands, microscopy has been shown to have considerable limitations, with well-designed studies reporting rates of false-negative results of >10%, rates of false-positive results of >20%, and a diagnostic specificity of ∼70% [4, 5]. Microscopic inaccuracies can result in the failure to use antimalarial drugs when they are needed or the inappropriate use of such drugs in patients with alternative diagnoses, resulting in the mistreatment of other potentially life-threatening infections [3–6]. The need for rapid and accurate diagnostic tools will become increasingly important as more-expensive antimalarial combination therapies are introduced in response to increasing drug resistance [3].

PCR-based methods have advantages over traditional microscopic methods for the diagnosis of malaria [4, 7]. However, many current PCR-based assays are labor-intensive, their results are not readily quantifiable, and they have the potential for contamination during postamplification handling of samples. Real-time PCR technology has the potential to overcome these limitations. Furthermore, this platform can also detect microbial-resistance determinants and monitor emerging drug resistance in returning travelers [8, 9].

For almost one-half of a century, chloroquine (CQ) was the drug of choice for the prevention and treatment of Plasmodium falciparum malaria because of its efficacy, affordability, ease of administration, and low toxicity [10–12]. The subsequent emergence and spread of CQ-resistant P. falciparum malaria has contributed to a resurgence of malaria, particularly in sub-Saharan Africa [10–12]. However, despite widespread resistance, CQ remains widely used in some regions of Africa because of a lack of an affordable alternative and because CQ seems to retain its effectiveness in individuals who have some degree of premunition to malaria [10–12].

Of interest is that a slow but steady return of CQ-susceptible P. falciparum malaria has been documented in areas where use of CQ was previously abandoned because of resistance [13–16]. Furthermore, studies of imported malaria cases among European and North American travelers to Africa indicate that a large proportion of P. falciparum isolates (40%–54%) are still responsive to CQ [8, 17, 18]. These data suggest that CQ would still be efficacious in the treatment of uncomplicated malaria, especially in imported cases, provided that there was a rapid and accurate assay to reliably detect CQ susceptibility.

CQ resistance, both in vivo and in vitro, has been linked to a series of point mutations in the P. falciparum CQ-resistance transporter gene (pfcrt). More specifically, a mutation in pfcrt encoding an amino acid substitution from Lys to Thr at position 76 (K76T) has been shown to be the key molecular marker for CQ resistance [19–21]. The absence of this mutation is highly predictive of CQ sensitivity in vitro and of chloroquine responsiveness in vivo [12, 14, 22]. Consequently, a rapid assay to detect the presence or absence of the K76T mutation may be useful, because the presence of the K76 allele would indicate effectiveness of treatment with CQ.

We developed and evaluated a real-time PCR assay for the diagnosis of CQ-susceptible and CQ-resistant P. falciparum malaria. We demonstrate that this assay is rapid, sensitive, and specific for the detection of P. falciparum malaria and the key K76T mutation in pfcrt associated with CQ resistance in febrile returned travelers.

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Materials And Methods

Patients and blood samples. Consecutive patients who presented or were referred to the Tropical Disease Unit of the University Health Network—Toronto General Hospital from July 1999 to March 2003 with fever (temperature, ⩾38°C) or a history of fever (within 48 h) and travel to a malaria-endemic area were eligible for inclusion in the study. All patients with P. falciparum infection (as detected by both microscopy and PCR) were enrolled. In addition, during the first 3 months of the study, patients who repeatedly had negative blood film results (i.e., patients who had a diagnosis of a febrile illness other than malaria) were enrolled to provide a comparable negative control group. Consecutive patients with Plasmodium vivax, Plasmodium malariae, or Plasmodium ovale infection (detected by both microscopy and PCR) were also included to assess the specificity of the assay for the detection of the K76T mutation associated with CQ resistance in P. falciparum. The prevalence of P. falciparum malaria in returned travellers during the study period was 10%.

Microscopy, nested PCR, and real-time PCR amplification and species identification were performed independently and in a blinded fashion. This study was reviewed and approved by the Ethical Review Committee of the University Health Network—Toronto General Hospital.

PCR for malaria species identification and detection of K76T. PCR detection and malaria species identification were performed as described elsewhere [23–25]. Genomic DNA was extracted from 200-µL whole blood samples using Qiagen columns (Qiagen) and eluted in 200 µL of water, in accordance with the manufacturer's instructions. A 5-µL aliquot of the DNA extract was used in a nested PCR assay to amplify segments of the Plasmodium 18S rRNA gene characteristic for each of the 4 human malarial species [24]. The resulting PCR product was analyzed on a 2% agarose gel stained with ethidium bromide. Another 5-µL aliquot of DNA (from the same extraction) was also used in a nested PCR reaction using primers TCRP1 (5′-CCG TTA ATA ATA AAT ACA CGC AG-3′) and TCRP2 (5′-CGG ATG TTA CAA AAC TAT AGT TAC C-3′) for the primary PCR and TCRD1 (5′-TGT GCT CAT GTG TTT AAA CTT-3′) and TCRD2 (5′-CAA AAC TAT AGT TAC CAA TTT TG-3′) for the nested PCR to amplify a region of the P. falciparum pfcrt gene spanning codon 76. After amplification, alleles carrying the K76 or T76 codon were discriminated by ApoI restriction, as described elsewhere [26]. The digest mixtures were analyzed on a 2% polyacrylamide gel stained with ethidium bromide. The results were verified further by direct sequencing of the PCR amplicon.

Real-time PCR. DNA aliquots (5 µL) from the same extraction used in the nested PCR amplifications were used in the real-time PCR amplification of a genomic region spanning codon 76. A proprietary set of primers (Artus GmBH) has been developed to recognize and amplify the region around codon 76 of the pfcrt gene of P. falciparum. Probes were also designed to bind to both mutant (76T) and wild-type (K76) alles at codon 76 and to be differentiated on the basis of differing melting temperatures (figure 1). A heterologous internal control was included in the assay to monitor both DNA extraction quality and potential PCR inhibition during the real-time PCR run. Furthermore, each run included a known positive control for each of 3 PfCRT haplotypes (CVMNK, SVMNT, and CVIET). The assay was designed using fluorescence resonance energy transfer technology on a LightCycler platform (Roche).

Figure 1
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Figure 1

A, Example of a typical real-time PCR run showing the melting curve results of 20 samples. The line indicates an example of a sample containing both CQ-susceptible and CQ-resistant parasite populations. B, Sequence alignment of selected samples showing the amino acid haplotype differences relative to the position of codon 76 (box) and of the probe. The Tm values indicate the melting temperature ranges of the probe in each haplotype.

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Results

During the study period, 200 individuals who presented with fever after travel to a malaria-endemic area were enrolled. Before treatment, whole blood samples obtained from these individuals were coded, processed, and analyzed in a blinded fashion by nested PCR for species identification; nested PCR followed by restriction fragment—length polymorphism (RFLP) and sequencing for pfcrt mutations were performed; and real-time PCR for pfcrt mutations was performed. Travelers from whom malaria isolates were obtained included 125 infected with P. falciparum (48 wild-type [K76] and 77 mutant [T76] at position 76, as defined by nested PCR followed by RFLP and sequencing as the reference standard). To assess the specificity of the assay, samples obtained from 22 travelers infected with P. vivax, 10 travelers infected with P. ovale, and 3 travelers infected with P. malariae were included. In addition, samples obtained from 40 controls known to be uninfected with malaria (i.e., subjects who repeatedly had negative malaria smear results and febrile illnesses other than malaria) were also included. The ratio of male to female patients was 1.6, with a mean age (± SD) of 31 ± 1.6 years (range, 4 months–73 years). Countries and regions of acquisition included Africa (78%), the Indian subcontinent (10%), South and Central America (5%), Oceania (4%), and Southeast Asia (3%). Parasitemia loads ranged from 20 parasites/µL to 500,000 parasites/µL, with 8% of patient samples containing <100 parasites/µL. Patients with malaria did not differ significantly from other patients with respect to age, sex, or duration of illness.

Figure 1 illustrates an example of a typical run of the real-time assay. Several pfcrt haplotypes (defined by mutations around positions 72–76: S[tct]VMNT, S[agt]VMNT, CVIET, CVMNK) were observed in the patient population. Previous studies have reported that African and Southeast Asian CQ-resistant isolates are characterized by the presence of the CVIET amino acid haplotype, which in our real-time assay occurred at a melting temperature of ∼63°C. In contrast, CQ-resistant isolates from South America in our assay have been primarily characterized by the presence of the SVMNT haplotype across the same region and by their clustering around a melting temperature of 54°C [27]. All susceptible isolates in this study carried the CVMNK haplotype across positions 72–76 and clustered around a melting temperature of 49°C (figure 1) [19–21, 27].

Table 1 shows the results of the real-time PCR system compared with results of nested PCR-RFLP assay for the detection of K76T. Compared with nested PCR-RFLP, the real-time PCR assay was 100% sensitive and 96.2% specific for detection of the P. falciparum and the K76T mutation. Assuming a 10% prevalence of P. falciparum malaria in the study population, the positive and negative predictive values of the assay were 82% and 100%, respectively. Of note were 3 cases (2 in patients with P. vivax infection, and 1 in a patient with P. malariae infection) identified by microscopy and nested PCR as single-species infections that were identified as mixed infections (P. vivax or P. malariae plus P. falciparum) by the real-time assay, which indicated that each of these cases also involved CQ-resistant P. falciparum isolates of the CVIET haplotype. In all 3 cases, direct sequencing of the respective amplicons verified the presence of a mixed infection with P. falciparum; however, because not all isolates were reassessed with this method, these findings were not included in the specificity calculations to avoid the introduction of verification bias (discrepant analysis, as described in [28, 29]). If the sequence results had been included as the reference standard, the specificity and positive predictive value of the assay would have improved to 100%.

Table 1
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Table 1

Results of the real-time pfcrt assay, compared with results of nested PCR—restriction fragment—length polymorphism (RFLP) as the reference standard, for the detection of chloroquine (CQ)—resistant and CQ-susceptible Plasmodium falciparum malaria in febrile returned travelers.

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Discussion

It is currently estimated that 50–80 million individuals from industrialized countries visit malaria-endemic areas each year and that at least 30,000 individuals contract malaria [30]. Despite receiving treatment, 1%–4% of travellers who acquire P. falciparum malaria die as a result of infection. This fatality rate increases to ⩾20% for patients who develop severe malaria or patients who are elderly [1, 31]. Because ∼90% of travellers who contract malaria will not become ill until returning home, accurate diagnosis depends on the expertise of clinical laboratories in areas where the illness is not endemic [32, 33]. Delays and inaccuracies in diagnosis are associated with increased case-fatality rates [1–3]. Although microscopic detection of parasites on Giemsa-stained blood smears has been the diagnostic reference standard for more than a century, it is an imperfect standard that is highly dependent on the technical expertise of the microscopist [4]. The ability to maintain competency is problematic, especially in medical centers in countries where the disease is not endemic [1, 4, 5, 32, 34]. Moreover, routine microscopy and newer malaria antigen detection assays (reviewed in [35]) provide little information on drug susceptibility at presentation, and they do not contribute meaningfully to global surveillance efforts to detect and monitor drug resistance.

In this study, we describe a real-time PCR assay for the detection of P. falciparum malaria and pfcrt alleles associated with CQ susceptibility and resistance. Geographically diverse malaria isolates obtained from returned febrile travelers were analyzed to ensure that the assay would reliably detect multiple pfcrt haplotypes associated with resistance. We demonstrate that this real-time assay is rapid, sensitive, and specific for the detection of P. falciparum malaria and for the characterization of a genetic marker of CQ resistance. The assay detected multiple pfcrt haplotypes and clearly discriminated between CQ-susceptible and CQ-resistant isolates associated with both the classic old world (CVIET) and new world (SVMNT) haplotypes [27] in <1 h. Its speed and performance characteristics may eliminate the need for more-complicated approaches and make it an attractive strategy for the detection and surveillance of CQ-resistant P. falciparum malaria in returned travelers.

Amplification-based methods currently utilized for malaria diagnosis, particularly nested PCR—based methods [24], are sensitive and specific, but they are also labor intensive, with turnaroundre also labor intensive, with turnaround times that are generally too long for routine clinical use. For determination of CQ resistance, additional time- and labor-intensive steps, such as RFLP analysis or sequencing of the pfcrt gene, are currently necessary to differentiate between the sensitive and mutant allele at position 76. Furthermore, most current PCR assays, especially nested assays, require considerable preamplification and postamplification sample handling (e.g., open PCR assays), and special efforts need to be employed to prevent false-positive results of assays caused by amplicon contamination. In contrast, real-time PCR technology has the potential to overcome these obstacles, offering a simple, closed amplification system (i.e., amplification and detection occurs in capped capillaries, thus reducing the potential for contamination), time efficiency (requiring ∼1 h), and an automated diagnostic system. In-house, real-time, quantitative PCR assays using SYBR green and fluorescence resonance energy transfer techniques for the detection of CQ resistance have been previously reported in malaria research settings [35, 36]. However, these studies were limited by the lack of a head-to-head comparison with an accepted reference standard and a lack of negative controls or other Plasmodium species to assess the specificity of the assay. Furthermore, these assays were validated using samples from geographically limited areas (primarily of African origin), and it remains to be demonstrated how well they will detect and distinguish other pfcrt haplotypes. To our knowledge, the real-time assay we describe is the only standardized CQ-resistance assay developed under Good Manufacturing Practices, a manufacturing method suitable for use in a routine clinical diagnostic laboratory, although it is not currently approved by the US Food and Drug Administration.37

Although our assay identifies P. falciparum infection and rapidly differentiates between CQ-resistant and CQ-sensitive isolates, it has limitations. This assay does not detect human malaria species other than P. falciparum and is not a quantitative assay. At present, it would need to be combined with a quantitative, standardized, real-time assay for other malaria species (which has been described elsewhere [37]), expert microscopy, or another accepted diagnostic method. Future generations of this assay would be improved by incorporating a multiplexed platform capable of quantitative discrimination of the 4 malarial species and by including additional genetic determinants of drug resistance, such as the ability to detect mutations in cytochrome b associated with atovaquone resistance [38–40].

A slow but steady return of CQ-susceptible P. falciparum malaria has been reported in malarious areas where use of CQ was previously discontinued because of resistance [13–16]. This fact, combined with studies indicating that many cases of imported malaria in travelers are still responsive to CQ [8, 17, 18], suggests that a rapid assay, such as the one we describe, could be used to determine whether CQ would be effective in the treatment of uncomplicated malaria. However, this assay needs additional clinical and field validation, especially in low-prevalence populations, to more fully define its performance characteristics. Furthermore, it is possible that this assay may miss a CQ-resistant parasite subpopulation that causes only a minor proportion of initial infections. Therefore, we do not advocate the use of CQ as therapy for P. falciparum malaria in travelers returning from the majority of malaria-endemic areas. Even in the presence of the CQ-susceptible allele, the current standard of care would support combining CQ with a second agent to avoid the emergence of resistance [41].

In summary, we describe a rapid, real-time PCR assay for the diagnosis of P. falciparum malaria in febrile returned travelers. This assay is sensitive and specific for the detection of P. falciparum malaria and the key K76T mutation in pfcrt associated with CQ resistance, compared with nested PCR-RFLP analysis and sequencing, as the molecular reference standard. The assay is automated, standardized, manufactured according to Good Manufacturing Practices, and suitable for use in routine clinical diagnostics laboratories. Although the relatively high cost of real-time PCR technology may preclude its use in resource-poor clinics, its performance characteristics, combined with its rapid results, suggest that it may be a useful diagnostic adjunct in developed countries.

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Acknowledgments

Financial support. Canadian Institutes of Health Research (MT-13701) to K.C.K., CIHR to K.C.K and G.F., and Genome Canada.

Potential conflicts of interest. R.S. is employed by Artus and helped to develop the assay and provided kits for evaluation. However, R.S. and Artus did not have a role in the design, conduct, or reporting of the study or in the decision to submit the manuscript for publication. G.F., K.Z., A.Z., and K.C.K.: no conflicts.

  • Received June 4, 2005.
  • Accepted October 16, 2005.
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  1. Clin Infect Dis. (2006) 42 (5): 622-627. doi: 10.1086/500134
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