Comparison of the parasite density using antigen detection, quantitative polymerase chain reaction, and microscopy in children with mild and severe malaria

Abstract

Malaria and its related complications are a major problem in most African countries. Accurate diagnosis of the disease is key to proper management and control and an ideal diagnostic parameter that correlates to disease outcome would be helpful in correctly identifying patients that need hospitalization versus those that can be managed at home. This study used Plasmodium falciparum histidine rich protein-2 (PfHRP-2) and Plasmodium specific lactate dehydrogenase (PLDH) ELISAs, quantitative polymerase chain reaction (qPCR) for total nucleic acids and microscopy to quantify the parasite load and thereafter correlate the load to disease severity. The World Health Organization recommends a malaria parasite density of 10,000 parasites per microlitre of blood as threshold for patient hospitalization. Unfortunately, many studies have questioned the use of parasite density as determined by microscopy as a method of identifying children with severe malaria from those with mild malaria. To re-evaluate the importance of parasite density in classifying malaria patients into severe or mild disease categories, this study compared the ability of three diagnostic methods to correctly categorize patients enrolled with a clinical diagnosis of either mild malaria or severe malaria. Parasite load was determined by microscopy, antigen ELISA for secreted parasite antigens, namely PfHRP-2 and PLDH and quantitative qPCR for total nucleic acids of the 18s ribosomal RNA (18s rRNA) gene. The study utilized archived samples (blood smear slides, serum, and nucleic acids previously collected in a hospital based case control study of children presenting with P. falciparum malaria at Kisumu District Hospital, Western Kenya. In that study, children (n=60; age<5 years presenting with severe malaria anemia (Hb < 6 gm/dl) were enrolled and matched by age and gender to children with mild malaria (Hb > 6 gm/dl). Statistical significance was considered at P≤0.05. The median parasite load and the 25th and the 75th percentile by microscopy in children with severe malaria (SM) was 49958 parasites/ul (12013-128695) and 24,233 (6122-103886) in the group with mild malaria (MM). This difference was not statistically significant P=0.103. The median parasite load and the 25th & 75th percentile obtained by the pLDH assay in the SM group was 2736 parasites/ul (546.2-12803) compared to 226 (79.06-428.0) in the MM group, which was statistically significant, P=0.0003. Using the PfHRP2 assay, children with SM had a higher median 628775 parasites/ul (332222-1.165e+006) compared to 150,453 (94292-399100) in children with MM. This difference was statistically significant P< 0.0001. By qPCR, the median parasite count was 31550 parasites/ul (4106-196640) in the group with SM compared to 24365 parasites/ul( 5512 – 93401) in the MM group. This difference was not statistically significant P=0.7336. The correlation of the parasite density by the PfHRP-2, PLDH and qPCR assays to the parasite density by microscopy was positive and significant in the MM group. However, the PfHRP-2 assay had the poorest correlation to the parasite density by microscopy in the SM group contrary to the PLDH assay and qPCR. The correlation of the parasite density to the total RBC count was negative and statistically significant only in the MM group for the PfHRP-2, PLDH and qPCR assays. It is concluded that the parasite load detected by antigen detection relates well to the disease outcome and can therefore be used to correctly assign malaria as either severe or mild.