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

Abstract

Background The unexpected high proportion of submicroscopic malaria infections in areas with low transmission intensity challenges the control and elimination of malaria in the Americas. The current PCR-based assays present limitations as most protocols still rely on amplification of few-copies target gene. Here, the hypothesis was that amplification of different plasmodial targets—ribosomal ( 18S rRNA ) and non-ribosomal multi-copy sequences (Pvr47 for Plasmodium vivax and Pfr364 for Plasmodium falciparum )—could increase the chances of detecting submicroscopic malaria infection. Methods A non-ribosomal real-time PCR assay targeting Pvr47/Pfr364 ( NR - qPCR ) was established and compared with three additional PCR protocols, two of them based on 18S rRNA gene amplification ( Nested - PCR and R - qPCR ) and one based on Pvr47/Pfr364 targets ( NR - cPCR ). The limit of detection of each PCR protocol, at single and artificial mixed P. vivax / P. falciparum infections, was determined by end-point titration curves. Field samples from clinical (n = 110) and subclinical (n = 324) malaria infections were used to evaluate the impact of using multiple molecular targets to detect malaria infections. Results The results demonstrated that an association of ribosomal and non-ribosomal targets did not increase sensitivity to detect submicroscopic malaria infections. Despite of that, artificial mixed-malaria infections demonstrated that the NR-qPCR was the most sensitive protocol to detect low-levels of P. vivax / P. falciparum co-infections. Field studies confirmed that submicroscopic malaria represented a large proportion (up to 77%) of infections among asymptomatic Amazonian residents, with a high proportion of infections (~ 20%) identified only by the NR-qPCR. Conclusions This study presents a new species-specific non-ribosomal PCR assay with potential to identify low-density P. vivax and P. falciparum infections. As the majority of subclinical infections was caused by P. vivax , the commonest form of malaria in the Amazon area, future studies should investigate the potential of Pvr47/Pfr364 to detect mixed-malaria infections in the field. Electronic supplementary material The online version of this article (10.1186/s12936-019-2781-3) contains supplementary material, which is available to authorized users.

Sensitive universal detection of blood parasites by selective pathogen-DNA enrichment and deep amplicon sequencing

“…The UPDx assay also detected parasites in one replicate at a concentration of 0.058 parasites/µL and failed to detect parasites at a concentration of 0.0058 parasites/µL. The LOD of our UPDx assay is therefore comparable to other recently published qPCR assays, which possess LODs ranging from 0.03 to 0.3 parasites per microliter of blood [ 35 – 37 ]. In addition to its consistent performance and comparatively low LOD, this assay has the added benefit that all blood parasites present in a sample may be detected.…”

Ultrasensitive CRISPR-based diagnostic for field-applicable detection of Plasmodium species in symptomatic and asymptomatic malaria

“…RPA primer targets were identified by reviewing the literature for the best-performing NAATs and searching for conserved and specific sequences from alignment of species-specific strains available from the National Center for Biotechnology Information (NCBI). For P. falciparum 18S rRNA, mitochondrial (cytochrome oxidase III, cytochrome B), and subtelomeric (Pfr364) targets were screened ( 23 – 29 ). The Pfr364 target, which is a species-specific, noncoding subtelomeric repeat sequence present in 41 copies on the P. falciparum genome, had the best signal in comparison to the other targets ( SI Appendix , Fig.…”

Dynamics of IgM and IgG responses to the next generation of engineered Duffy binding protein II immunogen: Strain-specific and strain-transcending immune responses over a nine-year period

“…The Giemsa-stained thick blood smears were prepared and examined by experienced local microscopists, according to the malaria diagnosis guidelines of the Brazilian Ministry of Health (2009) [41]. Species-specific PCR assays targeting different plasmodial targets (18S rRNA gene and non-ribosomal Pvr47/Pfr364 sequences) were carriedout essentially as previously described [42]. For this, genomic DNA was extracted from either whole blood samples collected in EDTA, or from dried blood spots on filter paper using the Puregene blood core kit B (Qiagen, Minneapolis, MN, USA) or the QIAmp DNA mini kit (Qiagen), respectively, according to manufacturers' instructions.…”