CORRESPONDENCE • CID 2019:69 (15 October) • 1463 suggested, we admit that it is the way sci- ence perpetuates itself. Given the present situation and information, we do not see any justification to reinterpret our data. We sincerely hope that our responses reassure the readership of the validity and robustness of the evidence regarding ar- temisinin resistance. Note Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Sabyasachi Das, 1 Bhaskar Saha, 2 Amiya Kumar Hati, 3 and Somenath Roy 1 1 Immunology and Microbiology Laboratory, Department of Human Physiology with Community Health, Vidyasagar University, Midnapore, West Bengal, 2 National Centre for Cell Science, Ganeshkhind, Pune, and 3 Calcutta School of Tropical Medicine, Kolkata, West Bengal, India References 1. Das S, Saha B, Hati AK, Roy S. Evidence of arte- misinin-resistant Plasmodium falciparum malaria in Eastern India. N Engl J Med 2018; 379:1962–4. 2. Global Malaria Programme. Artemisinin and artemisinin-based combination therapy resistance. (WHO) Status report, October 2016. Available at: https://apps.who.int/iris/bitstream/handle/ 10665/250294/WHO-HTM-GMP-2016.11- eng.pdf;jsessionid=2F5FD02CBA6BDD0D- 55E11F252B58691B?sequence=1 3. WWARN K13 Genotype-Phenotype Study Group. Association of mutations in the Plasmodium fal- ciparum Kelch13 gene (Pf3D7_1343700) with parasite clearance rates after artemisinin-based treatments a WWARN individual patient data meta-analysis. BMC Med 2019; 17:1. doi:10.1186/ s12916-018-1207-3 4. World Health Organization. Artemisinin resistance and artemisinin-based combination therapy effi- cacy: Status report, August 2018. Geneva: World Health Organization, 2018. Available at: http:// apps.who.int/iris/bitstream/handle/10665/274362/ WHO-CDS-GMP-2018.18-eng.pdf?ua=1. 5. Das S, Manna S, Saha B, Hati AK, Roy S. Novel pfkelch13 gene polymorphism associates with arte- misinin resistance in eastern India. Clin Infect Dis 2018. doi: 10.1093/cid/ciy1038. 6. Phyo AP, Nkhoma S, Stepniewska K, et al. Emergence of artemisinin-resistant malaria on the western border of Thailand: a longitudinal study. Lancet 2012; 379:1960–6. 7. Ahmed A, Lumb V, Das MK, Dev V, Wajihullah, Sharma YD. Prevalence of mutations associated with higher levels of sulfadoxine-pyrimethamine resistance in Plasmodium falciparum isolates from Car Nicobar Island and Assam, India. Antimicrob Agents Chemother 2006; 50:3934–8. 8. Das S, Chakraborty SP, Tripathy S, Hati A, Roy S. Novel quadruple mutations in dihydropteroate synthase genes of Plasmodium falciparum in West Bengal, India. Trop Med Int Health 2012; 17:1329–34. 9. Das MK, Lumb V, Mittra P, Singh SS, Dash AP, Sharma YD. High chloroquine treatment failure rates and predominance of mutant genotypes as- sociated with chloroquine and antifolate resistance among falciparum malaria patients from the island of Car Nicobar, India. J Antimicrob Chemother 2010; 65:1258–61. Correspondence: S. Roy, Vidyasagar University, Midnapore, West Bengal, India (roysomenath@hotmail.com). Clinical Infectious Diseases ® 2019;69(8):1462–3 © The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com. DOI: 10.1093/cid/ciz167 Ultralow-density Plasmodium falciparum Infections in African Settings To the Editor—As countries accelerate towards elimination, an increasing pro- portion of infections may be of low par- asite densities. In a recent report, Girma and colleagues [1] deployed ultrasensitive diagnostics to characterize asymptomatic infections in Ethiopia. The Plasmodium falciparum prevalence was 1.3% by mi- croscopy, 3.6% by conventional rapid diagnostic tests (RDT), 8.5% by ultrasen- sitive Alere RDT, 22.2% by loop-medi- ated isothermal amplification and 21.5% by ultrasensitive quantitative reverse transcription-polymerase chain reaction (qRT-PCR). These findings are in line with a growing body of evidence demon- strating the superiority of ultrasensitive diagnostics in detecting low-density infec- tions, when compared to microscopy and standard RDTs [2]. The reported qRT-PCR prevalence is considerably higher than prevalence estimates from a meta-analy- sis tool that relates microscopy and PCR prevalence data from population sur- veys [3]. Based on the meta-analysis, one would expect a P. falciparum PCR preva- lence in the range of 2.9% to 10.6%. The higher prevalence in the study by Girma and colleagues [1] may be explained by their approach to targeting highly abun- dant RNA targets instead of DNA targets. Their finding thus suggests that there may be a reservoir of infections that is too low to be detected by conventional diagnostics or even conventional PCR [4]. Our own findings, from cross-sectional surveys in pre-elimination settings of South Africa, are in line with the findings of Girma and colleagues [1], in the sense that we also detected infections with ultralow parasite densities, below the limit of detection of conventional PCR. Our study observed no RDT-positive infections or 18S nest- ed-PCR–positive infections among 1475 individuals, whilst 3.9% of the study pop- ulation was positive for P. falciparum par- asites by sensitive, telomere-associated repetitive element 2–based quantitative PCR (qPCR), sometimes with genetically complex infections (Table 1). e real challenge of the study by Girma and colleagues [1], as well as of our own work, lies in the interpretation of such parasite survey data in relation to transmission patterns, particularly in low-transmission settings. Ethiopia and South Africa have both set targets for malaria elimination. It is unclear to what extent the presence of ultralow-density infections may challenge these ambi- tions. e authors correctly point out the Table 1. Plasmodium falciparum Infection and Multiplicity of Infection Outcomes in South Africa Local Subjects Migrant Subjects RDTs (First Response Malaria) 0 (0/933) 0 (0/542) 18S rRNA PCR 0 (0/933) 0 (0/542) TARE-2 qPCR % (n/N) 2.6% (24/993) 6.1% (33/542) Mean multiplicity of infections (range) 1.8 (1–3) 2.8 (1–5) Subjects were recruited in 2 community-wide, cross-sectional surveys among asymptomatic participants in 2014 and 2015. The 18S rRNA PCR [5], TARE-2 qPCR [6], and multiplicity of infections [7] were based on established protocols, using 4.2 µL of blood from filter paper bloodspots. Abbreviation: PCR, polymerase chain reaction; RDT, rapid diagnostic tests; rRNA, ribo- somal RNA; TARE-2 qPCR, telomere-associated repetitive element-2 quantitative PCR. Downloaded from https://academic.oup.com/cid/article-abstract/69/8/1463/5320191 by guest on 07 June 2020