Correlating quantitative real-time PCR to rapid diagnostic test and RNA transcript expression in isolated gametocytemia and asexual parasitemia of Plasmodium falciparum malaria

Rachel Lau; Melissa Phuong; Filip Ralevski; Andrea K Boggild

doi:10.1186/s40794-015-0008-3

Correlating quantitative real-time PCR to rapid diagnostic test and RNA transcript expression in isolated gametocytemia and asexual parasitemia of Plasmodium falciparum malaria

Trop Dis Travel Med Vaccines. 2015 Sep 8:1:8. doi: 10.1186/s40794-015-0008-3. eCollection 2015.

Authors

Rachel Lau¹, Melissa Phuong², Filip Ralevski¹, Andrea K Boggild^{1

3

4}

Affiliations

¹ Public Health Ontario Laboratories, Public Health Ontario, Toronto, Canada.
² Faculty of Health Sciences, McMaster University, Hamilton, Canada.
³ Tropical Disease Unit, Division of Infectious Diseases, UHN-Toronto General Hospital, 200 Elizabeth Street, 13EN-218, Toronto, ON M5G 2C4 Canada.
⁴ Department of Medicine, University of Toronto, Toronto, Canada.

Abstract

Background: At present, only microscopic examination of stained thick and thin blood smears for malaria can differentiate clinically relevant asexual parasitemia from clinically irrelevant isolated gametocytemia. Microscopy is time consuming, labour intensive, and requires significant technical expertise to perform. Simple and rapid tests that can distinguish asexual from isolated sexual parasitemia are needed.

Methods: To determine if parasitemia and cycle threshold (C_T) values on Plasmodium genus and P. falciparum-specific quantitative polymerase chain reaction (qPCR) assays correlate to positivity of rapid diagnostic test (RDT), and 18S rRNA gene copy number, we analyzed blood samples from Ontario patients with isolated P. falciparum gametocytemia or asexual stages. RNA transcripts were evaluated to determine whether there is correlation of expression to different life cycle stages of P. falciparum.

Results: 45 specimens containing isolated P. falciparum gametocytes, and 40 specimens containing isolated asexual stages by microscopy were identified and analyzed. By RDT, 40 of 45 (88.9 %) isolated gametocytemia specimens and 40 of 40 (100 %) asexual-stage specimens were positive for Plasmodium falciparum-specific histidine rich protein-2 (HRP-2). Fourteen of 45 (31.1 %) isolated gametocytemia specimens, and 36 of 40 (90 %) asexual-stage specimens were positive for Plasmodium genus aldolase T2 band. Positivity of the aldolase T2 band was associated with lower mean Plasmodium genus and P. falciparum-specific C_T values, and to higher mean 18S rRNA gene copy by qPCR for both isolated gametocytemia and asexual-stage specimens. There was also a negative correlation of asexual parasitemia to both C_T values, and positive correlation to 18S rRNA gene copy number. Analysis of asexual stage-specific erythrocyte binding antigen (eba-175) transcripts on 25 isolated gametocytemia and 20 asexual-stage specimens gave a positive predictive value of 62.5 % and negative predictive value of 100 % for asexual parasitemia. Thus, an absence of eba-175 transcripts excluded the presence of asexual (clinically relevant) parasitemia.

Conclusions: Positivity of the aldolase T2 band of BinaxNow RDT correlated to higher parasite load in both isolated gametocytemia and asexual-stage specimens. Asexual stage-specific eba-175 RNA transcript expression provided reasonable negative predictive value for exclusion of asexual parasitemia in clinical samples, but was present in both isolated gametocytemia and asexual stage specimens.