Comparison of real-time PCR and microscopy for malaria parasite detection in Malawian pregnant women

Abstract

BackgroundNew diagnostic tools for malaria are required owing to the changing epidemiology of malaria, particularly among pregnant women in sub-Saharan Africa. Real-time PCR assays targeting Plasmodium falciparum lactate dehydrogenase (pfldh) gene may facilitate the identification of a high proportion of pregnant women with a P. falciparum parasitaemia below the threshold of microscopy. These molecular methods will enable further studies on the effects of these submicroscopic infections on maternal health and birth outcomes.MethodsThe pfldh real-time PCR assay and conventional microscopy were compared for the detection of P. falciparum from dried blood spots and blood smears collected from the peripheral blood of 475 Malawian women at delivery. A cycle threshold (Ct) of the real-time PCR was determined optimizing the sensitivity and specificity of the pfldh PCR assay compared to microscopy. A real-time PCR species-specific assay was applied to identify the contribution to malaria infections of three Plasmodium species (P. falciparum P. ovale and P. malariae) in 44 discordant smear and pfldh PCR assay results.ResultsOf the 475 women, P. falciparum was detected in 11 (2.3%) by microscopy and in 51 (10.7%) by real-time PCR; compared to microscopy, the sensitivity of real-time PCR was 90.9% and the specificity 91.2%. If a Ct value of 38 was used as a cut-off, specificity improved to 94.6% with no change in sensitivity. The real-time PCR species-specific assay detected P. falciparum alone in all but four samples: two samples were mixed infections with P. falciparum and P. malariae, one was a pure P. malariae infection and one was a pfldh PCR assay-positive/species-specific assay-negative sample. Of three P. malariae infections detected by microscopy, only one was confirmed by the species-specific assay.ConclusionsAlthough microscopy remains the most appropriate method for clinical malaria diagnosis in field settings, molecular diagnostics such as real-time PCR offer a more reliable means to detect malaria parasites, particularly at low levels. Determination of the possible contribution of these submicroscopic infections to poor birth outcomes and maternal health is critical. For future studies to investigate these effects, this pfldh real-time PCR assay offers a reliable detection method.

Etiology of Pediatric Fever in Western Kenya: A Case–Control Study of Falciparum Malaria, Respiratory Viruses, and Streptococcal Pharyngitis

“…PCR was subsequently performed on whole blood from all patients who provided a whole blood sample (N = 401) using a real-time PCR assay targeting the P. falciparum lactate dehydrogenase (pfldh) gene on genomic DNA (gDNA) extracted from whole blood using Chelex-100 (Sigma-Aldrich, St. Louis, MI). 21 Cycle threshold lines were set manually by personnel masked to other clinical data; all plates included water as a negative control, reaction plates were assembled in a PCR hood, and a randomly selected subset of 20% samples were also tested in a real-time PCR assay targeting human RNAse P to ensure adequate gDNA extraction.…”

Plasmodium falciparum infection in pregnant women attending antenatal care in Luanda, Angola

“…Our limitation resides in the fact that in high transmission areas, many P. falciparum infections during pregnancy stay undetected when only microscopy of Giemsa-stained blood smears of peripheral blood is used for diagnosis. These sub-microscopic infections can only be diagnosed by detection of circulating parasitic antigens or detection of parasite-specific DNA using conventional polymerase chain reaction (PCR), nested PCR, or the more recently developed real-time PCR 5,[35][36][37] .…”

Molecular Detection of Malaria at Delivery Reveals a High Frequency of Submicroscopic Infections and Associated Placental Damage in Pregnant Women from Northwest Colombia

“…13,14 However, the epidemiology and clinical significance of submicroscopic malaria infections during pregnancy are not well-understood. 13,[15][16][17][18][19][20] The present pilot study evaluated the frequency of submicroscopic plasmodial infections in parturient women in the Urabá-Altos Sinú -San Jorge-Bajo Cauca region of Colombia and their associations with clinical outcomes of pregnancy.…”