Prevalence and density-related concordance of three diagnostic tests for malaria in a region of Tanzania with hypoendemic malaria

Abstract

Accurate malaria diagnosis has dual roles in identification of symptomatic persons for effective malaria treatment and also enumeration of asymptomatic persons who contribute to the epidemiologic determinants of transmission. Three currently used diagnostic tests, microscopy, rapid diagnostic tests (RDTs), and real-time PCR, all have different sensitivities and specificities, which are parasite density dependent. Here, we compare their concordance among 451 febrile episodes in a cohort of 2,058 children and adults followed over 6 months in a region in central Tanzania with hypoendemic malaria. Microscopy, a histidine-rich protein-based RDT, and two different real-time PCR gene probes detected Plasmodium falciparum in 20, 54, 41, and 78 episodes of fever, respectively. They had complete concordance in only 9 episodes. Real-time PCR with an 18S probe was more sensitive than with a mitochondrial probe for cytochrome b despite higher copy numbers of mitochondrial DNA. Both PCR yields were increased 4-fold by glycogen/acetate precipitation with low-speed centrifugation. Duplicate PCR increases low-density malaria detection. RDT had the highest number of unique positives, presumably from persistent antigen despite the absence of parasites, although RDT did not detect 3 parasitemias with over 1,000 parasites/μl. In a latent class analysis, real-time PCR had significantly higher sensitivity than did microscopy or RDT. Agreement between real-time PCR, RDT, and microscopy was highest in March and April, when both the P. falciparum parasite rate and parasite densities are highest. Real-time PCR is more sensitive and specific than RDT and microscopy in low-prevalence, low-parasite-density settings.

Fig. 1.

ROC curves show glycogen precipitation, and using the highest of 2 RT-PCR replicates as a cutoff improves the AUC. The data are from 6 P. falciparum -positive patient samples diluted 4-fold (22 samples, with the highest 2 samples removed) and 20 P. falciparum -negative patient samples from regions where malaria is not endemic. The yellow dots represent the 650-RFU cutoff used to distinguish positive and negative samples. The diagonal red (A) and gray (B) dashed lines show where sensitivity plus specificity is equal to 1. (A) ROC curve showing effects of glycogen precipitation. Solid gray line, nonprecipitated DNA; dashed gray line, nonprecipitated DNA with >1,000 parasites/μl; solid black line, precipitated DNA; dashed black line, precipitated DNA with >1,000 parasites/μl. (B) ROC curve showing call schemes. Solid green line, C_T-based cut points; blue dashed line, average of the last 5 cycles and average of both RT-PCR replicates; pink solid line, average of cycles and highest of the RT-PCR replicates; red solid line, last cycle of the high RT-PCR replicate; black diamond, Bio-Rad default cutoff where both replicates are positive; and blue triangle, Bio-Rad cutoff where either replicate is positive.

Fig. 2.

The standard curve of the 18S target performs better than the standard curve of the cytochrome b target. Shown is RT-PCR (C_T) versus microscopy (log₁₀ parasites/μl). The open circles show Tanzania microscopy, and the crosses show JHH validation data. (A) 18S target with the standard curve (gray line) fitted to Tanzania microscopy (r² = 0.92) and the 18S standard curve compared to JHH validation data (mean squared error [MSE] = 1.22). (B) Cytochrome b target, where the standard curve (black line) is fitted to Tanzania microscopy (r² = 0.52) and the cytochrome b standard curve is compared to JHH validation data (MSE = 1.48).

Fig. 3.

Duplicate amplification of RT-PCR samples improves detection at low parasite densities. (A and C) Fluorescence (log₁₀ RFU) for replicate 2 versus replicate 1 for all 451 febrile patients for RT-PCR_18S (A) and RT-PCR_Cyto (C). Quadrant boundaries are at log₁₀ 650 RFU (A) and 1,000 RFU (B) and represent the cutoff for RT-PCR positivity. The shaded quadrants contain the patient blood samples where one replicate is positive and a second replicate is negative. The unshaded quadrants contain patient samples where duplicate RT-PCRs were both positive or both negative. Shown are RDT-positive (red dots), microscopy-positive (blue dots), microscopy-positive and RDT-positive (purple dots), and microscopy-negative and RDT-negative (white dots) samples. (B and D) Plot of RT-PCR quantification compared to high and low ranges of RT-PCR replicates. The line segments represent fluorescence ranges for 41(RT-PCR_18S) (B) and 55 (RT-PCR_cyto) (D) RT-PCR-positive patient samples and include RDT-positive (red lines), microscopy-positive (blue lines), microscopy-positive and RDT-positive (purple lines), and microscopy-negative and RDT-negative (black lines) samples. The tan-shaded insets show magnified views of the region that contains patient samples where one replicate is positive and the other is negative. qPCR, quantitative PCR.

Fig. 4.

In March and April, the P. falciparum (Pf) parasite rate and parasite densities by RT-PCR and microscopy are highest, and the ages of those infected are low. (A) P. falciparum parasite rates over time for positive tests. The lines indicate P. falciparum rates by RT-PCR (green), microscopy (blue), and RDT (red). (B and D) Parasite densities by time. Shown are quantities by RT-PCR (green) and microscopy (blue); the circles indicate patient sample parasite densities, and the lines indicate the locally weighted least-squares regression line for parasite densities over time. (C) Box plot of the ages of patients with RT-PCR-positive tests over time. From left to right, the boxes show the ages of patients with positive tests in January-February, March-April, and May-June-July.

Fig. 5.

Venn diagram for positive P. falciparum tests. RT-PCR_18S (left), microscopy (Micro) (center), and RDT (right) are shown (13).