Molecular Testing for Plasmodium falciparum by Use of Serum or Plasma and Comparison with Microscopy and Rapid Diagnostic Testing in Febrile Nigerian Patients

Abstract

Plasmodium nucleic acids have been detected in serum and plasma, but there is little published data describing the diagnostic performance of malaria nucleic acid amplification tests (NAATs) using these specimen types. Previously, our group described a multiplex NAAT for the detection of dengue virus, Leptospira, and Plasmodium species with a callout for P. falciparum (the DLM assay) that demonstrated sensitive detection of P. falciparum from plasma samples during initial evaluation. In this study, we evaluated the sensitivity and specificity of P. falciparum detection in febrile Nigerian patients using the DLM assay, microscopy, and a rapid diagnostic test (BinaxNOW Malaria). Assay performances were compared using a composite reference, which was considered positive if malaria was detected by two or more methods. Serum (n = 182) or plasma (n = 148) from 317 patients was tested; the average sample volume was 70 μl (range, 5 to 300 μl). The sensitivity and specificity of the DLM assay were 97.1% and 93.5%, respectively. The sensitivity of the malaria rapid diagnostic test (98.1%) was similar to that of the DLM assay, and both proved significantly more sensitive than microscopy (79%; P < 0.0001). When analysis was limited to samples with ≥75 μl of serum or plasma, the sensitivity of the DLM assay improved to 99% and specificity was 97.5%. For P. falciparum cases, cycle threshold values in the DLM assay correlated with the parasite density detected by microscopy (Spearman's rank correlation coefficient, P < 0.0001). In conclusion, malaria detection using the DLM assay on serum or plasma is more sensitive than and equal in specificity to microscopy in patients with P. falciparum malaria.

INTRODUCTION

The entirety of Nigeria is considered an area of moderate to high malaria transmission by the WHO (≥1 case per 1,000 residents), and virtually all cases result from infection with Plasmodium falciparum, the most common cause of severe malaria (1). Despite the high prevalence of malaria in Nigeria, there is concern that it is overdiagnosed in patients presenting with an undifferentiated systemic febrile illness (2). While traditional microscopy remains the primary malaria diagnostic method worldwide, malaria rapid diagnostic tests (mRDTs) are also being deployed for confirmation of suspected malaria cases. However, nucleic acid amplification tests (NAATs) for malaria, which have proven more sensitive than microscopy or mRDTs, are not routinely performed (3, 4). Therefore, it is possible that estimates of overtreatment may be inflated due to the relatively poor sensitivity of microscopy for malaria diagnosis. Although Plasmodium nucleic acids have been detected in serum and plasma, there is little published data describing the performance of malaria NAATs using these specimen types (4–7). Rather, most studies have used whole blood or dried blood spots (DBS) (8–12). While whole blood and DBS provide ample amounts of Plasmodium nucleic acids, they present significant challenges for (i) long-term storage and/or (ii) nucleic acid extraction with removal of inhibitors for optimal NAAT performance (13, 14).

Recently, our group described the development and evaluation of a single-reaction, internally controlled multiplex NAAT for the detection of dengue virus (DENV), Leptospira, and the five Plasmodium species known to cause human disease, with a specific callout for P. falciparum (referred to here as the DLM assay; previously referred to as the undifferentiated febrile illness [UFI] assay) (15). During initial evaluation of this assay, P. falciparum DNA was detected in paired whole-blood and plasma samples down to a parasitemia level of 0.0037% (estimated parasite density of 185 parasites/μl) (15). The purpose of the current study was to determine the sensitivity and specificity of P. falciparum detection in the DLM assay using serum or plasma and compare the values obtained with those determined by microscopy and the use of the BinaxNOW Malaria RDT in clinically ill Nigerian patients.

MATERIALS AND METHODS

Ethics statement.

Adults and guardians of children aged <18 years provided written, informed consent. Children aged 12 to 18 also provided assent. The research protocol for the current study was reviewed and approved by the Stanford University Institutional Review Board. Protocols for each cohort study from which samples were obtained were reviewed and approved by the Research Grants and Experimentation Ethical Committee of the University of Lagos, College of Medicine, and the Ministry of Health of the Lagos state government.

Study population and sample collection.

Precollected and deidentified samples collected as part of two cohort studies based at the University of Lagos were used for the current study. The cohort studies were designed to (i) determine the clinical performance of a urine-based mRDT and (ii) evaluate wild-type P. falciparum isolates from patients in Nigeria. In both studies, patients who presented with an acute febrile illness were enrolled. Whole blood was obtained for microscopy and testing using the BinaxNOW Malaria RDT (Alere). For the current study, samples were selected from similar numbers of patients who tested positive for malaria by microscopy and/or mRDT and those who tested negative. Aliquots of serum or EDTA-plasma were prepared in Nigeria from samples from all enrolled patients. Whole blood collected for serum or EDTA-plasma testing was stored under refrigerated (8°C) conditions and transported in cool boxes (maintained at 8 to 12°C) to a central laboratory and was again refrigerated before separation. Serum was obtained by allowing whole blood, collected in red-top tubes, to clot at room temperature (20 to 25°C). For the separation of plasma, whole blood was centrifuged at 1,500 × g for 20 min. Aliquots of serum and plasma were stored at −20°C until use.

Blood smear and rapid diagnostic testing.

Giemsa-stained thick and thin smears were prepared from whole blood at the time of patient enrollment. Thick smears were used for malaria detection. A thick smear was considered negative if no parasites were observed after review of 200 high-powered fields. Thin smears were used to confirm positive test results for thick smears and for species determination. Parasite density (determined as the number of parasites per microliter of blood) was calculated in relation to the number of white blood cells observed on the thick smear. A minimum of 200 white blood cells were counted on each smear to calculate this value.

The BinaxNOW Malaria RDT (referred to here as the mRDT) provides detection of two antigens: the histidine-rich protein II antigen specific to P. falciparum (PfHRP-2) and a pan-malarial antigen. This test was performed and interpreted according to the manufacturer's recommendations.

Nucleic acid extraction and molecular testing.

Total nucleic acids were extracted at Stanford University from serum and plasma using a protocol for cell-free fluids on an easyMAG instrument (bioMérieux) according to manufacturer recommendations. All available serum or plasma was used in the extraction (5 to 300 μl), and the elution volume was 60 μl. Samples were tested using the DLM assay, which was performed as previously described (15). This multiplex assay contains primers and probes for two possible malaria targets: the Pfr364 repetitive element, which is specific to P. falciparum, and a region of the 18S rRNA gene that is conserved across the five Plasmodium species known to cause human disease. Given that more copies of the Pfr364 element are present in the P. falciparum genome than in the 18S rRNA target, samples with detectable signal for Pfr364 were interpreted as positive for P. falciparum, even in the absence of signal for the 18S target (16). Samples with detectable signal only for the 18S target were considered positive for “Plasmodium, non-falciparum.” DENV serotyping was performed using the DENV multiplex assay, as previously described (17).

Statistics.

Sensitivity and specificity were calculated for each diagnostic test and compared to those of a composite reference. A patient was considered positive for malaria by the composite reference if the patient tested positive by two or more methods (microscopy, mRDT, and/or the DLM assay). A sample positive by only one method was considered negative by the composite reference. Basic statistics were determined using Excel software (Microsoft). The sensitivity and specificity values determined for the different methods were compared using Fisher's exact tests. Continuous variables, including cycle threshold (C_T) values, were compared using t tests. Fisher's exact tests, t tests, and Spearman's correlation coefficient calculations were performed using GraphPad software (GraphPad).

RESULTS

Samples were collected at study sites in Lagos between January 2013 and February 2014. A total of 330 serum (n = 182) and plasma (n = 148) samples from 317 patients were tested. Thirteen patients had duplicate specimens. A total of 163 patients were female, 131 were male, and 23 did not have gender information recorded. The average age of the members of the population was 16.9 years (standard deviation, 13.15). The mean specimen volume was 70 μl (range, 5 to 300 μl). All samples had a positive signal for the internal control in the DLM assay, indicating successful nucleic acid extraction and the absence of PCR inhibitors. A single patient had DENV detected from both serum and plasma (DENV serotype 1), but no other arboviral infections were detected. No patient had a documented Leptospira monoinfection. Two patients had evidence of mixed Leptospira-P. falciparum coinfections, though the Leptospira infections could not be confirmed by other methods.

The results of malaria testing for all patients are shown in Table 1. Among the species detected in the DLM assay, 200/204 (98.0%) were P. falciparum. Patients who tested positive for malaria by the composite reference were younger (14.0 years old; standard deviation, 11.4) than patients who tested negative (21.9 years old; standard deviation, 14.6; P < 0.0001). The gender distributions were similar for malaria-positive cases (100 female, 88 male) and malaria-negative cases (63 female, 43 male; P = 0.33). Compared to the composite reference, the sensitivity and specificity of the DLM assay were 97.1% and 93.5%, respectively. The sensitivities of the DLM assay and mRDT (98.1%) were similar, and both tests proved significantly more sensitive than microscopy (79%; P < 0.0001). The specificity of microscopy was significantly greater than the specificity of mRDT (P ≤ 0.0007). The specificity of the DLM assay did not differ significantly from the specificity of either microscopy or mRDT (P ≤ 0.065 or P ≤ 0.167, respectively). When the performance of the DLM assay was compared to the composite reference data for serum versus plasma samples, there was no significant difference in sensitivity (96.6% for serum versus 97.8% for plasma; P = 0.70) or specificity (93.0% for serum versus 94.0% for plasma; P = 0.68).

TABLE 1.

Malaria test results for 317 patients determined using the DLM assay on plasma or serum, microscopy, and the mRDT compared to a composite reference standard

Assay and result	No. of patients with indicated composite reference assay result			Sensitivity	Specificity
	Positive	Negative	Total
DLM
Positive	204	7	211	97.1	93.5
Negative	6	100	106
Microscopy
Positive	166	1	167	79.0	99.1
Negative	44	106	150
mRDT
Positive	206	14	220	98.1	86.9
Negative	4	93	97
Total	210	107	317

Six samples were positive by microscopy and mRDT but negative by the DLM assay. Parasite densities for these samples ranged from 2,387 to 97,480 parasites/μl. Five samples had <75 μl of volume available for nucleic acid extraction. When the analysis was limited to samples with ≥75 μl, the sensitivity of the DLM assay was 99% and specificity was 97.5% (Table 2). For this subset of samples, the DLM assay remained more sensitive than microscopy (71%; P < 0.0001) and proved more specific than the mRDT (83.5%; P ≤ 0.005). The cycle threshold (C_T) values for the Pfr364 target for samples consisting of ≥75 μl (mean, 24.7; standard deviation, 3.8) did not differ significantly from the C_T values for the entire set of positive samples (mean, 25.0; standard deviation, 3.9; P = 0.55). The single sample missed in the DLM assay had a parasite density of 4,258 parasites/μl and was positive only for PfHRP-2 in the mRDT.

TABLE 2.

Malaria test results for 179 patients who had ≥75 μl of plasma or serum available for extraction

Assay and result	No of patients with indicated composite reference assay result			Sensitivity	Specificity
	Positive	Negative	Total
DLM
Positive	99	2	101	99.0	97.5
Negative	1	77	78
Microscopy
Positive	71	0	71	71.0	100
Negative	29	79	108
mRDT
Positive	97	13	110	97.0	83.5
Negative	3	66	69
Total	100	79	179

When the two antigens in the mRDT were evaluated separately, detection of the pan-malarial antigen was 80% sensitive (168/210) and 97.2% specific (104/107) compared to the composite reference for all cases. For P. falciparum cases, detection of PfHRP-2 proved more sensitive (98.6%; 204/207) but less specific (87.3%; 96/110) than detection of the pan-malarial antigen (P < 0.01 for both comparisons). Fourteen samples were positive only by the mRDT, and all 14 samples (100%) were positive for PfHRP-2. Eleven samples (78.6%) were positive only for PfHRP-2, compared to 38/204 P. falciparum samples (18.6%) positive by the composite reference (P < 0.0001).

For P. falciparum cases that tested positive by both the DLM assay and microscopy, C_T values for the Pfr364 target correlated with the parasite density detected by microscopy (Fig. 1; Spearman's rank correlation coefficient, −0.555; P < 0.0001). Two patients had only gametocytes detected on smears and were removed from this analysis; both patients were also positive by the mRDT. Forty-four cases, including 43 cases of P. falciparum infection and 1 non-falciparum case, that were positive by the composite reference were missed by microscopy. The mean C_T value for P. falciparum cases positive by both the DLM assay and microscopy (23.9; standard deviation, 2.9) was lower than the mean C_T value for cases positive in the DLM assay but negative by microscopy (27.4; standard deviation, 4.0; P < 0.0001). However, there was considerable overlap in the two distributions (Fig. 2).

FIG 1.

DLM assay (Pfr364 target) C_T values for serum or plasma samples positive for P. falciparum versus the parasite density detected by microscopy (displayed as the log₁₀ of the parasite density). The solid black line represents the linear regression line.

FIG 2.

DLM assay (Pfr364 target) C_T values for patients who were positive for P. falciparum by the composite reference and tested positive by both the DLM assay and microscopy (Microscopy Positive) or positive in the DLM assay and negative by microscopy (Microscopy Negative). The solid black lines represent the mean C_T values.

DISCUSSION

In the current study, we compared the performance of the DLM assay with the performances of microscopy and the mRDT for detecting P. falciparum infections among symptomatic Nigerian patients. Although the detection of Plasmodium nucleic acids in serum or plasma was first reported in 2001, only two previous studies had evaluated the sensitivity and specificity of this technique using specimens from symptomatic cases (6, 7). Those reports provided useful, proof-of-concept data, but the sample numbers (a total of 33 samples) were too small to provide meaningful comparisons with standard malaria diagnostics. In 2012, Lamikanra et al. compared real-time PCR using plasma and DBS to microscopy in Mozambican children (4). Significantly more infections were detected by PCR using DBS, whereas PCR performed on plasma appeared no more sensitive than microscopy. The clinical utility of these findings for the diagnosis of acute, symptomatic malaria is not clear, however, as patients were not necessarily ill at the time of sample acquisition. Furthermore, only 50 μl of plasma was used for nucleic acid extraction, which may have limited clinical sensitivity (4).

In contrast, our results indicate that PCR using the DLM assay on serum or plasma is significantly more sensitive than microscopy for the detection of P. falciparum in symptomatic cases, and as one might expect, clinical sensitivity is improved by using higher specimen volumes. For the current study, available specimen volumes were lower than those used during the initial evaluation of the DLM assay (140 μl), and it is possible that a greater number of malaria cases would have been detected if higher volumes had been used (15). Similarly to the study by Lamikanra et al., we did find a significant correlation between C_T values in the DLM assay for patients with P. falciparum malaria and parasite density determined by microscopy (4). Although the clinical utility of quantitative malaria PCR remains unclear, a recent study by Imwong et al. showed that the plasma concentration of P. falciparum DNA correlates with disease severity (18). However, quantitative PCR was performed only on patients who had positive microscopy results in this study.

To our knowledge, the current report describes the first comparison of malaria PCR using serum or plasma to the mRDT. In this patient population with predominantly P. falciparum malaria (98.0% of cases), the mRDT demonstrated sensitivity similar to that of PCR at the possible expense of decreased specificity. The 14 samples that were detected only by the mRDT may have represented false-positive detection, as 78.6% of these samples were positive only for PfHRP-2, and detection of PfHRP-2 has been demonstrated to result in decreased specificity compared to other antigens (19). Alternatively, those patients may have had a recent P. falciparum infection, since PfHRP-2 can remain positive for weeks following treatment (20, 21).

Given the nature of the cohort studies from which samples were obtained, correlations between quantitative PCR results and severity or treatment outcomes could not be evaluated in our patient population. However, the performance of quantitative malaria PCR for both diagnosis and risk stratification warrants evaluation in future studies. Also, P. falciparum caused the overwhelming majority of infections in this population, which is consistent with malaria epidemiology in Nigeria. Our findings cannot necessarily be generalized to cases of non-falciparum malaria and will need to be confirmed for infections with other Plasmodium species.

In conclusion, we have demonstrated that malaria detection using the DLM assay on serum or plasma is more sensitive than and equal in specificity to microscopy in patients with falciparum malaria. Given the potential utility of quantitative PCR analysis of these specimen types for risk stratification, serum or plasma should be further evaluated for the molecular diagnosis of malaria in suspected cases.