Norihito Tarumoto

A novel diagnostic method for malaria using loop-mediated isothermal amplification (LAMP) and MinION™ nanopore sequencer

BackgroundA simple and accurate molecular diagnostic method for malaria is urgently needed due to the limitations of conventional microscopic examination. In this study, we demonstrate a new diagnostic procedure for human malaria using loop mediated isothermal amplification (LAMP) and the MinION™ nanopore sequencer.MethodsWe generated specific LAMP primers targeting the 18S–rRNA gene of all five human Plasmodium species including two P. ovale subspecies (P. falciparum, P. vivax, P. ovale wallikeri, P. ovale curtisi, P. knowlesi and P. malariae) and examined human blood samples collected from 63 malaria patients in Indonesia. Additionally, we performed amplicon sequencing of our LAMP products using MinION™ nanopore sequencer to identify each Plasmodium species.ResultsOur LAMP method allowed amplification of all targeted 18S–rRNA genes of the reference plasmids with detection limits of 10–100 copies per reaction. Among the 63 clinical samples, 54 and 55 samples were positive by nested PCR and our LAMP method, respectively. Identification of the Plasmodium species by LAMP amplicon sequencing analysis using the MinION™ was consistent with the reference plasmid sequences and the results of nested PCR.ConclusionsOur diagnostic method combined with LAMP and MinION™ could become a simple and accurate tool for the identification of human Plasmodium species, even in resource-limited situations.

Non-invasive diagnosis of cutaneous leishmaniasis by the direct boil loop-mediated isothermal amplification method and MinION™ nanopore sequencing

Cutaneous leishmaniasis (CL) is gaining attention as a public health problem. We present two cases of CL imported from Syria and Venezuela in Japan. We diagnosed them as CL non-invasively by the direct boil loop-mediated isothermal amplification method and an innovative sequencing method using the MinION™ sequencer. This report demonstrates that our procedure could be useful for the diagnosis of CL in both clinical and epidemiological settings.

A novel detection procedure for mutations in the 23S rRNA gene of Mycoplasma pneumoniae with peptide nucleic acid-mediated loop-mediated isothermal amplification assay

Rapid and easy detection of a single nucleotide point mutation of bacterial genes, which is directly linked to drug susceptibility, is essential for the proper use of antimicrobial agents. Here, we established a detection method using a peptide nucleic acid mediated loop-mediated amplification (LAMP) assay for macrolide (ML)-susceptible Mycoplasma pneumoniae. This assay specifically detected the absence of missense mutations encoding the central loop of domain V in the gene encoding 23S rRNA, which can reduce the affinity for MLs and subsequently generate ML-resistant strains of M. pneumoniae. Reactions were performed at 62°C for 60min and targeted gene amplifications were detected by real-time turbidity with a turbidimeter and naked-eye inspection of a color change. The assay had an equivalent detection limit of 100.0fg of DNA with the turbidimeter and showed specificity against 54 types of pathogens, whereas amplification was completely blocked, even at 100.0pg of DNA per reaction, in the presence of point mutations at 2063A and 2064A. The expected LAMP products were confirmed through identical melting curves in real-time LAMP procedures. This method would be a simple and rapid protocol for single nucleotide polymorphism genotyping as point-of-care testing technology without amplification of the sequences carrying the point mutations 2063A and 2064A in ML-resistant M. pneumoniae strains.

Identification of Disseminated Cryptococcosis Using MALDI-TOF MS and Clinical Evaluation

Disseminated cryptococcosis is rare but can often become severe with a poor outcome. Given recent reports that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyser is useful for Cryptococcus species identification, it was applied retrospectively to past cases of disseminated cryptococcosis at our hospital over the past 10 years, and their clinical courses were reviewed. For each case, the retained Cryptococcus spp. were used for identification using both MALDI-TOF MS and genetic sequencing, as well as for drug susceptibility testing. A total of eight cases were found. Cryptococcus spp. were found in cerebrospinal fluid in 3 cases and blood in 5 cases; anti-HIV antibody was either negative or untested. MALDI-TOF MS identified Cryptococcus neoformans as the pathogen in all 8 cases, but genetic testing identified one of these as Cryptococcus curvatus. The outcome was death within 30 days in 5 of the total 8 cases and in 2 of the 3 cases in which C. neoformans was detected in the cerebrospinal fluid, despite regimens and dosages that followed IDSA Guidelines in all 3 cases. Drug susceptibility testing showed no drug resistance that would have affected the therapy. In conclusion, the outcomes were very poor in these drug-susceptible cases, despite treatment in full accordance with standard guidelines. This study confirmed the need to develop newer therapies as well as the high capability of MALDI-TOF MS for the identification of C. neoformans. Genetic testing, however, may be necessary if non-neoformans Cryptococcus is suspected.

Rapid identification of pathogens from positive blood culture bottles with the MinION nanopore sequencer

Purpose. Bloodstream infections are major causes of morbidity and mortality that lead to prolonged hospital stays and higher medical costs. In this study, we aimed to evaluate the MinION nanopore sequencer for the identification of the most dominant pathogens in positive blood culture bottles. Methodology. 16S and ITS1‐5.8S‐ITS2 rRNA genes were amplified by PCR reactions with barcoded primers using nine clinical isolates obtained from positive blood bottles and 11 type strains, including five types of Candida species. Barcoded amplicons were mixed, and multiplex sequencing with the MinION sequencer was performed. In addition, barcoded PCR amplicons were sequenced by Sanger sequencing to validate the performance of the MinION. Results. The bacterial and Candida spp. identified by MinION sequencing, based on the highest homology of reference sequences from the NCBI gene databases, agreed with the matrix‐assisted laser desorption ionization time of flight mass spectrometry results, excepting the closely related species Streptococcus and Escherichia coli. The ‘pass’ reads obtained within about 10 min of sequencing were sufficient to identify the pathogens. The average values of sequence identities with 1D2 chemistry and the R9.5 flow cell were around 99 %; thus, frequent sequence errors did not affect species identification based on amplicon sequencing. Conclusion. We have established a rapid, portable and economical technique for the identification of pathogens in positive blood culture bottles through a novel MinION nanopore sequencer amplicon sequencing scheme, which replaces traditional Sanger sequencing.

Single nucleotide polymorphisms in genes encoding penicillin-binding proteins in β-lactamase-negative ampicillin-resistant Haemophilus influenzae in Japan

Objectiveβ-Lactamase-negative ampicillin-resistant Haemophilus influenzae is a common opportunistic pathogen of hospital- and community-acquired infections, harboring multiple single nucleotide polymorphisms in the ftsI gene, which codes for penicillin-binding protein-3. The objectives of this study were to perform comprehensive genetic analyses of whole regions of the penicillin-binding proteins in H. influenzae and to identify additional single nucleotide polymorphisms related to antibiotic resistance, especially to ampicillin and other cephalosporins.ResultsIn this genome analysis of the ftsI gene in 27 strains of H. influenzae, 10 of 23 (43.5%) specimens of group III genotype β-lactamase-negative ampicillin-resistant H. influenzae were paradoxically classified as ampicillin-sensitive phenotypes. Unfortunately, we could not identify any novel mutations that were significantly associated with ampicillin minimum inhibitory concentrations in other regions of the penicillin-binding proteins, and we reconfirmed that susceptibility to β-lactam antibiotics was mainly defined by previously reported SNPs in the ftsI gene. We should also consider detailed changes in expression that lead to antibiotic resistance in the future because the acquisition of resistance to antimicrobials can be predicted by the expression levels of a small number of genes.