Real-time PCR versus conventional PCR for malaria parasite detection in low-grade parasitemia

Experimental Parasitology 116 (2007) 427–432 www.elsevier.com/locate/yexpr

Real-time PCR versus conventional PCR for malaria parasite detection in low-grade parasitemia Bianca E. Gama a, Felipe do E.S. Silva-Pires a, Mauro N’Kruman R. Lopes a, Maria Ange´lica B. Cardoso b, Constanc¸a Britto b, Ka´tia L. Torres c, Leila de Mendonc¸a Lima d, Jose´ Maria de Souza e, Cla´udio T. Daniel-Ribeiro a, Maria de Fa´tima Ferreira-da-Cruz a,* a

e

Laboratory of Malaria Research, Department of Immunology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro (RJ), Brazil b Laboratory of Molecular Biology and Endemic Diseases, Department of Biochemistry and Molecular Biology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro (RJ), Brazil c Foundation of Hematology and Hemotherapy of the Amazonas State, Manaus (AM), Brazil d Laboratory of Molecular Biochemistry and Infectious Diseases Diagnosis, Department of Biochemistry and Molecular Biology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro (RJ), Brazil Ambulatory and Laboratory of Malaria Clinical Assays, Secretariat of Health Vigilance, Evandro Chagas Institute, Bele´m (PA), Brazil Received 10 October 2006; received in revised form 22 February 2007; accepted 23 February 2007 Available online 2 March 2007

Abstract We have optimized a faster and cheaper real-time PCR and developed a conventional genus specific PCR based on 18S rRNA gene to detect malaria parasites in low-grade parasitemias. Additionally, we compared these PCRs to the OptiMAL-IT test. Since there is no consensus on choice of standard quantitative curve in real-time assays, we decided to investigate the performance of parasite DNA from three different sources: ‘‘genome’’, amplicon and plasmid. The amplicon curve showed the best efficiency in quantifying parasites. Both PCR assays detected 100% of the clinical samples tested; the sensitivity threshold was 0.5 parasite/ll and no PCR positive reaction occurred when malaria parasites were not present. Conversely, if OptiMAL-IT were employed for malaria diagnosis, 30% of false-negative results could be expected. We conclude that PCR assays have potential for detecting malaria parasites in asymptomatic infections, in evaluation of malaria vaccine molecule candidates, for screening blood donors, especially in endemic areas, or even in monitoring malaria therapy.  2007 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: Malaria; Diagnosis; Real-time PCR, real-time polymerase chain reaction; PCR, polymerase chain reaction; DNA, deoxyribonucleic acid; dNTPs, deoxyribonucleotide triphosphates; EDTA, ethylenediaminetetraacetic acid; CT, threshold cycle; R2, determination coefficient; rRNA, ribosomal ribonucleic acid; FAM, 6-carboxy-fluoroscein; TAMRA, N,N,N,N-tetramethyl-6-carboxy-rhodamine; bp, base pair number; nM, nanomolar; TBS, thick blood smears; RBC, red blood cells; WBC, white blood cells

1. Introduction Malaria is still highly prevalent in tropical and subtropical regions, annually affecting 350–500 million people and causing around 1 million deaths (World Health Organiza*

Corresponding author. Fax: +55 21 3865 8145. E-mail address: mffcruz@ioc.fiocruz.br (M. de Fa´tima Ferreira-daCruz). 0014-4894/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2007.02.011

tion, 2005). Individuals from continuous transmission areas may, after several malaria infections, develop the premunition state, characterized by an immune response able to control the parasitemia but unable to purge all the circulating parasites. As a result, individuals can stay asymptomatic and act as a parasite reservoir since their blood is able to infect mosquito vectors and they may reintroduce malaria into certain regions (Vinetz & Gilman, 2002; Alves et al., 2005).

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Although the microscopic examination of Giemsastained TBS is referred as the gold standard method for malaria diagnosis, it is well known that this technique is not the choice for cases with low level parasitemia and for mixed infections (Speers et al., 2003; Torres et al., 2006). In Brazil, in meso- to hypo-endemic areas, asymptomatic infections had been also identified (Coura et al., 2006; Alves et al., 2002). Unlike hyperendemic regions, in these endemic areas asymptomatic infections are not usually followed by parasites detectable by microscopic examination. This could have a considerable impact on malaria control (Alves et al., 2005) and screening of blood donors (Torres et al., 2006). Considering the potential use of quantitative real-time PCR to follow-up patients for malaria recrudescence, we standardized a cheaper and faster real-time, genus specific, PCR based on the 18S rRNA gene (Lee et al., 2002) and for comparative sensitivity we develop a conventional PCR using the same set of primers. Additionally we compared both PCR based assays with microscopic examination and with the OptiMAL-IT dipstick test. 2. Materials and methods 2.1. Blood samples Blood samples (5 ml) from 33 patients with positive Giemsa-stained TBS were collected by venepuncture using EDTA vacutainer tubes (Becton Dickinson). These blood samples were from 15 Plasmodium falciparum and 18 P. vivax malaria cases from Paragominas city (Para´ state, Brazil). The samples were spun (350g, 10 min) and the pellets containing packed red blood cells (RBC) and white blood cells (WBC) were frozen with an equal volume of cryopreservation solution (0.9% NaCl, 4.2% sorbitol and 28% glycerol) and transported in liquid nitrogen container to the Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro. Blood samples (31) from clinically healthy individuals living in malaria endemic areas as well from HIV type 1 and HBV positive individuals were also collected, centrifuged and stored as described above. 2.2. Microscopic examination The diagnosis of malaria infection was performed by microscopic examination of 500 fields of Giemsa-stained thick and thin blood smears. Parasitemia was quantified by examination of at least 200 leukocytes in TBS. 2.3. DNA extraction methodology and previous PCR tests All parasite DNA blood samples (500 ll of packed RBC and WBC) were extracted by classical modified phenol– chloroform methodology to a 50 ll final volume as already described (Sallenave-Sales et al., 2003). The DNA parasite samples of malaria patients were first tested for P. vivax (Torres et al., 2006) and P. falciparum (Zalis et al., 1996)

as already standardized in our laboratory, to evaluate the presence of any polymerase inhibitor or damaged DNA. 2.4. Real-time PCR standardization procedures Primer sequences of M60 and M61 as well as the realtime probe M62 (labeled with FAM and TAMRA) have already been described (Lee et al., 2002), but we made modifications to minimize costs using low primer and DNA concentrations and reduce turnaround reaction times. Briefly, 2.5 ll of DNA were added to a 47.5 ll mixture containing the 1· TaqMan Universal PCR Master Mix (Applied Biosystems), 500 nM of each primer M60 (5 0 ACA TGG CTA TGA CGG GTA ACG3 0 ) and M61 (5 0 TGC CTT CCT TAG ATG TGG TAG CTA3 0 ), and 300 nM of M62 probe (5 0 FAM-TCA GGC TCC CTC TCC GGA ATC GA-TAMRA3 0 ). The thermal cycler was settled with two holds (50 C/2 min and 95 C/ 10 min) followed by 32 cycles of amplification (94 C/ 30 s, 60 C/30 s and 72 C/30 s). The real-time PCR was run at least in duplicate on the ABI PRISM Sequence Detection System 7000 (Applied Biosystems).The results were automatically analyzed by the real-time software (ABI Prism 7000 software v. 1.1 RQ study). 2.5. Determination of parasite DNA standard curve for quantification procedures Since there is not a consensus on the choice of standard quantitative curve in real-time assays, we also investigated the performance of parasite DNA from three different sources after two duplicate experiments using as references: (i) plasmid containing the target sequence, (ii) purified amplicon or (iii) genomic parasite DNA obtained from FCR3 P. falciparum strain ring stage parasites diluted with uninfected blood. The genomic P. falciparum DNA curve was performed using the same serial dilutions of FCR3 P. falciparum strain ring stage parasites that were utilized for sensitivity threshold determination. The concentration of plasmid and amplicon DNAs was determined by ultra violet spectrophotometry to determine DNA copy number, considering that a single copy of the PCR product has 9.2 · 108 pg of DNA (Applied Biosystems, 2003) in order to set up the 6-curve dilution points. As a result, the same DNA copy numbers, ranging from 0.25 · 1010 to 0.25 · 105 DNA copies, for both plasmid and amplicon DNAs were utilized in two separate experiments to build up the respective curves. To evaluate whether the DNA quantification estimated through plotted DNA amplified clinical blood samples was associated to its parasitemia estimated by TBS, we analyzed six P. falciparum ring-synchronized culture samples with a parasitemia ranging from 5 · 104 to 5 · 101 parasite/ll. For this purpose, we considered five copies of the gene by parasite based on the P. falciparum 3D7 genome sequence (Gardner et al., 2002). Thus, divid-

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ing DNA copy number by 5, the number of parasites could be estimated.

reproducibility of the threshold cycle (CT) in each parasitemia dilution were considered.

2.6. Conventional PCR standardization procedures

2.11. PCR specificity evaluation

This PCR was standardized using the same set of primers utilized in real-time PCR. The reactions were run with GeneAmp PCR System 9700 (Applied Biosystems) using 4 ll of DNA into a 46 ll mixture containing the 1· GeneAmp PCR Buffer II (Applied Biosystems), 2 mM of MgCl2, 1.25 U Amplitaq Gold (Applied Biosystems), 200 lM of each dNTP and 200 nM of each primers M60 and M61. The thermal cycler was set with an initial hold (95 C/10 min) followed by 32 cycles (94 C/30 s, 60 C/ 30 s and 72 C/30 s). The 84 bp amplicon was visualized by 2% ethidium bromide-stained agarose-gel electrophoresis.

The specificity of both PCR based assays was performed using HBV, HIV type 1 and clinically healthy human DNA templates.

2.7. PCR product cloning The small size of the amplified fragment required purification and cloning into a plasmid vector through the Wizard SV Gel and PCR Clean-Up System (Promega) and the Zero Blunt TOPO PCR Cloning Kit (Invitrogen), before DNA sequencing. These procedures were performed according to the manufacturer’s instructions. The electroporated and transformed One Shot TOP10 Escherichia coli cells (Invitrogen) were harvested for DNA extraction using the Wizard Plus SV Minipreps DNA Purification System (Promega). 2.8. Automatic sequencing The sequencing reaction was made according to the Big Dye Terminator Cycle Sequencing Ready Reaction version 3.1 (Applied Biosystems) instructions in the automatic capillary sequencing platform ABI PRISM DNA Analyzer 3730 (Applied Biosystems). 2.9. OptiMAL-IT tests This dipstick malaria rapid test was performed according to the manufacturer instructions (DiaMed). 2.10. Determination of PCR sensitivity thresholds The thresholds of both PCRs techniques were determined using serial dilutions of FCR3 P. falciparum strain ring stage parasites. These samples were generated from a 10-fold serial dilution into O Rh+ uninfected blood from 1% (50,000 parasites/ll) to 1 · 108% (0.0005 parasite/ll). For this purpose the parasites were cultured (Trager & Jensen, 1976), and highly synchronized as ring stage forms (Lambros & Vanderberg, 1979). DNA extraction was performed by the phenol–chloroform method. For the establishment of threshold in real-time PCR, variations and

2.12. Statistical analysis The variation values of CTs pairs obtained for each sample were assessed by Wilcoxon’s test. 3. Results 3.1. Sensitivity of real-time and conventional PCRs Both procedures were able to detect 100% of the 33 samples corresponding to 18 P. vivax and 15 P. falciparum samples. 3.2. DNA parasite sequences The sequence obtained from conventional PCR product matched exactly with the nucleotides chain presumed by an alignment software (MegAlign version 4.0, DNAStar Inc.) using the primer sequences and gene access numbers for each Plasmodium species (P. falciparum M19172, P. vivax X139172, P. malariae M54897, P. ovale L48987). The sequence obtained was 5 0 ACA TGG CTA TGA CGG GTA ACG GGG AAT TAG AGT TCG ATT CCG GAG AGG GAG CCT GAG AAA TAG CTA CCA CAT CTA AGG AAG GCA3 0 . 3.3. Determination of DNA quantification standard curve The genomic DNA and plasmid curves displayed very low efficient slopes and determination coefficients (R2) and consequently the number of parasites estimated by these curves had no association with microscopic counting independently of the parasite species analyzed (p > 0.05). Conversely, the purified amplicon curve generated slopes close to 100% of amplification efficiency and high determination coefficients (Fig. 1) that were maintained throughout 16 assays, with a low CT coefficient of variation and, consequently, was considered the best curve. When it was compared the parasite number detected by real-time PCR with TBS counts of 6 P. falciparum ring-synchronized samples with a parasitemia ranging from 5 · 104 to 5 · 101 parasite/ll, a positive correlation was found (p < 0.05). 3.4. Intra and inter-assay CT variation analysis in real-time PCR For this analysis, arithmetic mean, standard deviation and coefficient of variation to all parasite DNA duplicate

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sites were present in the reaction; it means that no amplicon was detected when DNA samples from healthy individuals or from individuals infected with HIV type 1 or HBV were tested in conventional or real-time PCRs. 3.7. Comparison of PCR based assays with OptiMAL-IT

Fig. 1. An example of a purified amplicon DNA quantitative standard curve: x-axis corresponds to the log of DNA initial quantity and the y-axis the CT (threshold cycle). This curve displayed an amplification efficiency of 100% and a determination coefficient (R2) of 0.99.

samples—CT pairs—were determined. Among low parasitized blood samples, only one generated a CT coefficient of variation above 0.2. No statistical differences between the CT pairs from each DNA sample were revealed (p > 0.05) and all CTs values generated from each DNA parasite sample in different experiments were positively associated (p < 0.05). 3.5. PCRs sensitivity threshold These thresholds were assessed using serial dilutions of ring-stage synchronous in vitro culture with known numbers of P. falciparum FCR3 strain parasites (Fig. 2). A positive PCR assay could be demonstrated at 0.00001% parasitemia (0.5 parasite/ll) for both 18S PCRs. 3.6. PCRs specificity Independently of PCR format, a PCR product amplified from the 18S gene was only obtained when malaria para-

Fig. 2. PCR sensitivity threshold. Ethidium-bromide stained 2% agarose gel from conventional PCR using P. falciparum FCR3 strain parasites. Samples were generated from a 10-fold serial dilution (Lanes 2–10: 5 · 104 parasites/ll to 5 · 104 parasite/ll) of highly synchronous ring stages; Lane 1: 50 bp DNA ladder.

In contrast to PCRs that presented 100% sensitivity when compared to microscopic examination, OptiMALIT was only able to diagnose malaria cases in 70% (20/ 28) of the malaria blood samples tested; the remaining 30% malaria cases with negative OptiMAL-IT diagnosis had parasitemias below to 37 parasites/ll. 4. Discussion The standardized 18S malaria genus conventional and real-time PCRs presented the same sensitivity (0.5 parasite/ll) and specificity including no DNA amplification of samples from the two most frequently encountered blood-borne viral diseases (Yee & Lee, 2005). The threshold sensitivity was 20 times more sensitive than TBS examination by experienced microscopists (10–50 parasites/ll; Berry et al., 2005) and 100 times more sensitive than OptiMAL-IT parasite detection (50–100 parasites/ll, according to the manufacturer). Except the original conventional nested PCR method for malaria parasite detection (Snounou et al., 1993) which had a threshold similar to that here reported (1 parasite/ ll), our results with both standardized PCR based assays were better than other methods using related 18S oligonucleotide primers, in conventional nested PCR assays (Kimura et al., 1997; Tham et al., 1999; Schindler et al., 2001) or single PCR followed by a membrane hybridization step (Ciceron et al., 1999), as well as in real-time PCRs (Monbrison et al., 2003; Perandin et al., 2004; Mangold et al., 2005). Conversely, two PCR conventional studies using single (Ciceron et al., 1999) or semi-nested formats (Schindler et al., 2001) reported a higher sensitivity threshold detection value than we found. However, since in these studies the sensitivity threshold value was calculated utilizing non-synchronized P. falciparum cultures which include schizont with up to 32 merozoites, the sensitivity value reported by these authors may be overestimated. Moreover, it is not possible to determine whether the reported differences represent intrinsic variability in assay sensitivities or are a result of calibration using different reference reagents that are poorly characterized and standardized. Although it has been reported that in real-time PCR assays increasing cycle numbers are related to an augment of variation at the threshold cycle—CT—(Klein, 2002), in our study this happened only with one clinical blood sample (75 parasites/ll) and with a purified amplicon sample containing 0.25 · 105 copies or 0.0025 pg of DNA. As a result, we concluded that low parasitemia values were not necessarily associated with an increased CT coefficient of variation.

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The choice of a standard DNA quantification curve was not obvious due to possible variation of rRNA gene copy number among parasite populations. For example, in P. vivax infections, clinical samples with similar TBS parasitemia but with different proportions of schizonts (14–21 genomes) will vary in copy number and consequently in realtime PCR parasite quantification. In view of this we investigated the performance of 3 different DNA parasite sources for defining the most appropriate one to be used for construction the standard DNA curve because this datum is lacking in the scientific literature. In this sense, the comparison of parasitemia determined by TBS with that assessed by genomic standard DNA quantification curve showed significantly divergence results among malaria samples. Conversely, in the amplicon standard DNA curve a positive correlation between P. falciparum TBS parasitemia values and PCR parasite determination was observed. As expected, in real-time PCR assays, the parasitemia levels were consistently higher than TBS count numbers, strongly suggesting that real-time PCR quantification is at least 10 times more sensitive than microscopic examination (TBS). Undoubtedly the dipstick tests, represented here by OptiMAL-IT, are faster and easier to perform but although these are comparable in cost to the conventional home made PCR, in our study it works poorly in blood samples with a parasitemia below 37 parasites/ll as assessed by microscopic examination. In this scenario, if rapid malaria tests were exclusively employed for malaria diagnosis, 30% of false-negatives could be expected. We successfully optimized the real-time PCR previously described by Lee et al. (2002). The modifications includes the use of less primer (M60) and DNA concentrations (500 nM of M60 instead of 900 nM and 2.5 ll of DNA instead of 5 ll for real-time PCR) as well as reduction of cycle number (32 cycles opposite to 45 cycles) rendering our method cheaper and faster whilst maintaining the same sensitivity e.g. 0.002 pg DNA which, in our experiments, corresponds to the positive end point of the purified amplicon standard titration curve (data not shown). In order to compare the performance of this optimized real-time PCR with the conventional one we needed to standardize it because no conventional PCR using these same set of primers has been previously reported. In this light both 18S Plasmodium PCR based assays presented the same specificity and sensitivity, and successfully detected submicroscopic parasitemias. These PCR based assays could be considered valuable tools where identification of low parasitemia malaria cases is required. For instance, these assays have potential use for detecting malaria parasites in asymptomatic infections, in the evaluation of malaria vaccine candidates, in the screening of blood donors, especially in endemic areas or even for monitoring malaria treatment. Finally, considering that both 18S PCR based assays had shown the same sensitivity and specificity, we are tempted to conclude that conventional PCR may be used instead of real-

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