Ribosomal and non-ribosomal PCR targets for the detection of low-density and mixed malaria infections

Malaria is a worldwide public health concern that is present in roughly 90 countries, mainly in tropical and subtropical regions [1]. While Plasmodium vivax is the most widely distributed parasite causing malaria, Plasmodium falciparum accounts for the most severe forms of the disease [2]. Although malaria incidence rate is estimated to have decreased by 18% globally between 2010 and 2016, a recent increase in case incidence occurred in the Americas, particularly in the Amazon rainforest [1, 3].

In order to progress towards malaria control and elimination, it is critical to understand the sources of transmission (the infectious reservoir) and those at risk of infection at the population level [4]. In this context, the molecular detection of Plasmodium infections in endemic areas have confirmed previous finding of high frequencies of malaria infections at densities below the limit of conventional microscopic diagnostics [5‐9]. It is particularly relevant as data from systematic reviews have shown that across different geographic areas low-density infections may represent the majority of malaria infections [10, 11]. Accordingly, a substantial proportion of asymptomatic and submicroscopic malarial infections has been described in peri-urban areas of the Brazilian Amazon [12]. Many are the implications of these findings as submicroscopic malaria carriers may be able to transmit the Plasmodium parasites, acting as reservoirs for malaria [13, 14]. Beyond the practical value of using molecular tools to identify submicroscopic carriers and mixed-species infections, malaria infections at any density may have significant health and socioeconomic consequences [15].

Historically, the small subunit 18S of the ribosomal RNA gene (18S rRNA) has been the most common target used for molecular diagnosis of malaria [16‐20]. As this gene is present in few copies (5 to 8) in the genome of Plasmodium parasites [21], low sensitivity and reproducibility of standard PCR protocols based on 18S rRNA gene amplification have been described [22, 23]. In the last decade, the genomic data mining of Plasmodium parasites has allowed the discovery of new species-specific multi-copy targets which show potential for molecular diagnosis of P. vivax and P. falciparum malaria [24‐26]. Among the promising targets include the non-coding subtelomeric repeat sequences Pvr47 and Pfr364 that are present in 14 and 41 copies in the genomes of P. vivax and P. falciparum, respectively [24]. While there is evidence for their location and distribution, the biological functions of Pvr47 and Pfr364 remains to be established. By using a single-step PCR assay to amplify Pvr47/Pfr364 targets, it was possible to demonstrate the relatively higher sensitivity of these targets as compared to the amplification of 18S rRNA gene by the conventional nested-PCR assay [24].

Since most malaria PCR-based protocols still relies on amplification of 18S rRNA gene, which has low sensitivity to detect low-density infections, we evaluated here how useful Pvr47/Pfr364 targets are to detect single and mixed P. vivax and P. falciparum infections in clinical and subclinical malaria. As the original PCR protocol to amplify Pvr47/Pfr364 involved DNA visualization on gel electrophoresis [24], here a new qPCR protocol targeting these high-copy non-ribosomal sequences was established. The experimental approach evaluated whether amplification of different plasmodial targets (Pvr47/Pfr364 and 18S rRNA gene) could increase the chances of detecting submicroscopic malaria infections. For that, field samples (clinical and subclinical malaria) were amplified by four different PCR assays, two of them targeting Pvr47/Pfr364 sequences [24] and two targeting the 18S rRNA gene [16, 17].

Nested-PCR assay for amplification of 18S rRNA gene was performed as described [22], using genus and species-specific primers previously described [16]. The real-time PCR (R-qPCR) method uses a single pair of genus-specific primers for the detection of the 18S rRNA gene, with two internal species-specific hydrolysis probes for P. falciparum and P. vivax [17]. Primers and cycling conditions for 18SrRNA gene amplification (Nested-PCR and R-qPCR) were included as Additional file 4.

Although major advances have been reached for the molecular detection of malaria parasites [25, 26, 36, 37], most sensitive PCR-based assays require high-volume of venous blood and complex sample processing [8, 23, 38, 39], being not feasible in the context of malaria routine surveillance. The current study involved investigate the hypothesis that the amplification of both ribosomal and non-ribosomal multi-copy PCR targets could increase the chances of detecting low parasite density and mixed P. falciparum and P. vivax infection. For that, a non-ribosomal (NR) qPCR targeting the multi-copy Pvr47/Pfr364 sequences was developed and this new protocol was compared with the original non-ribosomal gel-stained PCR-based protocol [24] as well as with two species-specific PCR assays based on the 18S rRNA gene amplification.

The end-point titration assays of field samples revealed that the NR-qPCR protocol was able to accurately detect both P. vivax and P. falciparum infections—in single and artificial mixed infections—producing reproducible results at the lowest parasite densities (1–3 parasites/µL). Although there was considerable variation between PCR protocols assayed, the non-ribosomal protocols (NR-cPCR and NR-qPCR) were more accurate than ribosomal (nested-PCR and R-qPCR) to detect mixed-species infections. Of interest, only NR-qPCR assay developed here were able to detect P. falciparum when this species was present in a proportion of 240-fold lower than P. vivax. As the sensitivity of any PCR protocol depends largely on the molecular target used [40], the high copy number of Pfr364 (around 20 copies of “subfamily 1” targeted by specific primers) probably facilitated the detection of low levels of P. falciparum in co-infections as compared to 18S rRNA (around 5–8 copies). Although different multi-copy targets have been described as sensitive for molecular diagnosis of malaria [23, 25, 36], those studies did not investigate the reliability of these targets in mixed-malaria infections, which precludes any potential comparison with results described here. In addition, most of the studies have been carried-out in endemic areas, such as Papua New Guinea, that currently does not represent an unstable and low-transmission endemic area [23]. More work needs to be done in this field of investigation. An apparent inability of 18S rRNA qPCR to detect low P. falciparum densities in situation where P. vivax was present in much higher densities was observed. The use of a single pair of primers to detect both species may have been a determinant factor in causing failure of R-qPCR to identify mixed infections. A similar phenomenon of primer competition was described in the original protocol [17], straightening that species-specific primers should be used in field studies in which malaria co-infection is expected to be relevant.

In clinical malaria suspects, the overall prevalence for P. vivax and P. falciparum detected by amplification of non-ribosomal Pvr47/Pfr364 targets was not significantly different than that of either conventional microscopy or 18S rRNA gene amplification. The predicted sensitivity and specificity of each PCR protocol assayed here were also similar, and it was independent of the parasite target. Although the clinical sample size limited the statistical power to detect small differences between protocols, these results were not completely unexpected as symptomatic patients usually present high parasite densities in the peripheral blood; consequently, it may facilitate the confirmation of malaria infection by less sensitive protocols such as microscopy and rapid diagnostic tests (RDTs) [41]. These findings reinforce that submicroscopic malaria infections are not prevalent among symptomatic patients, and LM and RDTs are adequate tools for case management [10, 42]. Nevertheless, the limited sensitivity of microscopy in correct identification of mixed-species malaria should be considered in areas where more than one Plasmodium species is circulating [43, 44], a result that was confirmed here.

While low frequencies of submicroscopic infections were observed in the group of clinical malaria cases (3–5%), screening for malaria in cross-sectional surveys demonstrated a large proportion (> 70%) of malaria cases in the study area that was not detected by conventional microscopy. The majority of subclinical infections were caused by P. vivax, the commonest malaria parasite in the Amazon basin, and frequently associated with low-density infections [27, 28, 45‐47]. These findings are in accordance with recent reports showing high proportions of submicroscopic P. vivax infections across different endemic settings, particularly areas with relatively low transmission intensity [10‐12, 26]. Although the reason for this high rate of asymptomatic P. vivax infections is unknown, it is probably associated with the unique biology of P. vivax that includes a fast acquisition of clinical immunity as compared with P. falciparum [47]. It is particularly relevant because in different epidemiological settings there are perspectives on treating asymptomatic infections for malaria elimination [48]. In the study area, the results demonstrated that, in general, multiple molecular targets (i.e., ribosomal plus non-ribosomal) did not increase sensitivity to detect subclinical malaria infections. Despite of that, the NR-qPCR developed here was the most sensitive protocol to detect submicroscopic asymptomatic malaria infections, which resulted in a significantly higher prevalence of submicroscopic infections (70 out of 73, 96%) when compared to that detected by ribosomal PCR assays (57 out of 73, 78%). While more sensitive amplification of 18S rRNA gene has been described [39, 49], the likelihood of amplify 18S rRNA gene was dependent on (i) large blood volume (2 mL); (ii) careful removal of plasma and buffy coat as prerequisite to avoid interference during PCR processing; (iii) concentration of purified DNA dehydrated in a centrifugal vacuum concentrator; additionally, these “high-volume” 18S rRNA PCR strategy did not allow the detection by species (only Plasmodium spp.) [39].

The apparent ability of Pvr47/Pfr364 NR-qPCR to increase sensitivity to investigate the true prevalence of malaria infection is relevant as an unexpectedly large reservoir of infections may hinder control and elimination of malaria in the Americas [3, 50]. These findings are critical as both subclinical and submicroscopic malaria carriers remain untreated in the Brazilian Amazon region and therefore might remain infective over long periods of time [12]. As parasite densities cannot be assumed as a static parameter and thus may fluctuate over time falling below the detection threshold of the assay [51], future studies should approach longitudinal PCR-malaria surveys. Although the NR-qPCR developed here may constitute powerful additive tools to identify endemic sites where relevant control measures have to be settled and monitored [52], the costs of PCR-based assays limited such type of study. In general, nucleic acid amplification tests (NAATs) require expensive equipment available, well-equipped laboratories, qualified personnel, and large quantities of disposable supplies that need to be frozen or refrigerated, which is sometimes difficult in the country [41, 53]. Currently, WHO recommends that the use of NAATs be considered only for epidemiological research or surveys mapping submicroscopic infections in low transmission areas [54]. Innovative and cost-effective strategies that identify the real burden of malaria infections (those detected by qPCR) are required to reach malaria elimination goals, but remain a challenge [48].

Assuming that the NR-qPCR developed here seems to be the most sensitive method—as it was positive in a number of samples not detected by other PCR protocols—the results suggested that NR-qPCR has a lower detection threshold. Despite of that, it is important to clarify the technical limitations that apply for the definition of “reference standard” for PCR-detection of submicroscopic malaria infections. In general, the estimative of test accuracy are based on the assumptions that the reference standard is 100% sensitive and that specific disagreements between the reference standard and the diagnostic test being evaluated (index test) result from incorrect classification by the index test [55]. However, this statement cannot be applied for the detection of low-density malaria infections because there is no “gold-standard”. While the conventional microscopy diagnostic present high number of false negatives at low parasite density [41], there is no consensus about a PCR assay able to detect all malaria infections [25]. Due to these inherent limitations, the “reference standard” for each molecular diagnostic method was defined as a combination of positive detections by any PCR assay, excluding the method under evaluation, as described before [23]. Consequently, “false positive” in this type of analysis is considered in the sense that no other PCR method found these infections. In this scenario, the findings unlikely represent a tendency to false positive by NR-qPCR as it was established by (i) end-point titration of well-characterized field samples, including mono and artificial mixed-infections; (ii) reproducibility of replicates at low levels of parasitaemia; (iii) no amplification with gDNA samples from malaria-free volunteers; (iv) no cross-reactivity with other Plasmodium species. Furthermore, considering the rules for quality assessment of diagnostic accuracy studies (QUADAS-2) [56], the risk of bias of the present study was reduced as methodological design involved: (i) structured sample size calculations and random selection of malaria-exposed individuals, with explicit exclusion criteria defined in methods; (ii) in the estimative of sensitivity and/or specificity, the diagnostic test being evaluated was clearly interpreted before the reference standard was known; (iii) the execution of the PCR-based assays and the definition of reference standard were described in sufficient detail to permit replication of the test. Consequently, valuable malaria information can be retrieved from the current study.

Finally, relatively low frequencies of mixed-malaria infections were detected here, which precluded a more detailed evaluation of the potential of Pvr47/Pfr364 to detect mixed-malaria infections in the field. In the Amazon area, besides P. vivax being the predominant malaria parasite [27, 29], the progress achieved in malaria control has decreased the number of P. falciparum cases in recent years [57]. Notwithstanding this study limitation, it is highly relevant the results from the end-point titration experiments showing the ability of Pvr47/Pfr364 to consistently detect P. vivax/P. falciparum co-infection, as the accurate detection of malaria mixed-infections seems to be critical for control and management of malaria [43, 44]. In fact, disease burden due to mixed species infections remains largely unknown, and this limitation have the potential to influence decisions on testing vaccines and new antimalarial drugs [58]. As malaria has been re-emerging in areas where it was previously controlled, dealing with mixed malaria infection cannot be bypassed, as recent evidence suggest that the frequency of these infections may be much higher than previously expected [44], including in the Amazon region [59]. Due to the risk of P. falciparum reemergence from Amazonian neighboring countries with high transmission rates, a cross-border malaria study to evaluate the relevance of NR-qPCR in mixed-malaria infections are on progress.