Development and Assessment of a Multiple-Analysis System for Diagnosing Malaria and Other Blood Parasite Infections in Humans and Non-Human Primates.

Background/

Objectives

Many tropical diseases such as malaria, Chagas, human African Trypanosomiasis, and Lymphatic filariasis coexist in endemic countries, affecting more than 1 billion people worldwide, and are recognised as major global vector-borne diseases. Tackling this disease requires an accurate diagnosis that is sensitive, specific, and rapid. This study aimed to describe and validate a new highly sensitive and specific multiple-analysis system that can effectively detect numerous etiological agents in a single test.

Methods

A total of 230 human blood samples were assessed retrospectively for parasite characterisation, as well as 58 stool samples from non-human primates. Primers and probes were designed in the small subunit ribosomal RNA gene, except for Plasmodium spp., for which the novel target was Cytochrome Oxidase Subunit 1.

Results

The analytical specificity of the presented method was 100%, with no unspecific amplifications or cross-reactions with other blood parasitic diseases. The detection limit obtained was between 0.6 and 3.01 parasites/µL for Plasmodium species, 1.8 parasites/mL for Trypanosomatidae, and 2 microfilariae/mL in the case of Filariae. The sensitivity, specificity, predictive values, and kappa coefficient reached almost 100%, except for Filariae, whose sensitivity dropped to 93.9% and whose negative predicted value dropped to 89.5%. The operational features described a turnaround and a hands-on time shorter than the compared methods with a lower cost per essay.

Conclusions

This work presents a cost-effective and highly sensitive multiplexed tool (RT-PCR-bp) capable of performing simultaneous detection for blood parasitic diseases using specific fluorescence probes, enabling the diagnosis of low parasite loads and coinfections.