Real-Time PCR for Detection and Identification of Plasmodium spp.
http://www.100md.com
微生物临床杂志 2005年第5期
Department of Pathology and Laboratory Medicine, Evanston Northwestern Healthcare, Evanston
Northwestern University Feinberg School of Medicine, Chicago, Illinois
Molecular Diagnosis Centre, National University Hospital, Singapore
Department of Pathology, National University of Singapore, Singapore
ABSTRACT
Rapid and accurate detection of malaria parasites in blood is needed to institute proper therapy. We developed and used a real-time PCR assay to detect and distinguish four Plasmodium spp. that cause human disease by using a single amplification reaction and melting curve analysis. Consensus primers were used to amplify a species-specific region of the multicopy 18S rRNA gene, and SYBR Green was used for detection in a LightCycler instrument. Patient specimens infected at 0.01 to 0.02% parasitemia densities were detected, and analytical sensitivity was estimated to be 0.2 genome equivalent per reaction. Melting curve analysis based on nucleotide variations within the amplicons provided a basis for accurate differentiation of Plasmodium falciparum, P. vivax, P. ovale, and P. malariae. For assay validation, 358 patient blood samples from the National University Hospital in Singapore and Evanston Northwestern Healthcare in Illinois were analyzed. Of 76 blinded patient samples with a microscopic diagnosis of P. falciparum, P. vivax, or P. ovale infection, 74 (97.4%) were detected by real-time PCR, including three specimens containing mixed P. falciparum-P. vivax infections. No Plasmodium DNA was amplified in any of the 82 specimens sent for malaria testing but that were microscopically negative for Plasmodium infection. In addition, 200 blood samples from patients whose blood was collected for reasons other than malaria testing were also determined to be negative by real-time PCR. Real-time PCR with melting curve analysis could be a rapid and objective supplement to the examination of Giemsa-stained blood smears and may replace microscopy following further validation.
INTRODUCTION
Malaria remains a global concern. The World Health Organization estimates 300 to 500 million cases of malaria infections resulting in over one million deaths occurring globally each year. Although the vast majority of these cases are found in the 100 countries in the tropical regions of Africa, Asia, Central and South Americas where the disease is endemic, the mobile nature of today's society results in ca. 1,400 cases of imported malaria reported each year in the United States as a result of international travel, immigration, and military service (10). The Centers for Disease Control and Prevention recommends that malaria should be considered in the differential diagnosis of febrile patients who have traveled to a region where malaria is endemic and in any patients who experience fevers of unknown origin regardless of their travel history (10). Provision of these diagnostic services and maintenance of competency may pose a challenge to many laboratories.
Species differentiation of Plasmodium is essential for selecting the proper treatment. Especially important is differentiating P. falciparum from the others, since this species is responsible for ca. 95% of the deaths due to malaria (33). The current standard for diagnosis is the microscopic examination of Giemsa-stained thick and thin blood smears (12, 16, 21, 22, 28). This procedure is time-consuming to prepare, read, and interpret the slides. Previous studies have shown that even with experienced microscopists, misdiagnosis occurs, particularly in cases of mixed infection or low parasitemia (12, 28). Immunochromatographic assays based on antigen detection have been developed but are also relatively insensitive in cases of low parasitemia (22, 24, 30). In addition, antigenemia may persist weeks beyond the actual infection, leading to the false diagnosis of malaria parasitemia (6, 22). Molecular detection for Plasmodium diagnosis using PCR has resulted in increased sensitivity and species discrimination compared to either microscopic or immunochromatographic diagnosis of malaria (2, 4, 12, 14, 22, 23, 25, 27). However, most published PCR assays are gel based with (4, 5, 15) or without (2, 3, 9, 23, 25-27, 29, 31, 35, 36) subsequent probe hybridizations, resulting in a lengthy procedure not optimal for clinical use. The need for a more sensitive and time-efficient assay has led to the development of molecular assays involving real-time PCR (7, 8, 13, 18). Real-time PCR assays have the potential to detect low levels of parasitemia, identify mixed infections, and allow for precise differentiation of species via melting curve analysis. In the present study, we developed and validated a real-time PCR assay to detect and identify Plasmodium spp. in a single reaction by using a simple collection method consisting of blood spotted on treated filter paper.
(This research was presented in part at the 43rd Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, Ill., 14 to 17 September 2003, and at the annual meeting of the Association for Molecular Pathology, Orlando, Fla., 22 November 2003.)
MATERIALS AND METHODS
Specimens. The study was approved by the Institutional Review Boards of Evanston Northwestern Healthcare (ENH) in Evanston, Ill., and the National University Hospital (NUH) in Singapore. Controls obtained from the American Type Culture Collection (ATCC; Manassas, Va.) included plasmids containing a partial 18S rRNA gene sequence from each species (P. falciparum [MRA-177], P. vivax [MRA-178], P. malariae [MRA-179], and P. ovale [MRA-180]). In addition, 10 DNA samples from P. falciparum-infected human red blood cell cultures and monkey blood samples infected with P. vivax (five strains) or P. malariae (one strain) were also obtained from the ATCC. During the development of the assay, DNA was isolated from eight blood specimens known to contain either P. falciparum or P. vivax from patients at ENH or NUH. For validation, 200 blood samples from patients whose blood was collected for reasons other than malaria diagnosis and 158 blinded samples from patients with fever and a history of recent travel within a region where malaria infection is endemic that were obtained from ENH and NUH, including specimens from sources in Malaysia, Myanmar, and Thailand, were tested. Microscopic analysis was performed by experienced microscopists at each hospital. Thin and thick smears from specimens with an initial determination of Plasmodium infection were again blinded and analyzed by a panel of experienced microscopists from ENH and Northwestern Memorial Hospital in Chicago, Ill. On the basis of enlarged infected red blood cells, the occasional presence of Schüffner's dots, and the rare identification of characteristic schizonts, four cases initially reported as either P. falciparum or P. malariae were identified as P. vivax in the second analysis; the results of the other blinded specimens were in agreement for both the initial and second microscopic analyses. The final microscopic determinations of species are listed in Table 1 and were compared to the real-time PCR results.
Blood specimen preparation. Since the present study involved an international collaboration, the use of ISOCODE cards (Schleicher & Schuell, Keene, N.H.) containing spotted samples of fresh or EDTA-anticoagulated blood was the requested method for specimen collection. Approximately 40 μl of blood was applied to each circle of an ISOCODE card and allowed to dry completely. DNA from a single 6-mm hole punch (equivalent to one-fourth to one-half of an ISOCODE card circle) was extracted utilizing one wash with 500 μl of nuclease-free water, followed by a 30-min incubation at 95 to 100°C in 50 μl of nuclease-free water. Twenty blood samples that were clotted or thickened by prolonged storage before application to the ISOCODE cards were alternatively processed by using a Puregene (Gentra Systems, Minneapolis, Minn.) procedure for tissue samples according to the manufacturer's instructions. The specimens chosen for the Puregene procedure were distinguishable as the dried blood and/or clot remained on the surface of the card and did not penetrate the paper efficiently.
Real-time PCR. Plasmodium detection was performed by using real-time PCR in the LightCycler (Roche Molecular Systems, Indianapolis, Ind.). The 18S rRNA gene was chosen as the target since it contains both highly conserved and variable regions, and at least five copies of the gene are dispersed on separate chromosomes of the Plasmodium genome (11, 20). Consensus primers were designed after comparing several partial 18S rRNA gene sequences for each of four Plasmodium species (PL1473F18 [5'-TAA CgA ACg AgA TCT TAA-3'] and PL1679R18 [5'-gTT CCT CTA AgA AgC TTT-3']; sequence numbering from GenBank accession number M19173). A BLAST analysis (1) of GenBank indicated that amplification with these two oligonucleotides would only occur when a Plasmodium spp. was available to act as a template (expected value of 0.31 for both oligonucleotides). Each 20-μl reaction mix contained 2 to 5 μl of sample DNA, 2 μl of 10x FastSTART DNA SYBR Green reagent (Roche), 6.5 mM MgCl2 (final concentration), and 0.5 mM concentrations of each primer. The PCR conditions consisted of an initial denaturation at 95°C for 10 min, followed by amplification for 40 cycles of 10 s at 95°C, 5 s at 50°C, and 20 s at 72°C, with fluorescence acquisition at the end of each extension step. Amplification was immediately followed by a melt program consisting of 2 min at 95°C, 2 min at 68°C, and a stepwise temperature increase of 0.2°C/s until 90°C, with fluorescence acquisition at each temperature transition. The fluorescence data were analyzed by using F1/F2 settings, which improved the detection of P. falciparum, and a cutoff of 35 cycles was used to define Plasmodium-positive samples in the present study. This assay for Plasmodium species differentiation required ca. 1 h to complete, in addition to the sample preparation time. Melt curve analysis was used to determine the species-specific mean melting temperature (Tm) based on values determined from the respective plasmid controls.
RESULTS
Typical amplification and melt curves depicting Tm detection for the various species are shown in Fig. 1 to 5. Based on the published genome size for P. falciparum (22.8 Mbp) (11), Avogadro's number (6.022 x 1023 molecules per mole), and a standard nucleic acid molar conversion (1 μg of 1-kb DNA = 1.54 μmol), we calculated that 1 μg of P. falciparum DNA is equal to 4.067 x 107 genome copies or one genome copy in 24.6 fg of DNA. Control Plasmodium DNA samples obtained from in vitro culture in human reticulocytes and diluted to 0.2 genome copies (5 fg) per reaction were detected and correctly identified in the melting curve analysis; this is possible due to the presence of more than one copy of the 18S rRNA gene per genome. An excess amount (150 ng) of human DNA previously tested negative for Plasmodium was included as a negative control to estimate the possible background fluorescence in this SYBR Green assay.
Melting curve analysis permitted the clear identification of each Plasmodium species control, as shown in Fig. 2. The Tm values for the control plasmids were highly reproducible on eight repeated melt curve runs. Table 2 contains the average melting curve peak Tm for each Plasmodium spp. identified in the control plasmids and the other genomic DNA specimens used to develop the real-time PCR assay. Mixed reactions were created to mimic a dual infection with P. falciparum and P. vivax. Detection of each species was achieved at a ratio equivalent to 10 P. falciparum: one P. vivax (data not shown), a ratio that should approximate the number of P. falciparum-infected red blood cells that is generally higher in peripheral blood smears. DNA obtained from P. vivax- or P. malariae-infected monkey blood samples and 10 separate isolates of P. falciparum genomic DNA from the ATCC confirmed the reproducibility of the melting curve (Fig. 3). Four P. falciparum patient samples and four P. vivax patient samples were also tested during assay development. We did observe a consistent slight shift to a higher Tm (up to 1°C) in the amplicons generated by the control plasmids compared to the other specimens tested (Table 2). The cause for this shift is currently unknown, although we assume sequence differences in the cloned fragments or variable salt concentrations in the DNA aliquots are responsible. Taking into consideration the Tm values for the control plasmids, the infected monkey blood, the infected human red blood cell cultures, and the eight patient samples, broad, nonoverlapping Tm ranges were selected to identify the Plasmodium species in the blinded patient specimens. The Tm values chosen were as follows: P. malariae, 73.5 to 75.5°C; P. falciparum, 75.5 to 77.5°C; P. ovale, 77.5 to 79.0°C; and P. vivax, 79.0 to 81.0°C. DNA prepared by using the Puregene procedure had Tm values universally lower by ca. 1°C than DNA prepared from identical specimens using the standard ISOCODE extraction protocol but was still at the lowest end of the broad ranges described above. These lower Tm values are most likely due to the salt content in the Puregene DNA hydration buffer compared to the nuclease-free water of the ISOCODE card protocol.
Parasites in 74 of 76 (97.4%) blinded patient samples with a positive microscopic diagnosis of malaria were detected by real-time PCR (Fig. 4), including three specimens containing mixed P. falciparum-P. vivax infections. Two samples that were diagnosed by microscopy as only P. falciparum infections were identified as mixed P. falciparum-P. vivax infections by real-time PCR (Fig. 5). A single patient infected with P. ovale was identified by both microscopic analysis and real-time PCR (microscopic identification of P. ovale confirmed by the Illinois Department of Public Health). Two specimens microscopically diagnosed as containing P. falciparum with parasitemia levels of ca. 0.01% (data not shown) did not produce an amplification curve within the 35 cycle cutoff limit used for the present study, even when the amount of template DNA used in each reaction was increased from 2 to 5 μl. Thus, the detection sensitivity for patient samples was estimated to be in the range of 0.01 to 0.02% parasitemia. Using the normal range of 4.7 to 6.1 million red blood cells per ml of blood, 0.01 to 0.02% parasitemia would be equivalent to 470 to 1,220 parasites per ml, or approximately 1 parasite per μl. This sensitivity corresponds favorably to other published methods, specifically real-time PCR assays.
Four samples were determined by initial microscopy to be two P. malariae- and two P. falciparum-infected specimens; all four were identified as P. vivax by the real-time PCR assay, which is in agreement with the final consensus identification for these four samples. Eighty-two specimens from patients with clinical findings suggesting malaria but microscopically negative for Plasmodium and subsequently determined not to be infected were PCR negative. An additional 200 blood samples from patients collected for diagnoses unrelated to malaria (data not shown) were also determined to be negative by real-time PCR. Using results from the second microscopic analysis as the gold standard, the sensitivity, specificity, positive and negative predictive values, and accuracy of the real-time PCR assay for each detected Plasmodium spp. are summarized in Table 3.
DISCUSSION
Although there are many published studies showing the improved sensitivity and specificity of PCR-based assays over microscopic or immunochromatographic diagnosis of malaria (2, 4, 12, 14, 22, 23, 25, 27), only a handful take advantage of the even more sensitive and time-efficient real-time PCR technologies. In several studies in which real-time PCR was utilized, either a single-species was identified (13) or no distinction between the four human parasites was made (8, 18). This distinction is critical in the clinical management of patients, since the course of treatment varies depending on which species is the cause of the infection. Only in the study by de Monbrison et al. were the four human parasites identified by real-time PCR (7). This assay included five separate primer pairs amplified simultaneously in a LightCycler instrument, utilizing melting curve analysis to distinguish the Plasmodium spp. In the de Monbrison study, twenty-nine single infections and four dual infections were identified by real-time PCR. Twelve specimens were negative by both microscopy and real-time PCR.
The single reaction real-time PCR assay described in our study used only a single pair of primers to detect and identify Plasmodium spp. in 3 h, including standard DNA sample preparation, amplification, and detection, with sensitivities equivalent to microscopy. An additional 1 to 2 h was needed when the more extensive Puregene DNA preparation was required. As with several other studies, we chose to target a species-specific region of the Plasmodium 18S (small subunit) rRNA gene in our real-time PCR assay. By targeting a gene with multiple copies dispersed throughout the Plasmodium genome (11), the sensitivity of this assay is greater than if a single-copy gene was chosen. We also chose to target the partial 18S rRNA gene sequence included in the cloned plasmids available from the ATCC to permit easy access to individual species-specific controls. Although several fluorescence resonance energy transfer hybridization probes and different primer sets were tested in the development of this assay (data not shown), the described SYBR Green assay provided the best discrimination among the different species. The sequences bracketed by the primers are sufficiently divergent in the four species to provide at least a 1°C difference between the average Tm values to distinguish between the species during the melting curve analysis. Single base differences theoretically may also exist between strains within a species from different geographic regions, but our results indicate that the variances among individual strains or patient specimens prepared by the same DNA purification procedure are minor in comparison to the differences among species. Additional specimens from other regions around the world would be required to confirm these assumptions.
We did not address the analysis of blood samples collected by various methods in the present study. To obtain a diverse group of patient specimens, blinded samples from ENH and NUH were collected on ISOCODE cards and processed in the Molecular Diagnostics laboratory at ENH. We overcame potential problems posed by blood samples that were clotted or thickened due to long-term storage at 4°C before application to cards, thus failing to percolate into the filter paper adequately, by processing the "clumps" using the Puregene procedure for tissue samples. Samples with significant red blood cell lysis as a result of freezing were found to be unsuitable for analysis (data not shown). In the present study, DNA was isolated from ca. 10 μl of blood from each ISOCODE card specimen, and 1/50 to 1/20 of this DNA was used in each real-time PCR.
We analyzed 76 blood specimens from patients with clinical findings suggesting malaria for which a microscopic determination of Plasmodium infection had been identified. We, like others, had some difficulty in arriving at a consensus for the microscopic diagnosis. Our final microscopic diagnosis used for comparison was based on review by a panel of experienced microscopists and is reported in Table 1. Real-time PCR analysis identified P. falciparum, P. vivax, or P. ovale in 74 (97.4%) of these specimens, including 3 that contained a dual infection with P. falciparum and P. vivax, for which only 1 had been microscopically identified as such. Since it is often difficult to diagnose mixed infections by microscopy, the distinct dual peaks observed in Fig. 5 indicate that the real-time PCR assay may be more accurate than the gold standard of microscopy in these two cases. To date, only a single patient with a P. ovale infection had been identified by microscopy and real-time PCR. Of the two specimens microscopically diagnosed as containing P. falciparum that did not produce a positive result for any Plasmodium infection in the real-time PCR assay, the amount of parasitemia in these samples was ca. 0.01%, at the lower threshold of sensitivity for this assay. In both cases, the blood had been stored for several days before aliquots were spotted onto the ISOCODE cards. It is also possible that the sensitivity of the assay was decreased by the delay in specimen preparation, in spite of using the Puregene procedure.
Of the 74 infected specimens detected by both microscopy and real-time PCR, 4 had discrepant species identification based on the initial microscopic analysis. Two specimens with P. malariae and two with P. falciparum, as originally determined by microscopic analysis, were all identified as P. vivax positive by the real-time PCR assay. GenBank sequence comparisons and the results of preliminary testing using ATCC controls would suggest that misidentification of either P. falciparum or P. malariae as P. vivax is unlikely using this sequence-based assay and may indicate an inaccurate microscopic diagnosis. The four discrepant specimens were included in a blinded group reviewed by a panel of experienced microscopists. The results of this second review were in agreement with the real-time PCR results, indicating the real-time PCR assay may provide accurate identification of Plasmodium spp. when the microscopic analysis is uncertain.
Recently, P. knowlesi, a species that is morphologically similar to P. malariae has been identified as a human pathogen in patients from Malaysia (32). Sequence analysis indicates that this species should also be amplified by using the conserved primers described in this assay. Whether the differences in sequence composition between the primers are sufficient to result in a Tm closer to that of P. vivax than to P. malariae needs to be determined by testing confirmed P. knowlesi specimens.
A drawback common to all DNA-based malaria assays is the real possibility of detecting persistent DNA in the bloodstream after the infection has subsided. Several studies reported the posttreatment detection of Plasmodium DNA up to 4 days after microscopic detection (4, 25), and another purports that detection at 5 to 8 days after initiation of treatment indicates therapeutic failure, possibly due to parasite resistance (17). However, this persistence of Plasmodium DNA is still less than the persistent antigenemia reported with immunochromatographic assays (6, 22). Also, DNA-based molecular assays have demonstrated a possible chronic detection in regions of the world where malaria is endemic, which may be due to very low parasitemias in immune individuals (3, 19, 34).
In the present study, we developed and validated a real-time PCR assay that can accurately detect and identify Plasmodium spp. in a single reaction by using a simple method for sample collection consisting of blood spotted on treated filter paper (Table 3). The entire process, including specimen preparation using the standard ISOCODE card procedure, PCR amplification, and melting curve identification of Plasmodium spp., was completed in less than 3 h. This real-time PCR assay for the detection and identification of Plasmodium spp. can be used to confirm microscopic findings and in many settings can be used for the primary identification of an infected patient without the need for multiple blood specimens or even microscopic analysis.
ACKNOWLEDGMENTS
We acknowledge the assistance of Kevin S. W. Tan, Department of Microbiology, National University of Singapore; May Ann Lee, Defense Ministry Research Institute, Singapore; and Thiha Nyunt, Yangon, Myanmar, in obtaining the patient blood specimens used in this study. We appreciate the time-consuming microscopic analysis performed by Linda Kuksuk of Northwestern Memorial Hospital, Chicago, Ill. We also acknowledge the technical assistance of Wooi Loon Ng and Mui Joo Khoo of National University Hospital, Singapore.
This study was funded by the Department of Pathology and Laboratory Medicine at Evanston Northwestern Healthcare, Evanston, Ill.
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Northwestern University Feinberg School of Medicine, Chicago, Illinois
Molecular Diagnosis Centre, National University Hospital, Singapore
Department of Pathology, National University of Singapore, Singapore
ABSTRACT
Rapid and accurate detection of malaria parasites in blood is needed to institute proper therapy. We developed and used a real-time PCR assay to detect and distinguish four Plasmodium spp. that cause human disease by using a single amplification reaction and melting curve analysis. Consensus primers were used to amplify a species-specific region of the multicopy 18S rRNA gene, and SYBR Green was used for detection in a LightCycler instrument. Patient specimens infected at 0.01 to 0.02% parasitemia densities were detected, and analytical sensitivity was estimated to be 0.2 genome equivalent per reaction. Melting curve analysis based on nucleotide variations within the amplicons provided a basis for accurate differentiation of Plasmodium falciparum, P. vivax, P. ovale, and P. malariae. For assay validation, 358 patient blood samples from the National University Hospital in Singapore and Evanston Northwestern Healthcare in Illinois were analyzed. Of 76 blinded patient samples with a microscopic diagnosis of P. falciparum, P. vivax, or P. ovale infection, 74 (97.4%) were detected by real-time PCR, including three specimens containing mixed P. falciparum-P. vivax infections. No Plasmodium DNA was amplified in any of the 82 specimens sent for malaria testing but that were microscopically negative for Plasmodium infection. In addition, 200 blood samples from patients whose blood was collected for reasons other than malaria testing were also determined to be negative by real-time PCR. Real-time PCR with melting curve analysis could be a rapid and objective supplement to the examination of Giemsa-stained blood smears and may replace microscopy following further validation.
INTRODUCTION
Malaria remains a global concern. The World Health Organization estimates 300 to 500 million cases of malaria infections resulting in over one million deaths occurring globally each year. Although the vast majority of these cases are found in the 100 countries in the tropical regions of Africa, Asia, Central and South Americas where the disease is endemic, the mobile nature of today's society results in ca. 1,400 cases of imported malaria reported each year in the United States as a result of international travel, immigration, and military service (10). The Centers for Disease Control and Prevention recommends that malaria should be considered in the differential diagnosis of febrile patients who have traveled to a region where malaria is endemic and in any patients who experience fevers of unknown origin regardless of their travel history (10). Provision of these diagnostic services and maintenance of competency may pose a challenge to many laboratories.
Species differentiation of Plasmodium is essential for selecting the proper treatment. Especially important is differentiating P. falciparum from the others, since this species is responsible for ca. 95% of the deaths due to malaria (33). The current standard for diagnosis is the microscopic examination of Giemsa-stained thick and thin blood smears (12, 16, 21, 22, 28). This procedure is time-consuming to prepare, read, and interpret the slides. Previous studies have shown that even with experienced microscopists, misdiagnosis occurs, particularly in cases of mixed infection or low parasitemia (12, 28). Immunochromatographic assays based on antigen detection have been developed but are also relatively insensitive in cases of low parasitemia (22, 24, 30). In addition, antigenemia may persist weeks beyond the actual infection, leading to the false diagnosis of malaria parasitemia (6, 22). Molecular detection for Plasmodium diagnosis using PCR has resulted in increased sensitivity and species discrimination compared to either microscopic or immunochromatographic diagnosis of malaria (2, 4, 12, 14, 22, 23, 25, 27). However, most published PCR assays are gel based with (4, 5, 15) or without (2, 3, 9, 23, 25-27, 29, 31, 35, 36) subsequent probe hybridizations, resulting in a lengthy procedure not optimal for clinical use. The need for a more sensitive and time-efficient assay has led to the development of molecular assays involving real-time PCR (7, 8, 13, 18). Real-time PCR assays have the potential to detect low levels of parasitemia, identify mixed infections, and allow for precise differentiation of species via melting curve analysis. In the present study, we developed and validated a real-time PCR assay to detect and identify Plasmodium spp. in a single reaction by using a simple collection method consisting of blood spotted on treated filter paper.
(This research was presented in part at the 43rd Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, Ill., 14 to 17 September 2003, and at the annual meeting of the Association for Molecular Pathology, Orlando, Fla., 22 November 2003.)
MATERIALS AND METHODS
Specimens. The study was approved by the Institutional Review Boards of Evanston Northwestern Healthcare (ENH) in Evanston, Ill., and the National University Hospital (NUH) in Singapore. Controls obtained from the American Type Culture Collection (ATCC; Manassas, Va.) included plasmids containing a partial 18S rRNA gene sequence from each species (P. falciparum [MRA-177], P. vivax [MRA-178], P. malariae [MRA-179], and P. ovale [MRA-180]). In addition, 10 DNA samples from P. falciparum-infected human red blood cell cultures and monkey blood samples infected with P. vivax (five strains) or P. malariae (one strain) were also obtained from the ATCC. During the development of the assay, DNA was isolated from eight blood specimens known to contain either P. falciparum or P. vivax from patients at ENH or NUH. For validation, 200 blood samples from patients whose blood was collected for reasons other than malaria diagnosis and 158 blinded samples from patients with fever and a history of recent travel within a region where malaria infection is endemic that were obtained from ENH and NUH, including specimens from sources in Malaysia, Myanmar, and Thailand, were tested. Microscopic analysis was performed by experienced microscopists at each hospital. Thin and thick smears from specimens with an initial determination of Plasmodium infection were again blinded and analyzed by a panel of experienced microscopists from ENH and Northwestern Memorial Hospital in Chicago, Ill. On the basis of enlarged infected red blood cells, the occasional presence of Schüffner's dots, and the rare identification of characteristic schizonts, four cases initially reported as either P. falciparum or P. malariae were identified as P. vivax in the second analysis; the results of the other blinded specimens were in agreement for both the initial and second microscopic analyses. The final microscopic determinations of species are listed in Table 1 and were compared to the real-time PCR results.
Blood specimen preparation. Since the present study involved an international collaboration, the use of ISOCODE cards (Schleicher & Schuell, Keene, N.H.) containing spotted samples of fresh or EDTA-anticoagulated blood was the requested method for specimen collection. Approximately 40 μl of blood was applied to each circle of an ISOCODE card and allowed to dry completely. DNA from a single 6-mm hole punch (equivalent to one-fourth to one-half of an ISOCODE card circle) was extracted utilizing one wash with 500 μl of nuclease-free water, followed by a 30-min incubation at 95 to 100°C in 50 μl of nuclease-free water. Twenty blood samples that were clotted or thickened by prolonged storage before application to the ISOCODE cards were alternatively processed by using a Puregene (Gentra Systems, Minneapolis, Minn.) procedure for tissue samples according to the manufacturer's instructions. The specimens chosen for the Puregene procedure were distinguishable as the dried blood and/or clot remained on the surface of the card and did not penetrate the paper efficiently.
Real-time PCR. Plasmodium detection was performed by using real-time PCR in the LightCycler (Roche Molecular Systems, Indianapolis, Ind.). The 18S rRNA gene was chosen as the target since it contains both highly conserved and variable regions, and at least five copies of the gene are dispersed on separate chromosomes of the Plasmodium genome (11, 20). Consensus primers were designed after comparing several partial 18S rRNA gene sequences for each of four Plasmodium species (PL1473F18 [5'-TAA CgA ACg AgA TCT TAA-3'] and PL1679R18 [5'-gTT CCT CTA AgA AgC TTT-3']; sequence numbering from GenBank accession number M19173). A BLAST analysis (1) of GenBank indicated that amplification with these two oligonucleotides would only occur when a Plasmodium spp. was available to act as a template (expected value of 0.31 for both oligonucleotides). Each 20-μl reaction mix contained 2 to 5 μl of sample DNA, 2 μl of 10x FastSTART DNA SYBR Green reagent (Roche), 6.5 mM MgCl2 (final concentration), and 0.5 mM concentrations of each primer. The PCR conditions consisted of an initial denaturation at 95°C for 10 min, followed by amplification for 40 cycles of 10 s at 95°C, 5 s at 50°C, and 20 s at 72°C, with fluorescence acquisition at the end of each extension step. Amplification was immediately followed by a melt program consisting of 2 min at 95°C, 2 min at 68°C, and a stepwise temperature increase of 0.2°C/s until 90°C, with fluorescence acquisition at each temperature transition. The fluorescence data were analyzed by using F1/F2 settings, which improved the detection of P. falciparum, and a cutoff of 35 cycles was used to define Plasmodium-positive samples in the present study. This assay for Plasmodium species differentiation required ca. 1 h to complete, in addition to the sample preparation time. Melt curve analysis was used to determine the species-specific mean melting temperature (Tm) based on values determined from the respective plasmid controls.
RESULTS
Typical amplification and melt curves depicting Tm detection for the various species are shown in Fig. 1 to 5. Based on the published genome size for P. falciparum (22.8 Mbp) (11), Avogadro's number (6.022 x 1023 molecules per mole), and a standard nucleic acid molar conversion (1 μg of 1-kb DNA = 1.54 μmol), we calculated that 1 μg of P. falciparum DNA is equal to 4.067 x 107 genome copies or one genome copy in 24.6 fg of DNA. Control Plasmodium DNA samples obtained from in vitro culture in human reticulocytes and diluted to 0.2 genome copies (5 fg) per reaction were detected and correctly identified in the melting curve analysis; this is possible due to the presence of more than one copy of the 18S rRNA gene per genome. An excess amount (150 ng) of human DNA previously tested negative for Plasmodium was included as a negative control to estimate the possible background fluorescence in this SYBR Green assay.
Melting curve analysis permitted the clear identification of each Plasmodium species control, as shown in Fig. 2. The Tm values for the control plasmids were highly reproducible on eight repeated melt curve runs. Table 2 contains the average melting curve peak Tm for each Plasmodium spp. identified in the control plasmids and the other genomic DNA specimens used to develop the real-time PCR assay. Mixed reactions were created to mimic a dual infection with P. falciparum and P. vivax. Detection of each species was achieved at a ratio equivalent to 10 P. falciparum: one P. vivax (data not shown), a ratio that should approximate the number of P. falciparum-infected red blood cells that is generally higher in peripheral blood smears. DNA obtained from P. vivax- or P. malariae-infected monkey blood samples and 10 separate isolates of P. falciparum genomic DNA from the ATCC confirmed the reproducibility of the melting curve (Fig. 3). Four P. falciparum patient samples and four P. vivax patient samples were also tested during assay development. We did observe a consistent slight shift to a higher Tm (up to 1°C) in the amplicons generated by the control plasmids compared to the other specimens tested (Table 2). The cause for this shift is currently unknown, although we assume sequence differences in the cloned fragments or variable salt concentrations in the DNA aliquots are responsible. Taking into consideration the Tm values for the control plasmids, the infected monkey blood, the infected human red blood cell cultures, and the eight patient samples, broad, nonoverlapping Tm ranges were selected to identify the Plasmodium species in the blinded patient specimens. The Tm values chosen were as follows: P. malariae, 73.5 to 75.5°C; P. falciparum, 75.5 to 77.5°C; P. ovale, 77.5 to 79.0°C; and P. vivax, 79.0 to 81.0°C. DNA prepared by using the Puregene procedure had Tm values universally lower by ca. 1°C than DNA prepared from identical specimens using the standard ISOCODE extraction protocol but was still at the lowest end of the broad ranges described above. These lower Tm values are most likely due to the salt content in the Puregene DNA hydration buffer compared to the nuclease-free water of the ISOCODE card protocol.
Parasites in 74 of 76 (97.4%) blinded patient samples with a positive microscopic diagnosis of malaria were detected by real-time PCR (Fig. 4), including three specimens containing mixed P. falciparum-P. vivax infections. Two samples that were diagnosed by microscopy as only P. falciparum infections were identified as mixed P. falciparum-P. vivax infections by real-time PCR (Fig. 5). A single patient infected with P. ovale was identified by both microscopic analysis and real-time PCR (microscopic identification of P. ovale confirmed by the Illinois Department of Public Health). Two specimens microscopically diagnosed as containing P. falciparum with parasitemia levels of ca. 0.01% (data not shown) did not produce an amplification curve within the 35 cycle cutoff limit used for the present study, even when the amount of template DNA used in each reaction was increased from 2 to 5 μl. Thus, the detection sensitivity for patient samples was estimated to be in the range of 0.01 to 0.02% parasitemia. Using the normal range of 4.7 to 6.1 million red blood cells per ml of blood, 0.01 to 0.02% parasitemia would be equivalent to 470 to 1,220 parasites per ml, or approximately 1 parasite per μl. This sensitivity corresponds favorably to other published methods, specifically real-time PCR assays.
Four samples were determined by initial microscopy to be two P. malariae- and two P. falciparum-infected specimens; all four were identified as P. vivax by the real-time PCR assay, which is in agreement with the final consensus identification for these four samples. Eighty-two specimens from patients with clinical findings suggesting malaria but microscopically negative for Plasmodium and subsequently determined not to be infected were PCR negative. An additional 200 blood samples from patients collected for diagnoses unrelated to malaria (data not shown) were also determined to be negative by real-time PCR. Using results from the second microscopic analysis as the gold standard, the sensitivity, specificity, positive and negative predictive values, and accuracy of the real-time PCR assay for each detected Plasmodium spp. are summarized in Table 3.
DISCUSSION
Although there are many published studies showing the improved sensitivity and specificity of PCR-based assays over microscopic or immunochromatographic diagnosis of malaria (2, 4, 12, 14, 22, 23, 25, 27), only a handful take advantage of the even more sensitive and time-efficient real-time PCR technologies. In several studies in which real-time PCR was utilized, either a single-species was identified (13) or no distinction between the four human parasites was made (8, 18). This distinction is critical in the clinical management of patients, since the course of treatment varies depending on which species is the cause of the infection. Only in the study by de Monbrison et al. were the four human parasites identified by real-time PCR (7). This assay included five separate primer pairs amplified simultaneously in a LightCycler instrument, utilizing melting curve analysis to distinguish the Plasmodium spp. In the de Monbrison study, twenty-nine single infections and four dual infections were identified by real-time PCR. Twelve specimens were negative by both microscopy and real-time PCR.
The single reaction real-time PCR assay described in our study used only a single pair of primers to detect and identify Plasmodium spp. in 3 h, including standard DNA sample preparation, amplification, and detection, with sensitivities equivalent to microscopy. An additional 1 to 2 h was needed when the more extensive Puregene DNA preparation was required. As with several other studies, we chose to target a species-specific region of the Plasmodium 18S (small subunit) rRNA gene in our real-time PCR assay. By targeting a gene with multiple copies dispersed throughout the Plasmodium genome (11), the sensitivity of this assay is greater than if a single-copy gene was chosen. We also chose to target the partial 18S rRNA gene sequence included in the cloned plasmids available from the ATCC to permit easy access to individual species-specific controls. Although several fluorescence resonance energy transfer hybridization probes and different primer sets were tested in the development of this assay (data not shown), the described SYBR Green assay provided the best discrimination among the different species. The sequences bracketed by the primers are sufficiently divergent in the four species to provide at least a 1°C difference between the average Tm values to distinguish between the species during the melting curve analysis. Single base differences theoretically may also exist between strains within a species from different geographic regions, but our results indicate that the variances among individual strains or patient specimens prepared by the same DNA purification procedure are minor in comparison to the differences among species. Additional specimens from other regions around the world would be required to confirm these assumptions.
We did not address the analysis of blood samples collected by various methods in the present study. To obtain a diverse group of patient specimens, blinded samples from ENH and NUH were collected on ISOCODE cards and processed in the Molecular Diagnostics laboratory at ENH. We overcame potential problems posed by blood samples that were clotted or thickened due to long-term storage at 4°C before application to cards, thus failing to percolate into the filter paper adequately, by processing the "clumps" using the Puregene procedure for tissue samples. Samples with significant red blood cell lysis as a result of freezing were found to be unsuitable for analysis (data not shown). In the present study, DNA was isolated from ca. 10 μl of blood from each ISOCODE card specimen, and 1/50 to 1/20 of this DNA was used in each real-time PCR.
We analyzed 76 blood specimens from patients with clinical findings suggesting malaria for which a microscopic determination of Plasmodium infection had been identified. We, like others, had some difficulty in arriving at a consensus for the microscopic diagnosis. Our final microscopic diagnosis used for comparison was based on review by a panel of experienced microscopists and is reported in Table 1. Real-time PCR analysis identified P. falciparum, P. vivax, or P. ovale in 74 (97.4%) of these specimens, including 3 that contained a dual infection with P. falciparum and P. vivax, for which only 1 had been microscopically identified as such. Since it is often difficult to diagnose mixed infections by microscopy, the distinct dual peaks observed in Fig. 5 indicate that the real-time PCR assay may be more accurate than the gold standard of microscopy in these two cases. To date, only a single patient with a P. ovale infection had been identified by microscopy and real-time PCR. Of the two specimens microscopically diagnosed as containing P. falciparum that did not produce a positive result for any Plasmodium infection in the real-time PCR assay, the amount of parasitemia in these samples was ca. 0.01%, at the lower threshold of sensitivity for this assay. In both cases, the blood had been stored for several days before aliquots were spotted onto the ISOCODE cards. It is also possible that the sensitivity of the assay was decreased by the delay in specimen preparation, in spite of using the Puregene procedure.
Of the 74 infected specimens detected by both microscopy and real-time PCR, 4 had discrepant species identification based on the initial microscopic analysis. Two specimens with P. malariae and two with P. falciparum, as originally determined by microscopic analysis, were all identified as P. vivax positive by the real-time PCR assay. GenBank sequence comparisons and the results of preliminary testing using ATCC controls would suggest that misidentification of either P. falciparum or P. malariae as P. vivax is unlikely using this sequence-based assay and may indicate an inaccurate microscopic diagnosis. The four discrepant specimens were included in a blinded group reviewed by a panel of experienced microscopists. The results of this second review were in agreement with the real-time PCR results, indicating the real-time PCR assay may provide accurate identification of Plasmodium spp. when the microscopic analysis is uncertain.
Recently, P. knowlesi, a species that is morphologically similar to P. malariae has been identified as a human pathogen in patients from Malaysia (32). Sequence analysis indicates that this species should also be amplified by using the conserved primers described in this assay. Whether the differences in sequence composition between the primers are sufficient to result in a Tm closer to that of P. vivax than to P. malariae needs to be determined by testing confirmed P. knowlesi specimens.
A drawback common to all DNA-based malaria assays is the real possibility of detecting persistent DNA in the bloodstream after the infection has subsided. Several studies reported the posttreatment detection of Plasmodium DNA up to 4 days after microscopic detection (4, 25), and another purports that detection at 5 to 8 days after initiation of treatment indicates therapeutic failure, possibly due to parasite resistance (17). However, this persistence of Plasmodium DNA is still less than the persistent antigenemia reported with immunochromatographic assays (6, 22). Also, DNA-based molecular assays have demonstrated a possible chronic detection in regions of the world where malaria is endemic, which may be due to very low parasitemias in immune individuals (3, 19, 34).
In the present study, we developed and validated a real-time PCR assay that can accurately detect and identify Plasmodium spp. in a single reaction by using a simple method for sample collection consisting of blood spotted on treated filter paper (Table 3). The entire process, including specimen preparation using the standard ISOCODE card procedure, PCR amplification, and melting curve identification of Plasmodium spp., was completed in less than 3 h. This real-time PCR assay for the detection and identification of Plasmodium spp. can be used to confirm microscopic findings and in many settings can be used for the primary identification of an infected patient without the need for multiple blood specimens or even microscopic analysis.
ACKNOWLEDGMENTS
We acknowledge the assistance of Kevin S. W. Tan, Department of Microbiology, National University of Singapore; May Ann Lee, Defense Ministry Research Institute, Singapore; and Thiha Nyunt, Yangon, Myanmar, in obtaining the patient blood specimens used in this study. We appreciate the time-consuming microscopic analysis performed by Linda Kuksuk of Northwestern Memorial Hospital, Chicago, Ill. We also acknowledge the technical assistance of Wooi Loon Ng and Mui Joo Khoo of National University Hospital, Singapore.
This study was funded by the Department of Pathology and Laboratory Medicine at Evanston Northwestern Healthcare, Evanston, Ill.
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