Gruenberg et al. Malar J (2018) 17:55 https://doi.org/10.1186/s12936-018-2201-0 Journal

REVIEW Open Access vivax molecular diagnostics in community surveys: pitfalls and solutions Maria Gruenberg1,2†, Clara Antunes Moniz1,2†, Natalie Ellen Hofmann1,2, Rahel Wampfer1,2, Cristian Koepfi3, Ivo Mueller3, Wuelton Marcelo Monteiro4, Marcus Lacerda4,5, Gisely Cardoso de Melo4,5, Andrea Kuehn4, Andre M. Siqueira4,6 and Ingrid Felger1,2*

Abstract A distinctive feature of infections is the overall low parasite density in peripheral blood. Thus, iden- tifying asymptomatic infected individuals in endemic communities requires diagnostic tests with high sensitivity. The detection limits of molecular diagnostic tests are primarily defned by the volume of blood analysed and by the copy number of the amplifed molecular marker serving as the template for amplifcation. By using mitochondrial DNA as the multi-copy template, the detection limit can be improved more than tenfold, compared to standard 18S rRNA targets, thereby allowing detection of lower parasite densities. In a very low transmission area in Brazil, application of a mitochondrial DNA-based assay increased prevalence from 4.9 to 6.5%. The usefulness of molecular tests in malaria epidemiological studies is widely recognized, especially when precise prevalence rates are desired. Of concern, how- ever, is the challenge of demonstrating test accuracy and quality control for samples with very low parasite densities. In this case, chance efects in template distribution around the detection limit constrain reproducibility. Rigorous assessment of false positive and false negative test results is, therefore, required to prevent over- or under-estimation of parasite prevalence in epidemiological studies or when monitoring interventions. Keywords: Plasmodium vivax, Surveillance, Molecular diagnostics, Mitochondrial DNA, 18S rRNA transcripts, LAMP, Quantifcation,

Background which infects only reticulocytes that account for less than Parasite densities in Plasmodium vivax infections are 1% of all erythrocytes. P. falciparum is less restricted generally lower compared to in selection and, thus, can reach higher densi- densities. For example, in (PNG), ties. Moreover, age trends in infection prevalence and among children living in an area with similar P. falcipa- clinical incidence suggest an earlier acquisition of clini- rum and P. vivax transmission rates, the diference in cal immunity and more efective control of parasitaemia mean parasite density between both species was ten-fold for P. vivax compared to P. falciparum [3]. In a cohort of by light microscopy (LM) and 30-fold by quantitative young children from PNG (1–4 years), the incidence of PCR (qPCR) (Fig. 1) [1]. A similar diference in densities clinical P. vivax episodes decreased signifcantly by the between both species was observed in the general popu- second year of , whereas the incidence of clinical P. lation [2]. Te lower parasite densities of P. vivax can be falciparum episodes continued to rise up to 4 years of age explained by the strict host cell preference of this species, [3]. Tis indicates that young children in endemic areas acquire the ability to efectively control P. vivax parasitae- mia early in life. *Correspondence: [email protected] Te overall lower density of P. vivax compared to P. fal- †Maria Gruenberg and Clara Antunes Moniz contributed equally to this work ciparum plays a critical role in limiting the test sensitivity 1 Swiss Tropical and Institute, Socinstrasse 57, 4002 Basel, of diagnostic methods used to measure parasite preva- Switzerland lence, such as light microscopy (LM), rapid diagnostic Full list of author information is available at the end of the article

© The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Gruenberg et al. Malar J (2018) 17:55 Page 2 of 10

a b

P. falciparum (qPCR) P. falciparum (LM) Median (IQR): 335 (42-2142) copies/µl blood Median (IQR): 805 (272-9680) parasites/µl blood Geometricmean: 364 copies/µl blood Geometricmean: 1412 parasites/µl blood

P. vivax (qPCR) P. vivax (LM) Median (IQR): 12 (4-26) copies/µl blood Median (IQR): 89 (57-213) parasites/µl blood Geometricmean: 9 copies/µl blood Geometricmean: 119 parasites/µl blood Fig. 1 Parasite densities of P. falciparum and P. vivax measured by qPCR (a) and light microscopy (b) in community samples from PNG (5–9 years old children from 6 neighbouring villages) (Data taken from [41])

test (RDT) and quantitative PCR (qPCR). A systematic to molecular diagnosis derives from the very small vol- review of sub-microscopic P. vivax infections showed ume of blood (0.025–0.0625 µL whole blood) examined that in cross-sectional surveys from diverse transmission per blood slide for parasite counts in feld studies [6]. settings, an average 67% of all P. vivax infections were Molecular techniques permit examination of an equiva- sub-microscopic and would thus remain undetected by lent of 5–10 µL whole blood, which increases test sen- LM [4]. As for P. falciparum, a negative relationship was sitivity substantially. However, using increasingly high observed between the proportion of sub-microscopic blood volumes in molecular diagnostic tests would not infections and prevalence by LM. In view of the lower P. necessarily result in a linear increase in sensitivity, as vivax densities overall, molecular-based diagnostic tools large amounts of human genomic DNA will act as PCR are even more relevant for detection of P. vivax than for inhibitor. Attempts to maximize molecular test sensi- P. falciparum, particularly in areas of low transmission. tivity by increasing the input material to several mL of In this paper, diagnostics for detecting P. vivax blood venous blood would require depletion of human white stage infections are discussed. Hypnozoites, another hall- blood cells [7]. mark of P. vivax infections, cannot be detected by current To increase the comparability of molecular-epidemi- diagnostic methods. ological data generated across diferent feld sites and laboratories, a defned set of experimental details should Diagnostic tools for surveillance be included in any report. Tese recommended specifca- LM has traditionally been the gold standard for malaria tions are presented in Box 1. , while prevalence by LM has been used For diagnosing community samples, the desired pro- to describe malaria transmission levels globally. Having fle of a diagnostic test difers from that of clinical man- made substantial progress in malaria control, interven- agement. For example, control interventions targeting tions have shifted focus from targeting clinical cases only all individuals who may contribute to malaria transmis- towards identifying and treating asymptomatic parasite sion require robust diagnosis of low density infections in carriers, as well. Hence, the extent of sub-microscopic asymptomatic parasite carriers. In response to this need, Plasmodium infections and the ability of molecular diag- experts in P. vivax diagnostics and epidemiology recently nostic tools to detect them have increasingly attracted defned target product profles (TPP) for P. vivax diag- attention [4, 5]. Te limited sensitivity of LM compared nosis in malaria epidemiological feld work [8]. Tree Gruenberg et al. Malar J (2018) 17:55 Page 3 of 10

Box 1 Recommended reporting of experimental details in molecular-epidemiological studies Requirement for publication Experimental information to be reported

Imperative Sampling details of fnger prick or venous blood sampling; e.g. type of flter paper (treated or not), microtainer/tubes (heparin, EDTA), storage solution (RNAprotect/Trizol) Description of extraction method; e.g. extracted blood volume, spin columns, chelex, type of DNAse treatment of puri- fed RNA Resuspension/elution volume for extracted DNA or RNA Description of molecular target; e.g. Gene ID, amplicon size, primer and probe sequences Reagent concentrations and total reaction volume; e.g. concentrations of primer/probe, template volume added to amplifcation reaction Standard used for quantifcation; e.g. parasite trendline (with stage composition), NIBSC WHO reference standard, plasmid (linearized/supercoiled) Clear defnition of quantifcation results; e.g. method used for conversion of copy numbers into parasites/µL blood, clear denominator for “template copy number/µL whole blood or DNA” Assay performance parameters; e.g. specifcity, assay LOD, PCR efciency Optional Storage conditions and time prior to extraction, e.g. of flter papers or whole blood, temperature, desiccant, particularly for flter papers Reproducibility; e.g. results from duplicates or triplicates Comparison to LM; e.g. correlation of parasite counts/µL blood to copy numbers/µL blood

Technical details should be reported according to MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments [50] distinct TPPs for the next generation of P. vivax diagnos- contain 16–24 genomes, a direct conversion from copy tic tests for control and elimination were generated under number to parasite counts will not be accurate. Tis issue the leadership of the Foundation for Innovative Diagnos- has been investigated using digital droplet PCR (ddPCR), tics (FIND). Each TPP addressed a particular diagnostic a technology that permits absolute quantifcation of tem- task: (i) a point-of-care tool for clinical case management plate DNA [12]. A very strong correlation (R = 0.86) was (e.g. an ultra-sensitive RDT for P. vivax); (ii) a molecular found for P. vivax quantifcation by the two molecular ultra-sensitive test for mobile teams engaged in surveil- methods, ddPCR and standard Pv18S rRNA qPCR [12]. lance-response activities targeting asymptomatic carriers Te correlation between P. vivax microscopy counts and that can be performed rapidly, in a single tube and at the quantifcation by ddPCR and qPCR was good (R = 0.72 point-of-care; and (iii) a molecular ultra-sensitive test for and R = 0.73, P < 0.0001) [12]. Similar correlations were large-scale surveillance activities or research where the observed for P. falciparum, thus, it seems that the occa- time to result is not critical, and that can be performed at sional presence of P. vivax late stages in fnger-prick high throughput and low cost at a core facility [8]. Molec- blood samples does not substantially afect molecular ular assays that target multiple copies per genome have quantifcation. Te number of Pv18S rRNA gene copies the potential to increase test sensitivity sufciently to detected per parasite was determined by comparing with allow pooling of several samples without compromising LM data. On average, one Pv18S rRNA copy per parasite test sensitivity. Using pooling for the last two tasks can was measured by ddPCR. As multiple genomes should be reduce costs, particularly in areas of low P. vivax preva- detected per schizont, a loss or damage of genomic cop- lence (< 2%). ies during DNA extraction has to be assumed [12]. Te same Pv18S rRNA assay can also be used to target Plasmodium vivax 18S rRNA as marker gene Pv18S rRNA transcripts instead of the genes themselves for DNA‑ and RNA‑based detection [10, 13, 14]. Targeting RNA transcripts amplifes sen- 18S rRNA genes are the standard molecular markers for sitivity, since each ribosome carries one copy of rRNA, diferentiating Plasmodium species. In the two P. vivax which amounts to thousands of 18S rRNA transcripts per reference genomes sequenced, Sal1 and P01, three dis- cell. For P. falciparum, a factor to convert Pf18S rRNA tinct 18S rRNA copies exist and are expressed in difer- transcripts into parasite counts was established using ent developmental stages (Additional fle 1: Table S1) [9]. synchronous cultured parasites [13]. ­104 18S rRNA tran- However, a widely used Pv18S RNA assay [10] targets scripts were measured per ring stage parasite; this num- only one of the three Pv18S rRNA copies by qPCR. ber remained constant for the initial 24-h of the life cycle. In contrast to P. falciparum, schizont stages of P. vivax As P. vivax cannot be readily cultured in vitro, a conver- are found in the peripheral blood [11]. As schizonts can sion factor for P. vivax only could be estimated using Gruenberg et al. Malar J (2018) 17:55 Page 4 of 10

parasite counts by LM from feld samples [10]. Correla- a tion between microscopic P. vivax counts and the num- 2 ber of Pv18S rRNA transcripts was moderate ­(r = 0.44) blood

[10]. Te discrepancies between LM and molecular quan- hole

tifcation might derive from between-sample variation Lw

/µ Cut-off >10 copies/µL in parasite stage composition or from RNA content per s parasite. Additionally, variable conditions of RNA preser- vation and sample storage in the feld afect the quality number of extracted RNA, making RNA-based quantifcation less copy

reliable compared to DNA-based quantifcation. or Samples (n=128) Problems caused by targeting transcripts

of Plasmodium vivax 18S rRNA Transcript During nucleic acid extraction, there is an inherent risk of b

contaminating parasite negative samples handled along- lood side parasite positive samples. Cross-contamination can eb

occur even without pipetting errors, by spreading aero- whol sols when handling highly concentrated nucleic acids. Tus, the greatest care needs to be taken when working with clinical samples for both DNA and RNA template molecules. In view of the exceedingly high copy numbers numbers/µl of ribosomal RNA transcripts compared to genomic 18S copy rRNA copies, this contamination threat is potentiated by or working at RNA level, leading to false positivity [10, 15]. Samples (n=124) Tis risk of cross-contamination was addressed in a Transcript cross-sectional survey of 315 children from PNG, where Fig. 2 Detection of 18S rRNA genomic copies compared to 18S rRNA DNA-based and RNA-based detection and quantifca- transcripts. Data used for this blot derived from earlier published work tion were compared for P. vivax and P. falciparum [10]. [10]. Dashed line: choice of cut-of (> 10 transcripts per reaction) Figure 2 shows the number of P. falciparum and P. vivax 18S rRNA transcripts in study participants, plotted by decreasing number of 18S rRNA transcripts. For P. fal- positivity was required when detecting Pf18S rRNA tran- ciparum, transcript numbers trailed of over a substan- scripts, to exclude false positive results caused by low-key tial number of samples at the lower density end (< 10 contamination from the few intermittent high density transcripts, 40% of all positive samples). Tis was not infections. Based on the distribution of Pv18S rRNA tran- observed for P. vivax, which may be explained by the script copy numbers (Fig. 2b), a cut-of for P. vivax RNA- lower median parasite density (8 times lower by LM and based parasite detection and quantifcation did not seem by qPCR) in P. vivax positive samples compared to P. fal- necessary. In summary, the pitfalls of RNA-based diag- ciparum infections of the same study. nosis do not reject parasite detection based on 18S rRNA Te potential for cross-contamination between the wells transcripts, but rather call for awareness, utmost caution of an RNA extraction plate was investigated by analysing and well-controlled experimental procedures. large numbers of negative controls (phosphate-bufered saline, PBS) in parallel with interspersed wells contain- Plasmodium vivax assays targeting multi‑copy ing high density P. falciparum 3D7 culture, mimicking templates high density clinical infections. False positive results were High-copy genomic sequences can serve as new PCR observed in some of the wells neighbouring high-density targets for the detection of malaria infections, providing samples. Tese confrmed false positives were typically increased sensitivity over single- or low-copy 18S rRNA characterized by transcript numbers < 10 transcript cop- genes, without the pitfalls of RNA-based amplifcation. ies/µL and in very few exceptions < 50 copies/µL. Such Furthermore, multi-copy markers have the potential to high parasitaemia, as used in these control experiments, allow sample pooling without jeopardizing test sensitiv- might be reached only rarely in community samples, yet, ity. Tis would be particularly favourable in the context this observation calls for great care during extraction of increasingly large study sizes required for community and pipetting. Analysis of the PNG feld samples (Fig. 2) surveys conducted in elimination settings with low prev- led to the conclusion that for P. falciparum, a cut-of for alence rates. Gruenberg et al. Malar J (2018) 17:55 Page 5 of 10

Te P. vivax genome was mined to identify species- cytochrome B gene (cytB) and cox1 [30]; nested PCR, tar- specifc, repetitive sequences. Te best target identifed geting cytochrome C oxidase III (cox3) [31]; and genus- was the non-coding subtelomeric repeat sequence Pvr47, specifc nested PCR, targeting the cyt B gene, followed by which occurs in 14 copies per P. vivax Sal1 genome [16]. sequencing of the PCR product or restriction fragment A Pvr47-based single-step PCR assay was almost as sen- length polymorphism (PCR–RFLP) for species identifca- sitive as nested PCR targeting the P. vivax 18S rRNA tion [32, 33]. when visualized in an agarose gel [16]. Attempts to use Pvr47 for designing a LAMP assay failed due to speci- Targeting mitochondrial DNA by qPCR fcity problems [17]. When the Pvr47 assay was used to in cross‑sectional samples from Brazil detect P. vivax in spp. mosquitoes, non-spe- A qPCR assay was designed to target the P. vivax mito- cifc bands and sequences were produced [18]. chondrial cox1 gene (Pv-mtCOX1 qPCR, Additional A number of attempts were made to identify other fle 2: Table S2). Tis assay showed performance char- multi-copy markers for detection of P. vivax. Similar to acteristics superior to Pv 18S rRNA qPCR (Additional a qPCR assay developed for ultra-sensitive detection fle 3: Table S3, Additional fle 4: Table S4). Some 604 of P. falciparum that targets the conserved C-terminus samples collected from a cross-sectional survey in the of the var gene family [19], P. vivax candidates were Amazonas region, Brazil, in 2014 were re-analysed with sought among the vir/pir multigene family [20, 21]. How- Pv-mtCOX1 qPCR to investigate the efect of applying ever, genetic diversity among members of this family is highly sensitive DNA-based parasite detection to com- extremely large, such that no DNA stretches of sufcient munity samples and asymptomatic parasite carriers. Te sequence conservation and size for primer and probe number of P. vivax positive samples difered substan- design were identifed [22]. Recently, a revised P. vivax tially by assay and 23.8% of positive samples were only reference genome (P01) with improved assembly of the detected by the Pv-mtCOX1 assay (Fig. 3a). Overall posi- subtelomeres became available [23]; new attempts are tivity was very low, with 4.9% (CI­ 95 [3.4–6.9%]) of sam- currently underway to identify multi-copy targets. ples testing positive by 18S rRNA qPCR and 6.5% (CI­ 95 In view of the high genetic variability in repeated [4.7–8.7%]) by Pv-mtCOX1 qPCR. In samples deemed genomic regions and in the vir genes of P. vivax, mitochon- positive by both assays, the correlation of template copy drial DNA (mtDNA) ofers relatively conserved regions numbers obtained by the two assays was good (Spear- for primer design, as well as sufcient diversity for distin- man’s rho = 0.85, red data points in Fig. 3b). guishing the diferent Plasmodium species. Te mitochon- To investigate the relationship between template copy drial genome of malaria parasites is present in multiple number and positivity, copy numbers were plotted for copies per cell, contained in a single . For all positive samples for both assays (Fig. 4; Additional P. falciparum, the total number per ring stage parasite is fle 5: Figure S1). Te median gene copy number for about 20 mitochondrial genomes [24]. Te bulk of these Pv-mtCOX1 was about ten times higher than for Pv18S copies are present in linear tandem arrays of 3–4 units rRNA. Infections of very low parasitaemia were detected [25]. Replication occurs simultaneously with the nuclear by Pv-mtCOX1 qPCR but not by Pv18S rRNA qPCR. genome, about 24-h post invasion. For P. falciparum with Tese results confrm the mitochondrial genome as a sequestered late stages, the gain in sensitivity from using suitable target for achieving a substantially more sensi- a mitochondrial marker compared to nuclear markers is tive qPCR assay, enabling detection of scarce mitochon- potentially limited, as the multiple copies of mitochondrial drial template copies in very low density infections. DNA (mtDNA) are not distributed independently, but in six molecules, each composed of the 3–4 tandem repeat Loop‑mediated isothermal amplifcation (LAMP) units of mtDNA. In P. vivax, however, late stages with mul- LAMP assays amplify single-copy or multi-copy molecu- tiple genomes and the replicating mitochondrial genomes lar markers in an isothermal reaction. Tis method seems are also present in peripheral blood. While the organiza- optimally suited for application at the point-of-care tion of P. vivax mtDNA is not known, a substantial tem- (POC) in feld settings. LAMP requires little in the way plate multiplication factor can be expected. Tus, the gain of equipment and may be carried out by mobile labora- in sensitivity from targeting the mitochondrial genome tories. LAMP is suitable for detecting sub-microscopic might be greater for P. vivax than for P. falciparum. infections [34, 35]. However, LAMP cannot quantify A number of assays for diagnosing P. vivax have tar- parasitaemia and some protocols for measuring ampli- geted mtDNA: one-step PCR; loop-mediated isother- fcation are not very robust, such as hydroxynaphtol mal amplifcation (LAMP) or qPCR, targeting the blue detection (Additional fle 6: Figure S2). Te use of cytochrome C oxidase I gene (cox1) [26–29]; genus- fuorescent dyes to detect LAMP products can overcome specifc PCR, targeting non-coding regions between the some of the limitations of conventional LAMP detection. Gruenberg et al. Malar J (2018) 17:55 Page 6 of 10

Fig. 3 Comparison of Pv-mtCOX1 and Pv18S rRNA assays performed in parallel in 604 community samples from Brazil. a Overlap in positivity by Pv-mtCOX1 and Pv18S rRNA qPCR. b Correlation of log10 template copy numbers detected by Pv-mtCOX1 and Pv18S rRNA qPCR

at ultralow template concentrations around the limit of detection. Systematic validation is further complicated by chance efects in template distribution in low densities. To improve the specifcity of LAMP, several assay parameters were optimized, such as decreasing the primer concentration, reducing the incubation time of the LAMP reaction, testing diferent published primer sets, and optimizing primers (unpublished own results) using commercial (Mast Isoplex Malaria Lamp Kit; Mast Diagnostica) as well as home-made master mixes made up of individually purchased reagents (New England Bio- labs). False positive results were primarily obtained with primers targeting the 18S rRNA genes of the genus Plas- modium [39]. Using alternative primers that target the Fig. 4 Copy numbers of each marker gene detected per sample. mitochondrial genome of the genus Plasmodium [35], Each dot represents one sample, red indicates all samples positive false-positive results in negative controls were substan- for Pv18S rRNA qPCR, orange indicates samples detected only by Pv- tially reduced but not eliminated. Amplifcation of LAMP mtCOX1 qPCR. Dashed line: Molecular assays have a theoretical LOD, products can be tracked in real-time using a StepOne i.e., at least 1 template copy must be present per PCR reaction thermocycler to detect the fuorescent dye of a commer- cial master mix. In negative controls, signals from unspe- cifc amplifcation appeared later in the reaction than Unspecifc template-independent amplifcation is a signals from the true positive reaction observed when long-standing problem in LAMP that has been addressed a template was present. However, positive samples with by a number of authors [36–38]. Amplifcation artefacts low parasite densities equivalent to 1 parasite/µL could arise from primer complexes formed by the four to six not be distinguished from false-positives (Additional primers per reaction, two of which are very long prim- fle 7: Figure S3). Duration of incubation was a crucial ers pre-designed for generating loops. Primer dimers or determinant for false-positive results. Some published junk amplifcation products may be generated in nega- protocols incubate LAMP for 60 min, for example [39], tive controls. False-positive LAMP reactions reportedly whereas LAMP kit manuals allot 40 min. To avoid false- occurred at random [34]. Tis phenomenon leads to positive results, reaction time should not be extended, loss of confdence in results, as this type of amplifcation even though this might result in a potential loss of sen- artefact cannot be distinguished from reagent contami- sitivity by missing low density infections. When a com- nation. Moreover, it is difcult to validate true positivity mercial LAMP Kit with lyophilized primers (EIKEN Gruenberg et al. Malar J (2018) 17:55 Page 7 of 10

CHEMICAL CO., LTD) was used, unspecifc amplifca- than for P. falciparum. Te P. vivax-specifc diagnostic tion was only rarely observed. challenges include lower mean parasite densities, less sensitive RDTs and a greater need for diagnosis of all Consequences of false‑positive and false‑negative infections to prevent later relapses and, thus, continued test results transmission [8]. Tese challenges can be tackled to some To guide malaria control and surveillance, reliable preva- extent by molecular diagnostics, but all diagnostic meth- lence data is of great importance, particularly in areas ods, including NAAT, sooner or later reach a test-specifc with low endemicity or in regions recently declared limit of detection. Test sensitivity largely depends on the malaria-free. False-positive test results lead to overesti- volume of blood used for DNA or RNA extraction and mation of residual malaria transmission and may cause on the whole blood equivalent added to the amplifcation unnecessary concerns. In contrast, a large extent of false- reaction. Increasing test sensitivity beyond the current negative results would underestimate the true transmis- levels of detection would require venous blood samples sion intensity. However, such underestimation is usually and white blood cell depletion [40]. Tat option is not expected, as epidemiologists and public health workers considered feasible for large-scale feld surveys. Tus, are well aware of imperfect malaria diagnosis. detection of malaria parasites remains imperfect. What should be the guiding principle for selecting the However, if capacity, equipment and reagents were most suitable diagnostic test? Te dilemma consists in a available in P. vivax endemic areas, those facilities could trade-of between sensitivity and false-positivity, as seen act as reference laboratories for quality assurance. Tis in selecting the incubation time for a LAMP reaction, or would greatly help to improve diagnostic quality in in the use of RNA-based parasite detection by qRT-PCR. research and surveillance. Te answer to the question Evidently, the conservative and more stringent results are about molecular diagnostics being essential or not largely preferable because the detectability of parasites in ultra- depends on the specifc task, be it rapid reactive response low infections is always imperfect. Te most appropriate or general surveillance, research or clinical trial. nucleic acid amplifcation technique (NAAT) must be Te usage of molecular diagnosis for understanding the chosen in consideration of the task in question. For exam- reservoir of transmission and to guide interventions has ple, for focal screen and treat or surveillance-response been emphasized by many recent publications [41–45], activities, high sensitivity might be more important than but the epidemiological relevance of detecting submicro- an occasional false-positive result. Tus, any decision scopic P. vivax infections is not the primary focus of this about which diagnostic methods to use should be aligned publication. with each specifc task and consider the limitations of the diagnostics applied. Gametocytes in low density P. vivax infections It is important to keep in mind that stochastic variation Treatment of asymptomatic P. vivax infections has two in results is always observed when infections are around aims: frstly, to target gametocytes to prevent onward the limit of detection of a given assay. For example, when transmission to mosquitoes and, secondly, to target 150 samples collected in PNG were screened three times dormant liver stages to prevent relapses. Blood-stage with the same P. vivax 18S rRNA assay, only 14 infections infections originating from relapses frequently carry were detected in all three replicates, while 19 infections gametocytes and, thus, likely also contribute to transmis- were detected in only one or two replicates. Tis varia- sion [46]. tion was less pronounced for P. falciparum (24/31 infec- In the context of transmission control, the question tions detected in all three replicates), most likely due to arises whether all low intensity P. vivax infections carry the overall higher density of P. falciparum [12]. Tus, gametocytes and whether molecular tools are required to when results are compared between feld laboratories determine the prevalence of gametocytes in the popula- and reference laboratories, slightly diferent results are tion. Plasmodium vivax gametocytes are detected either expected, even when using the same protocols. Only if a by LM or by quantifying transcripts of genes that are laboratory repeatedly detects fewer infections compared specifcally expressed in P. vivax gametocytes. P. vivax to a reference laboratory do lab-specifc procedures have gametocytes are difcult to distinguish from trophozo- to be optimized. ites by LM. Molecular detection of gametocytes is more sensitive and more precise. Te standard marker gene Relevance of detecting ultralow parasite densities pvs25 encodes an ookinete surface protein. Quantitative Te limited resources in malaria endemic areas war- reverse transcription PCR (qRT-PCR) is performed on rant a discussion on whether molecular diagnostics and RNA extracted from a blood sample [10]. the establishment of qPCR assays in feld laboratories detection in feld surveys is complicated by the require- are needed. For P. vivax, NAAT seem more necessary ment for appropriate RNA-stabilizing procedures, such Gruenberg et al. Malar J (2018) 17:55 Page 8 of 10

as immediate transfer of a blood sample into a stabiliz- •• Te input blood volume determines test sensitivity. ing reagent [10]. When pvs25 transcript numbers were To improve test performance, the volume of fnger plotted against Pv18S rRNA gene copies, a moderate prick blood processed or DNA and RNA template correlation (R = 0.59) was observed in samples from two added to NAAT should always be maximized. cross-sectional community surveys conducted in PNG •• Multi-copy targets used for qPCR are superior for 2 (Fig. 5) [2, 41]. A much stronger correlation ­(r = 0.82) detection and necessary for pooling samples before was observed in a study from Tailand, using the same molecular analysis. Te tenfold increase of PCR tem- diagnostic methods [44]. P. vivax gametocytes can be plates per cell when using the Pv-mtCOX1 assay led detected within 3 days of the appearance of asexual para- to gains in positivity and more precise prevalence sites in the blood [47]. Tis also argues for using P. vivax estimates in a cross-sectional survey in Brazil. blood stage parasites as a surrogate marker for gameto- •• Te suitability of RNA-based assays is questionable cytaemia. Performing gametocyte detection and quantif- for processing large-scale feld samples with a wide cation assays are not required for surveillance. range of parasite densities. A fully enclosed system Asymptomatic infections were often found to carry for sample processing and tight controls seem critical gametocytes in studies in Brazil, Tailand and PNG [2, to avoid false-positivity. 44, 48]. Te global trend of declining P. vivax malaria •• Diferent numbers of genomes per P. vivax blood entails reduced mean population parasite densities and, stage do not permit simple quantifcation of parasi- thus, a lower proportion of infections carrying detectable taemia or gametocytaemia. Te most robust quantif- gametocytes [2]. How much these low density infections cation consists of the copy numbers of the molecular contribute to transmission remains unclear. An indica- marker detected per µL of whole blood equivalent. tion that low density infections may be relevant derives •• Tere is no need for specifc gametocyte assays in from feeding assays conducted in Tailand, surveillance and monitoring of interventions, as P. showing that P. vivax infections < 10 parasites/µL can be vivax asexual densities and gametocyte densities are infective, though only rarely and yielding few oocyts [49]. well correlated. Similarly, mosquito feeding assays will be required to •• Some limitations for NAAT cannot be resolved, such determine the infectivity of P. vivax gametocytes follow- as imperfect detection derived from restrictions in ing drug treatment. Transmission during convalescence blood volume, sampling procedures in the feld or might be relevant in elimination settings, where levels of chance efects in detecting a very low abundant PCR acquired immunity are low and therefore a large propor- template. tion of all those infected will seek treatment. •• It is important to investigate methodological limita- tions and shortfalls of the diagnostic techniques used Conclusions and consider their efects on clinical trial outcomes, Key points with particular relevance for P. vivax diagno- as well as on the planning of interventions. sis in community samples: Additional•• fles

Additional fle 1: Table S1. Gene IDs of P. vivax 18S rRNA (small subunit rRNA gene). Additional fle 2: Table S2. Assay conditions for P. vivax qPCR targeting cox1 (Pv-mtCOX1 assay). Additional fle 3: Table S3. Performance Pv-mtCOX1 qPCR. Additional fle 4: Table S4. Limit of detection of Pv-mtCOX1. Additional fle 5: Figure S1. Fold-diference in template copy numbers detected by molecular marker Pv-mtCOX1 versus marker Pv18S rRNA. Additional fle 6: Figure S2. LAMP reaction detected with hydroxynaph- tol blue (HNB). Additional fle 7: Figure S3. Real-time LAMP reaction with calcein detection.

Abbreviations Fig. 5 Correlation between P. vivax parasite density measured by cytB: cytochrome B; cox1: cytochrome C oxidase I; cox3: cytochrome C oxidase 18S rRNA qPCR and P. vivax gametocyte density determined as pvs25 III; ddPCR: digital droplet PCR; LM: light microscopy; LAMP: loop-mediated transcript numbers by qRT-PCR (graph based on data originally isothermal amplifcation; NAAT: nucleic acid amplifcation technique; mtCOX1: published in [2]) mitochondrial cytochrome C oxidase 1; mtDNA: mitochondrial DNA; PCR: Gruenberg et al. Malar J (2018) 17:55 Page 9 of 10

polymerase chain reaction; PNG: Papua New Guinea; POC: point-of-care; qPCR: 4. Cheng Q, Cunningham J, Gatton ML. Systematic review of sub-micro- quantitative PCR; qRT-PCR: quantitative reverse transcription PCR; RDT: rapid scopic P. vivax infections: prevalence and determining factors. PLoS Negl diagnostic test; RFLP: restriction fragment length polymorphism; TPP: target Trop Dis. 2015;9:e3413. product profle. 5. Moreira CM, Abo-Shehada M, Price RN, Drakeley CJ. A systematic review of sub-microscopic Plasmodium vivax infection. Malar J. 2015;14:360. Authors’ contributions 6. WHO. Basic malaria microscopy. Geneva: World Health Organization; MG, CAM, RW, NEH, CK, WMM, GCM, AK performed laboratory work and 2010. helped to edit the manuscript. ML, AMS, IM, IF contributed to conception, 7. Imwong M, Hanchana S, Malleret B, Renia L, Day NP, Dondorp A, et al. study design and supervision of feld projects. IF wrote the manuscript. All High-throughput ultrasensitive molecular techniques for quantifying authors read and approved the fnal manuscript. low-density malaria parasitemias. J Clin Microbiol. 2014;52:3303–9. 8. Ding XC, Ade MP, Baird JK, Cheng Q, Cunningham J, Dhorda M, et al. Author details Defning the next generation of Plasmodium vivax diagnostic tests for 1 Swiss Tropical and Public Health Institute, Socinstrasse 57, 4002 Basel, Swit- control and elimination: target product profles. PLoS Negl Trop Dis. zerland. 2 University of Basel, Basel, Switzerland. 3 Walter and Eliza Hall Institute 2017;11:e0005516. of Medical Research, Parkville, VIC, Australia. 4 Fundação de Medicina Tropical 9. Li J, Gutell RR, Damberger SH, Wirtz RA, Kissinger JC, Rogers MJ, et al. Dr. Heitor Vieira Dourado (FMT-HVD), Manaus, Brazil. 5 Universidade do Estado Regulation and trafcking of three distinct 18 S ribosomal RNAs during do Amazonas, Manaus, Brazil. 6 Instituto Nacional de Infectologia, Evandro development of the malaria parasite. J Mol Biol. 1997;269:203–13. Chagas, Fiocruz, Rio de Janeiro, Brazil. 10. Wampfer R, Mwingira F, Javati S, Robinson L, Betuela I, Siba P, et al. Strategies for detection of Plasmodium species gametocytes. PLoS ONE. Acknowledgements 2013;8:e76316. We thank the feld and laboratory teams in Brazil and PNG. 11. Lopes SC, Albrecht L, Carvalho BO, Siqueira AM, Thomson-Luque R, Nogueira PA, et al. Paucity of Plasmodium vivax mature schizonts in Competing interests peripheral blood is associated with their increased cytoadhesive poten- The authors declare that they have no competing interests. tial. J Infect Dis. 2014;209:1403–7. 12. 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Development of a highly sensitive genus-specifc quantitative reverse Ethics approval and consent to participate transcriptase real-time PCR assay for detection and quantitation of This study was approved by the Brazilian National Committee of Ethics Plasmodium by amplifying RNA and DNA of the 18S rRNA genes. J Clin (CONEP) (349.211/2013). All participants were informed about the objectives Microbiol. 2011;49:2946–53. of the study as well as the potential risks and benefts of their participation 15. Adams M, Joshi SN, Mbambo G, Mu AZ, Roemmich SM, Shrestha B, in the study. An informed consent form was signed by all study participants et al. An ultrasensitive reverse transcription polymerase chain reaction or by a parent or legal guardian if participants were younger than 18 years. assay to detect asymptomatic low-density Plasmodium falciparum and Children between 12 and 17 years signed an additional assent form. Plasmodium vivax infections in small volume blood samples. 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