ABSTRACT
BACKGROUND: In this work, a platform was developed and tested to allow to detect a variety of candidate viral, bacterial and parasitic pathogens, for acute fever of unknown origin. The platform is based on a centrifugal microfluidic cartridge, the LabDisk ("FeverDisk" for the specific application), which integrates all necessary reagents for sample-to-answer analysis and is processed by a compact, point-of-care compatible device. METHODOLOGY/PRINCIPAL FINDINGS: A sample volume of 200 µL per FeverDisk was used. In situ extraction with pre-stored reagents was achieved by bind-wash-elute chemistry and magnetic particles. Enzymes for the loop-mediated isothermal amplification (LAMP) were pre-stored in lyopellet form providing stability and independence from the cold chain. The total time to result from sample inlet to read out was 2 h. The proof-of-principle was demonstrated in three small-scale feasibility studies: in Dakar, Senegal and Khartoum, Sudan we tested biobanked samples using 29 and 9 disks, respectively; in Reinfeld, Germany we tested spiked samples and analyzed the limit of detection using three bacteria simultaneously spiked in whole blood using 15 disks. Overall during the three studies, the FeverDisk detected dengue virus (different serotypes), chikungunya virus, Plasmodium falciparum, Salmonella enterica Typhi, Salmonella enterica Paratyphi A and Streptococcus pneumoniae. CONCLUSIONS/SIGNIFICANCE: The FeverDisk proved to be universally applicable as it successfully detected all different types of pathogens as single or co-infections, while it also managed to define the serotype of un-serotyped dengue samples. Thirty-eight FeverDisks at the two African sites provided 59 assay results, out of which 51 (86.4%) were confirmed with reference assay results. The results provide a promising outlook for future implementation of the platform in larger prospective clinical studies for defining its clinical sensitivity and specificity. The technology aims to provide multi-target diagnosis of the origins of fever, which will help fight lethal diseases and the incessant rise of antimicrobial resistance.
Subject(s)
Fever , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Point-of-Care Systems , Chikungunya virus , DNA, Bacterial , Diagnosis, Differential , Germany , Humans , Prospective Studies , Salmonella/genetics , Salmonella paratyphi A , Senegal , SudanABSTRACT
Mycoplasma genitalium is a sexually transmissible, pathogenic bacterium and a significant cause of nongonococcal urethritis in both men and women. Due to the difficulty of the culture of M. genitalium from clinical samples, the laboratory diagnosis of M. genitalium infection is almost exclusively carried out using nucleic acid amplification tests. Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification technology, utilising a set of 4 primers specific to 6 distinct regions of the target DNA sequence, in order to amplify target DNA in a highly specific and rapid manner. A LAMP assay was designed to the pdhD gene of M. genitalium, and the limit of detection of the assay was determined as 10 fg of M. genitalium genomic DNA, equating to ~16 copies of the M. genitalium genome, which was equally sensitive as a gold standard 16S rRNA polymerase chain reaction assay.
Subject(s)
Molecular Diagnostic Techniques/methods , Mycoplasma Infections/diagnosis , Mycoplasma genitalium/isolation & purification , Nucleic Acid Amplification Techniques/methods , Bacterial Proteins/genetics , DNA Primers/genetics , Female , Humans , Male , Mycoplasma Infections/microbiology , Mycoplasma genitalium/genetics , Sensitivity and Specificity , Time FactorsABSTRACT
A loop-mediated isothermal amplification (LAMP) assay for open reading frame 1 (ORF1) of the glutamine synthetase gene of Neisseria gonorrhoeae was able to tolerate urea concentrations of ≤ 1.8 M, compared with a PCR assay that was functional at concentrations of <100 mM. The LAMP assay was as sensitive as the PCR assay while being faster and simpler to perform.