Comparative evaluation of fluorescent in situ hybridization and Giemsa microscopy with quantitative real-time PCR technique in detecting malaria parasites in a holoendemic region of Kenya

Abstract

BackgroundEarly and accurate diagnosis of malaria is important in treatment as well as in the clinical evaluation of drugs and vaccines. Evaluation of Giemsa-stained smears remains the gold standard for malaria diagnosis, although diagnostic errors and potential bias estimates of protective efficacy have been reported in practice. Plasmodium genus fluorescent in situ hybridization (P-Genus FISH) is a microscopy-based method that uses fluorescent labelled oligonucleotide probes targeted to pathogen specific ribosomal RNA fragments to detect malaria parasites in whole blood. This study sought to evaluate the diagnostic performance of P-Genus FISH alongside Giemsa microscopy compared to quantitative reverse transcription polymerase chain reaction (qRT-PCR) in a clinical setting.MethodFive hundred study participants were recruited prospectively and screened for Plasmodium parasites by P-Genus FISH assay, and Giemsa microscopy. The microscopic methods were performed by two trained personnel and were blinded, and if the results were discordant a third reading was performed as a tie breaker. The diagnostic performance of both methods was evaluated against qRT-PCR as a more sensitive method.ResultsThe number of Plasmodium positive cases was 26.8% by P-Genus FISH, 33.2% by Giemsa microscopy, and 51.2% by qRT-PCR. The three methods had 46.8% concordant results with 61 positive cases and 173 negative cases. Compared to qRT-PCR the sensitivity and specificity of P-Genus FISH assay was 29.3 and 75.8%, respectively, while microscopy had 58.2 and 93.0% respectively. Microscopy had a higher positive and negative predictive values (89.8 and 68.0% respectively) compared to P-Genus FISH (56.0 and 50.5%). In overall, microscopy had a good measure of agreement (76%, k = 0.51) compared to P-Genus FISH (52%, k = 0.05).ConclusionThe diagnostic performance of P-Genus FISH was shown to be inferior to Giemsa microscopy in the clinical samples. This hinders the possible application of the method in the field despite the many advantages of the method especially diagnosis of low parasite density infections. The P-Genus assay has great potential but application of the method in clinical setting would rely on extensive training of microscopist and continuous proficiency testing.

True malaria prevalence in children under five: Bayesian estimation using data of malaria household surveys from three sub-Saharan countries

“…In this respect, it can be noted that the values provided by the experts from Uganda were in line with some population-based survey studies that calculated the sensitivity of the microscopy using the PCR as gold standard (e.g., [ 5 ]). On the other hand, the values provided by the experts from Kenya and the Democratic Republic of the Congo seemed to be higher than those reported in population-based survey studies where PCR was used as gold standard (e.g., [ 28 , 29 ]). Nonetheless, the one-way sensitivity analyses showed that the true prevalence estimates in all three countries were robust against misspecification of this prior.…”

Sexual forms obtained in a continuous in vitro cultured Colombian strain of Plasmodium falciparum (FCB2)

“…The gametocytes were then tested regarding their sexual differentiation capability in vitro, using 100 µL gametocyte culture (stage V) at 27 °C for 20 min [31]. Samples were then spun at 2500 rpm for 3 min, pellets were analysed by Giemsa staining and gametes were visualized at 40× using a Primo Star Carl Zeiss microscope [32].…”