Reply to “Flow Cytometry for Antimalarial Drug Testing: More than Meets the Eye” Bruce Russell, a Laurent Rénia, b Benoît Malleret a,b Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System, Singapore a ; Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Biopolis, Singapore b T he desire to develop less expensive and easier-to-use antima- larial susceptibility assays is laudable, yet simplicity often comes at a cost, as is the case with flow cytometry assays using hemozoin (Hz) as the endpoint measure. Below we list some im- portant considerations regarding the use of Hz as the sole measure of parasite health in antimalarial susceptibility assays. 1. There are major intra- and interspecies differences in Hz levels. The development times, clumping patterns, and op- tical properties of Hz crystals vary widely among Plasmo- dium spp. (clearly understood by any basic malaria micros- copist). For example, we also see differences in the times and formats of Hz development in Asian versus African P. falciparum parasites. 2. Variations in red cell tropism of Plasmodium spp. can affect Hz production. For example, while Plasmodium falciparum invades normocytes, it prefers to invade reticulocytes. The development and formation of Hz in parasites invading re- ticulocytes differ from those in the mature erythrocyte. These factors may significantly interact with the effect of the antimalarial on Hz formation (1). 3. Different antimalarials affect the kinetics of Hz formation differently. Different antimalarials affect the kinetics of Hz formation differently. For example, while therapeutics such as atovaquone and cycloguanil allow for Hz development in sensitive strains of P. falciparum, the parasite is essentially dead, as it cannot develop to schizont stage. 4. Leukocytes significantly increase 50% inhibitory concen- trations, as leukocytes act as a sink for many antimalarial compounds. The current Hz assay, which does not remove leukocytes, is subject to serious confounders due to differ- ential leukocyte counts in ex vivo patient samples (2). The bottom line is that Hz-based assays (as with Plasmodium lactate dehydrogenase [PLDH] assays) use a secondary measure of development as an endpoint for parasite health. Unlike tests that directly measure the maturation of schizonts or parasitemia by microscopy or flow cytometry (measuring DNA and/or RNA), the Hz assay depends on a surrogate measure of antimalarial effect and is thus subject to a range of confounders (see the first three points above). Furthermore, the Hz assay detects less than 50% of mature P. falciparum (3D7) parasites after 24 h or 30 h of culture in the absence of drug pressure (3). In the past, flow cytometry approaches with only a single stain, such as Hoechst dye (4), Sybr green (5), or hydroethidine (6), were subject to poor sensitivity for detecting parasitemias below 1% (commonly present with field isolates). However, the potential of double stains to significantly increase assay sensitivity was demonstrated by Prof. Shapiro in the 1980s (7). We have tested the susceptibilities of 100s of field isolates (P. falciparum, P. vivax, P. malariae, P. knowlesi, and P. cynomolgi) to many standard and novel antimalarials using the method outlined in reference 8, combining a nucleic acid intercalating dye (Sybr green with a blue laser for detection [9] or Hoechst dye with a near-UV laser [10]) with a metabolized nucleic acid dye (dihydro- ethidium) in various field and laboratory settings (it is important to note that the identities of events in the gating strategy were confirmed by flow cytometry sorting [11]). While this method is not perfect (we still keep microtest thick films as a backup), it is still relatively inexpensive while retaining verifiable accuracy and reproducibility. We agree that Hz-based assays deserve some attention, yet they also need much more optimization and will almost certainly need another stain or marker to ensure their usefulness in determining the antimalarial susceptibility of malaria parasites in a clinical set- ting. REFERENCES 1. Lin JW, Spaccapelo R, Schwarzer E, Sajid M, Annoura T, Deroost K, Ravelli RB, Aime E, Capuccini B, Mommaas-Kienhuis AM, O’Toole T, Prins F, Franke-Fayard BM, Ramesar J, Chevalley-Maurel S, Kroeze H, Koster AJ, Tanke HJ, Crisanti A, Langhorne J, Arese P, Van den Steen PE, Janse CJ, Khan SM. 2015. Replication of Plasmodium in reticulocytes can occur without hemozoin formation, resulting in chloroquine resis- tance. J Exp Med 212:893–903. http://dx.doi.org/10.1084/jem.20141731. 2. Kaewpongsri S, Sriprawat K, Suwanarusk R, Kyle DE, Lek-Uthai U, Leimanis M, Lwin KM, Phyo AP, Zwang J, Russell B, Nosten F, Renia L. 2011. The presence of leukocytes in ex vivo assays signifi- cantly increases the 50-percent inhibitory concentrations of artesunate and chloroquine against Plasmodium vivax and Plasmodium falcipa- rum. Antimicrob Agents Chemother 55:1300 –1304. http://dx.doi.org /10.1128/AAC.01103-10. 3. Rebelo M, Shapiro HM, Amaral T, Melo-Cristino J, Hanscheid T. 2012. Haemozoin detection in infected erythrocytes for Plasmodium falcipa- rum malaria diagnosis—prospects and limitations. Acta Trop 123:58 – 61. http://dx.doi.org/10.1016/j.actatropica.2012.03.005. 4. van Vianen PH, van Engen A, Thaithong S, van der Keur M, Tanke HJ, van der Kaay HJ, Mons B, Janse CJ. 1993. Flow cytometric screening of blood samples for malaria parasites. Cytometry 14:276 –280. http://dx.doi .org/10.1002/cyto.990140307. 5. Smilkstein M, Sriwilaijaroen N, Kelly JX, Wilairat P, Riscoe M. 2004. Simple and inexpensive fluorescence-based technique for high- Citation Russell B, Rénia L, Malleret B. 2016. Reply to “Flow cytometry for antimalarial drug testing: more than meets the eye.” J Clin Microbiol 54:818 –819. doi:10.1128/JCM.03158-15. Editor: A. J. McAdam Address correspondence to Benoît Malleret, benoit_malleret@immunol.a-star.edu.sg. This is a response to a letter by Rebelo and Hänscheid (doi:10.1128/JCM.03017-15). Copyright © 2016, American Society for Microbiology. All Rights Reserved. AUTHOR REPLY crossmark 818 jcm.asm.org March 2016 Volume 54 Number 3 Journal of Clinical Microbiology on June 5, 2020 by guest http://jcm.asm.org/ Downloaded from