Measurement of parasitological data by quantitative real-time PCR from controlled human malaria infection trials at the Walter Reed Army Institute of Research.

BACKGROUND: The use of quantitative real-time PCR (qPCR) has allowed for precise quantification of parasites in the prepatent period and greatly improved the reproducibility and statistical power of controlled human malaria infection (CHMI) trials. Parasitological data presented here are from non-immunized, control-challenged subjects who participated in two CHMI trials conducted at the Walter Reed Army Institute of Research (WRAIR). METHODS: Standardized sporozoite challenge was achieved through the bite of five Anopheles stephensi mosquitoes infected with the 3D7clone of the NF54 strain of Plasmodium falciparum. Blood smears were scored positive when two unambiguous parasites were found. Analysis of parasitological PCR data was performed on log-transformed data using an independent sample t-test when comparing the two studies. The multiplication rate of blood-stage parasites was estimated using the linear model. RESULTS: On average, parasites were detected 4.91 days (95% CI = 4.190 to 5.627) before smears. The earliest parasites were detected within 120 hours (5.01 days) after challenge. Parasite densities showed consistent cyclic patterns of blood-stage parasite growth in all volunteers. The parasite multiplication rates for both studies was 8.18 (95% CI = 6.162 to 10.20). Data showed that at low parasite densities, a combination of sequestration and stochastic effects of low copy number DNA may impact qPCR detection and the parasite detection limit. CONCLUSION: Smear positive is an endpoint which antimalarial rescue is imperative whereas early detection of parasitological data by qPCR can allow for better anticipation of the endpoint. This would allow for early treatment to reduce clinical illness and risk for study participants. To use qPCR as the primary endpoint in CHMI trials, an algorithm of two positives by qPCR where one of the positives must have parasite density of at least 2 parasites/µL is proposed.

Sample-ready multiplex qPCR assay for detection of malaria.

BACKGROUND: Microscopy and antigen detecting rapid diagnostic tests are the diagnostic tests of choice in management of clinical malaria. However, due to their limitations, the need to utilize more sensitive methods such as real-time PCR (qPCR) is evident as more studies are now utilizing molecular methods in detection of malaria. Some of the challenges that continue to limit the widespread utilization of qPCR include lack of assay standardization, assay variability, risk of contamination, and the need for cold-chain. Lyophilization of molecular assays can overcome some of these limitations and potentially enable widespread qPCR utilization. METHODS: A recently published multiplex malaria qPCR assay was lyophilized by freezing drying into Sample-Ready™ format (MMSR). MMSR assay contained all the required reagents for qPCR including primers and probes, requiring only the addition of water and sample to perform qPCR. The performance of the MMSR assay was compared to the non-freeze dried, "wet" assay. Stability studies were done by maintaining the MMSR assays at four different ambient temperatures of 4°C, room temperature (RT), 37°C and 42°C over a period of 42 days, tested at seven-day intervals. Plasmodium falciparum and Plasmodium vivax DNAs were used for analysis of the MMSR assay either as single or mixed parasites, at two different concentrations. The CT values and the standard deviations (SD) were used in the analysis of the assay performance. RESULTS: The limit of detection for the MMSR assay was 0.244 parasites/µL for Plasmodium spp. (PLU) and P. falciparum (FAL) assay targets compared to "wet" assay which was 0.39 and 3.13 parasites/µL for PLU and FAL assay targets, respectively. The MMSR assay performed with high efficiencies similar to those of the "wet" assay and was stable at 37°C for 42 days, with estimated shelf-life of 5 months. When used to analyse field clinical samples, MMSR assay performed with 100% sensitivity and specificity compared to the "wet" assay. CONCLUSION: The MMSR assay has the same robust performance characteristics as the "wet" assay and is highly stable. Availability of MMSR assay allows flexibility and provides an option in choosing assay for malaria diagnostics depending on the application, needs and budget.

Multiplex qPCR for detection and absolute quantification of malaria.

We describe development of an absolute multiplex quantitative real-time PCR for detection of Plasmodium spp., P. falciparum and P. vivax targets in order to produce an assay amenable to high throughput but with reduced costs. Important qPCR experimental details and information that is critical to performance and reliability of assay results were investigated. Inhibition studies were performed to test and compare co-purification of PCR inhibitors in samples extracted from whole blood using either the manual or automated methods. To establish the most optimal qPCR reaction volume, volume titration of the reaction master mix was performed starting at 10 µl to 1 µl reaction master mix with 1 µl of template DNA in each reaction. As the reaction volume decreased, qPCR assays became more efficient with 1 µl reaction master mix being the most efficient. For more accurate quantification of parasites in a sample, we developed plasmid DNAs for all the three assay targets for absolute quantification. All of absolute qPCR assays performed with efficiency of more than 94%, R(2) values greater than 0.99 and the STDEV of each replicate was <0.167. Linear regression plots generated from absolute qPCR assays were used to estimate the corresponding parasite density from relative qPCR in terms of parasite/µl. One copy of plasmid DNA was established to be equivalent to 0.1 parasite/µl for Plasmodium spp. assay, 0.281 parasites for P. falciparum assay and 0.127 parasite/µl for P. vivax assay. This study demonstrates for the first time use of plasmid DNA in absolute quantification of malaria parasite. The use of plasmid DNA standard in quantification of malaria parasite will be critical as efforts are underway to harmonize molecular assays used in diagnosis of malaria.

Comparative evaluation of published real-time PCR assays for the detection of malaria following MIQE guidelines.

BACKGROUND: The use of malaria-specific quantitative real-time PCR (qPCR) is increasing due to its high sensitivity, speciation and quantification of malaria parasites. However, due to the lack of consensus or standardized methods in performing qPCR, it is difficult to evaluate and/or compare the quality of work reported by different authors for a cross-study and/or cross-platform assay analysis. METHODS: The performances of seven published qPCR assays that detect Plasmodium spp or Plasmodium falciparum were compared using standard DNA and samples from a clinical trial. Amplification and qPCR measurements were performed using the Applied Biosystems 7500 Fast Real-Time PCR System. All the analyses were automatically established using the default settings. For the TaqMan probe format, the assays were performed in the background of QuantiFast Probe Master Mix whereas in SYBR Green format, the assays were performed in the background of QuantiFast SYBR Green Master Mix and QuantiTect SYBR Green Master Mix background. RESULTS: Assays with high PCR efficiencies outperformed those with low efficiencies in all categories including sensitivity, precision and consistency regardless of the assay format and background. With the exception of one assay, all assays evaluated showed lower sensitivity compared to what have been published. When samples from a malaria challenge study were analysed, the qPCR assay with the overall best performance detected parasites in subjects earliest and with most consistency. CONCLUSION: The data demonstrate the need for increased consensus and guidelines that will encourage better experimental practices, allowing more consistent and unambiguous interpretation of qPCR results.

Use of qPCR and genomic sequencing to diagnose Plasmodium ovale wallikeri malaria in a returned soldier in the setting of a negative rapid diagnostic assay.

Plasmodium ovale is one of several clinically relevant malaria species known to cause disease in humans. However, in contrast to Plasmodium falciparum and Plasmodium vivax, which are responsible for most cases of human malaria, P. ovale has a wide distribution but low prevalence in tropical regions. Here, we report the case of a soldier returning from Liberia with P. ovale wallikeri malaria. This case highlights the limitations of both microscopy and the malaria rapid diagnostic test for diagnosing infection with P. ovale and for distinguishing P. ovale wallikeri from P. ovale curtisi. To our knowledge, this is the first case report in which quantitative real-time polymerase chain reaction amplification using the Cytochrome B gene, coupled with genomic sequencing of the potra locus, was used for definitive diagnosis of P. ovale wallikeri malaria.

Development of a TaqMan Allelic Discrimination assay for detection of single nucleotides polymorphisms associated with anti-malarial drug resistance.

BACKGROUND: Anti-malarial drug resistance poses a threat to current global efforts towards control and elimination of malaria. Several methods are used in monitoring anti-malarial drug resistance. Molecular markers such as single nucleotide polymorphism (SNP) for example are increasingly being used to identify genetic mutations related to anti-malarial drug resistance. Several methods are currently being used in analysis of SNP associated with anti-malarial drug resistance and although each one of these methods has unique strengths and shortcoming, there is still need to improve and/or develop new methods that will close the gap found in the current methods. METHODS: TaqMan Allelic Discrimination assays for detection of SNPs associated with anti-malarial drug resistance were designed for analysis on Applied Biosystems PCR platform. These assays were designed by submitting SNP sequences associated with anti-malarial drug resistance to Applied Biosystems website. Eleven SNPs associated with resistance to anti-malarial drugs were selected and tested. The performance of each SNP assay was tested by creating plasmid DNAs carrying codons of interests and analysing them for analysis. To test the sensitivity and specificity of each SNP assay, 12 clinical samples were sequenced at codons of interest and used in the analysis. Plasmid DNAs were used to establish the Limit of Detection (LoD) for each assay. RESULTS: Data from genetic profiles of the Plasmodium falciparum laboratory strains and sequence data from 12 clinical samples was used as the reference method with which the performance of the SNP assays were compared to. The sensitivity and specificity of each SNP assay was establish at 100%. LoD for each assay was established at 2 GE, equivalent to less than 1 parasite/µL. SNP assays performed well in detecting mixed infection and analysis of clinical samples. CONCLUSION: TaqMan Allelic Discrimination assay provides a good alternative tool in detection of SNPs associated with anti-malarial drug.

Conformational equilibria of bulged sites in duplex DNA studied by EPR spectroscopy.

Conformational flexibility in nucleic acids provides a basis for complex structures, binding, and signaling. One-base bulges directly neighboring single-base mismatches in nucleic acids can be present in a minimum of two distinct conformations, complicating the examination of the thermodynamics by calorimetry or UV-monitored melting techniques. To provide additional information about such structures, we demonstrate how electron paramagnetic resonance (EPR) active spin-labeled base analogues, base-specifically incorporated into the DNA, are monitors of the superposition of different bulge-mismatch conformations. EPR spectra provide information about the dynamic environments of the probe. This information is cast in terms of "dynamic signatures" that have an underlying basis in structural variations. By examining the changes in the equilibrium of the different states across a range of temperatures, the enthalpy and entropy of the interconversion among possible conformations can be determined. The DNA constructs with a single bulge neighboring a single-base mismatch ("bulge-mismatches") may be approximately modeled as an equilibrium between two possible conformations. This structural information provides insight into the local composition of the bulge-mismatch sequences. Experiments on the bulge-mismatches show that basepairing across the helix can be understood in terms of purine and pyrimidine interactions, rather than specific bases. Measurements of the enthalpy and entropy of formation for the bulge-mismatches by differential scanning calorimetry and UV-monitored melting confirm that the formation of bulge-mismatches is in fact more complicated than a simple two-state process, consistent with the base-specific spectral data that bulge-mismatches exist in multiple conformations in the premelting temperature region. We find that the calculations with the nearest-neighbor (NN) model for the two likely conformations do not correlate well with the populations of structures and thermodynamic parameters inferred from the base-specific EPR dynamics probe. We report that the base-specific spin probes are able to identify a bistable, temperature dependent, switching between conformations for a particular complex bulged construct.

Influence of buffer species on the thermodynamics of short DNA duplex melting: sodium phosphate versus sodium cacodylate.

Thermodynamic parameters of the melting transitions of 53 short duplex DNAs were experimentally evaluated by differential scanning calorimetry melting curve analysis. Solvents for the DNA solutions contained approximately 1 M Na+ and either 10 mM cacodylate or phosphate buffer. Thermodynamic parameters obtained in the two solvent environments were compared and quantitatively assessed. Thermodynamic stabilities (deltaG(o) (25 degrees C)) of the duplexes studied ranged from quite stable perfect match duplexes (approximately -30 kcal/mol) to relatively unstable mismatch duplexes (approximately -9 kcal/mol) and ranged in length from 18 to 22 basepairs. A significant difference in stability (average free energy difference of approximately 3 kcal/mol) was found for all duplexes melted in phosphate (greater stability) versus cacodylate buffers. Measured effects of buffer species appear to be relatively unaffected by duplex length or sequence content. The popular sets of published nearest-neighbor (n-n) stability parameters for Watson-Crick (w/c) and single-base mismatches were evaluated from melting studies performed in cacodylate buffer (SantaLucia and Hicks, Annu. Rev. Biophys. Biomol. Struct. 2004, 33, 415). Thus, when using these parameters to make predictions of sequence dependent stability of DNA oligomers in buffers other than cacodylate (e.g., phosphate) one should be mindful that in addition to sodium ion concentration, the type of buffer species also provides a minor but significant contribution to duplex stability. Such considerations could potentially influence results of sequence dependent analysis using published n-n parameters and impact results of thermodynamic calculations. Such calculations and analyses are typically employed in the design and interpretation of DNA multiplex hybridization experiments.

DNA multiplex hybridization on microarrays and thermodynamic stability in solution: a direct comparison.

Hybridization intensities of 30 distinct short duplex DNAs measured on spotted microarrays, were directly compared with thermodynamic stabilities measured in solution. DNA sequences were designed to promote formation of perfect match, or hybrid duplexes containing tandem mismatches. Thermodynamic parameters DeltaH degrees , DeltaS degrees and DeltaG degrees of melting transitions in solution were evaluated directly using differential scanning calorimetry. Quantitative comparison with results from 63 multiplex microarray hybridization experiments provided a linear relationship for perfect match and most mismatch duplexes. Examination of outliers suggests that both duplex length and relative position of tandem mismatches could be important factors contributing to observed deviations from linearity. A detailed comparison of measured thermodynamic parameters with those calculated using the nearest-neighbor model was performed. Analysis revealed the nearest-neighbor model generally predicts mismatch duplexes to be less stable than experimentally observed. Results also show the relative stability of a tandem mismatch is highly dependent on the identity of the flanking Watson-Crick (w/c) base pairs. Thus, specifying the stability contribution of a tandem mismatch requires consideration of the sequence identity of at least four base pair units (tandem mismatch and flanking w/c base pairs). These observations underscore the need for rigorous evaluation of thermodynamic parameters describing tandem mismatch stability.