A New Method to Detect Variants of SARS-CoV-2 Using Reverse Transcription Loop-Mediated Isothermal Amplification Combined with a Bioluminescent Assay in Real Time (RT-LAMP-BART).

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), of which there are several variants. The three major variants (Alpha, Delta, and Omicron) carry the N501Y, L452R, and Q493R/Q498R mutations, respectively, in the S gene. Control of COVID-19 requires rapid and reliable detection of not only SARS-CoV-2 but also its variants. We previously developed a reverse transcription loop-mediated isothermal amplification assay combined with a bioluminescent assay in real time (RT-LAMP-BART) to detect the L452R mutation in the SARS-CoV-2 spike protein. In this study, we established LAMP primers and peptide nucleic acid probes to detect N501Y and Q493R/Q498R. The LAMP primer sets and PNA probes were designed for the N501Y and Q493R/Q498R mutations on the S gene of SARS-CoV-2. The specificities of RT-LAMP-BART assays were evaluated using five viral and four bacterial reference strains. The sensitivities of RT-LAMP-BART assays were evaluated using synthetic RNAs that included the target sequences, together with RNA-spiked clinical nasopharyngeal and salivary specimens. The results were compared with those of conventional real-time reverse transcription-polymerase chain reaction (RT-PCR) methods. The method correctly identified N501Y and Q493R/Q498R. Within 30 min, the RT-LAMP-BART assays detected up to 100-200 copies of the target genes; conventional real-time RT-PCR required 130 min and detected up to 500-3000 copies. Surprisingly, the real-time RT-PCR for N501Y did not detect the BA.1 and BA.2 variants (Omicron) that exhibited the N501Y mutation. The novel RT-LAMP-BART assay is highly specific and more sensitive than conventional real-time RT-PCR. The new assay is simple, inexpensive, and rapid; thus, it can be useful in efforts to identify SARS-CoV-2 variants of concern.

An improved primer design for the loop-mediated isothermal amplification (LAMP) method to detect oxacillinase (OXA)-48 ß-lactamase genes in Gram-negative bacteria for clinical applications.

INTRODUCTION: Recently, increased frequencies of carbapenemase-producing Enterobacteriaceae have been reported worldwide. Among multiple genetic subtypes, oxacillinase (OXA)-48 ß-lactamase-producing strains have been associated with inbound infection because they have been detected predominantly in patients who traveled outside of Japan. However, a recent case report of OXA-48 ß-lactamase-producing Enterobacteriaceae suggested the latent spread of domestic infections. Due to a lack of specific inhibitors, culture-based detection of OXA-48 ß-lactamase-producing bacteria is difficult. Thus, DNA-based detection methods, including PCR, direct sequencing and loop-mediated isothermal amplification (LAMP), have been employed. Among these methods, LAMP detection is more favorable than other methods because of its technical simplicity and low cost. METHODS: We designed novel LAMP primers to detect OXA-48 ß-lactamase-producing bacteria and investigated their possible clinical applications with bacterial genome-spiked human materials (cerebrospinal fluid, blood, feces, urine, and sputum). We evaluated the specificity of the LAMP primers using 37 bacterial strains: 8 standard, 9 reference, and 20 clinical Gram-negative strains. RESULTS: Our LAMP primers detected 10 copies of the OXA-48 type ß-lactamase gene and exhibited no cross reactivity with other ß-lactamase genes. Sensitivity was not influenced in any clinical sample, in contrast to PCR detection, which was strongly inhibited by substances in fecal samples. CONCLUSIONS: These results suggest the superior performance of LAMP compared with conventional PCR for detecting the OXA-48 type ß-lactamase gene in various clinical samples.

Recent advances in tuberculosis diagnostics in resource-limited settings.

Smear-negative and drug-resistant cases of tuberculosis (TB) disease necessitate the development of new diagnostic methods, especially in resource-limited settings. To improve the current TB situations, sensitive and specific TB point-of-care tests (POCTs) should be developed. This review addresses the current status of TB, novel diagnostic methodologies for TB, and the impact of those new diagnostics on TB control in such situations. Moreover, the perspective of TB management based on laboratory examinations is described. Smear microscopy with sputum samples is the only laboratory examination available in many resource-limited settings and is still used globally. Several nucleic acid amplification tests (NATs) have been developed. The World Health Organization (WHO) endorsed novel diagnostics based on NATs and updated their definition of a bacteriologically confirmed case requiring the biological specimen to be positive by smear microscopy, culture, or the WHO-recommended rapid diagnostic protocols. The use of new diagnostics increased the number of bacteriologically confirmed TB cases. Novel diagnostics are now available, but their sensitivity is still lower than that of conventional liquid culture method. To address the increasing incidence of TB, more resources including novel diagnostics as POCTs with higher sensitivity must be allocated to healthcare systems.

Frequent isolation of extended-spectrum beta-lactamase-producing bacteria from fecal samples of individuals with severe motor and intellectual disabilities.

Extended-spectrum beta-lactamase (ESBL) producing bacteria spread worldwide and became major concern for antibiotic treatment. Although surveillance reports in general hospitals and long-term care facilities are increasing, their frequencies in individuals with severe motor and intellectual disabilities (SMID) are so far unknown. In this study, we examined the frequency of ESBL in stool samples collected from 146 asymptomatic SMID subjects hospitalized in a single institution. With their clinical information, we evaluated possible risk factors for ESBL colonization. From 146 fecal samples, ESBL-producing bacteria were isolated in 45 cases (31%). Drug sensitivity testing showed that 82% of the isolates were resistant to levofloxacin but were sensitive to tazobactam/piperacillin and cefmetazole. The most frequent genotype was CTX-M-9 detected in 36/45 (80%). A high degree of disability, antibiotic use within three months before sampling and post-tracheostomy were statistically significant risk factors. Tube feeding was also strongly correlated with ESBL colonization (p < 0.001) and associated with lower micro-organismic diversities. Our findings are the first to reveal a high prevalence of ESBL in the fecal samples of SMID individuals and suggest possible relationships between high degree disability, tube feeding and latest histories of antibiotic use.

Pan-Lineage Mycobacterium tuberculosis Reference Genome for Enhanced Molecular Diagnosis.

In Mycobacterium tuberculosis (MTB) control, whole genome sequencing-based molecular drug susceptibility testing (molDST-WGS) has emerged as a pivotal tool. However, the current reliance on a single-strain reference limits molDST-WGS's true potential. To address this, we introduce a new pan-lineage reference genome, "MtbRf". We assembled "unmapped" reads from 3,614 MTB genomes (751 L1; 881 L2; 1,700 L3; and 282 L4) into 35 shared, annotated contigs (54 CDSs). We constructed MtbRf through: 1) searching for contig homologs among genome database that precipitating results uniquely within Mycobacteria genus; 2) comparing genomes with H37Rv ("lift-over") to define 18 insertions; and 3) filling gaps in H37Rv with insertions. MtbRf adds 1.18% sequences to H37rv, salvaging >60% of previously unmapped reads. Transcriptomics confirmed gene-expression of new CDSs. The new variants provided a moderate DST predictive value (AUROC 0.60-0.75). MtbRf thus unveils previously hidden genomic information, and lays the foundation for lineage-specific molDST-WGS.

SARS-CoV-2 detection in pediatric dental clinic wastewater reflects the number of local COVID-19 cases in children under 10 years old.

This was the first longitudinal study to analyze dental clinic wastewater to estimate asymptomatic SARS-CoV-2 infection trends in children. We monitored wastewater over a 14-month period, spanning three major COVID-19 waves driven by the Alpha, Delta, and Omicron variants. Each Saturday, wastewater was sampled at the Pediatric Dental Clinic of the only dental hospital in Japan's Saitama Prefecture. The relationship between the weekly number of cases in Saitama Prefecture among residents aged < 10 years (exposure) and wastewater SARS-CoV-2 RNA detection (outcome) was examined. The number of cases was significantly associated with wastewater SARS-CoV-2 RNA positivity (risk ratio, 5.36; 95% confidence interval, 1.72-16.67; Fisher's exact test, p = 0.0005). A sample from Week 8 of 2022 harbored the Omicron variant. Compared to sporadic individual testing, this approach allows continuous population-level surveillance, which is less affected by healthcare seeking and test availability. Since wastewater from pediatric dental clinics originates from the oral cavities of asymptomatic children, such testing can provide important information regarding asymptomatic COVID-19 in children, complementing clinical pediatric data.

Corrigendum: Molecular Serotype-Specific Identification of Non-type b Haemophilus influenzae by Loop-Mediated Isothermal Amplification.

[This corrects the article DOI: 10.3389/fmicb.2017.01877.].

Detection of SARS-CoV-2 and the L452R spike mutation using reverse transcription loop-mediated isothermal amplification plus bioluminescent assay in real-time (RT-LAMP-BART).

The new coronavirus infection (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be fatal, and several variants of SARS-CoV-2 with mutations of the receptor-binding domain (RBD) have increased avidity for human cell receptors. A single missense mutation of U to G at nucleotide position 1355 (U1355G) in the spike (S) gene changes leucine to arginine (L452R) in the spike protein. This mutation has been observed in the India and California strains (B.1.617 and B.1.427/B.1.429, respectively). Control of COVID-19 requires rapid and reliable detection of SARS-CoV-2. Therefore, we established a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay plus a bioluminescent assay in real-time (BART) to detect SARS-CoV-2 and the L452R spike mutation. The specificity and sensitivity of the RT-LAMP-BART assay was evaluated using synthetic RNAs including target sequences and RNA-spiked clinical nasopharyngeal and saliva specimens as well as reference strains representing five viral and four bacterial pathogens. The novel RT-LAMP-BART assay to detect SARS-CoV-2 was highly specific compared to the conventional real-time RT-PCR. Within 25 min, the RT-LAMP-BART assay detected 80 copies of the target gene in a sample, whereas the conventional real-time RT-PCR method detected 5 copies per reaction within 130 min. Using RNA-spiked specimens, the sensitivity of the RT-LAMP-BART assay was slightly attenuated compared to purified RNA as a template. The results were identical to those of the conventional real-time RT-PCR method. Furthermore, using a peptide nucleic acid (PNA) probe, the RT-LAMP-BART method correctly identified the L452R spike mutation. This is the first report describes RT-LAMP-BART as a simple, inexpensive, rapid, and useful assay for detection of SARS-CoV-2, its variants of concern, and for screening of COVID-19.

Development of a novel loop-mediated isothermal amplification assay for ß-lactamase gene identification using clinical isolates of Gram-negative bacteria.

Rapid evaluation of antimicrobial susceptibility is important in the treatment of nosocomial infections by Gram-negative bacteria, which increasingly carry carbapenemases and metallo-ß-lactamases. We developed loop-mediated isothermal amplification (LAMP)-based assays for four ß-lactamase genes (bla KPC, bla NDM-1, bla IMP-1 group, and bla VIM). The assays were evaluated using eight reference bacterial strains (Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter bereziniae) harboring six ß-lactamase genes. A total of 55 Gram-negative bacterial strains, including 47 clinical P. aeruginosa isolates, fully characterized by next-generation sequencing (NGS), were used to evaluate the LAMP assays. The results were compared to those of conventional PCR. The LAMP assays were able to detect as few as 10 to 100 copies of a gene, compared to 10 to 104 copies for conventional PCR. The LAMP assay detected four ß-lactamase genes with a sensitivity similar to that using purified DNA as the template in DNA-spiked urine, sputum, and blood specimens. By contrast, the sensitivity of PCR was 1- to 100-fold lower with DNA-spiked clinical specimens. Therefore, the LAMP assays were proved to be an appropriate tool for the detection of four ß-lactamases.

Loop-mediated isothermal amplification assay for detection of Haemophilus influenzae type b in cerebrospinal fluid.

Haemophilus influenzae type b (Hib) is one of the leading causes of meningitis in developing countries. To establish and evaluate a novel loop-mediated isothermal amplification (LAMP) assay for Hib, we designed a LAMP primer set targeting the Hib-specific capsulation locus. LAMP detected 10 copies of purified DNA in a 60-min reaction. This indicated that the detection limit of LAMP was >100-fold lower than the detection limits of both a PCR for the detection of bexA and a nested PCR for Hib (Hib PCR). No H. influenzae, other than Hib or control bacteria, was detected. Linear determination ranged from 10 to 1,000,000 microorganisms per reaction mixture using real-time turbidimetry. We evaluated the Hib LAMP assay using a set of 52 randomly selected cerebrospinal fluid (CSF) specimens obtained from children with suspected meningitis. For comparison, the CSF specimens were tested using a conventional Hib PCR assay. Hib was detected in 30 samples using LAMP and in 22 samples using the Hib PCR assay. The Hib PCR showed a clinical sensitivity of 73.3% and a clinical specificity of 100% relative to the Hib LAMP assay. These results suggest that further development and evaluation of the Hib LAMP will enhance the global diagnostic capability for Hib detection.

Effect of xylitol gum on the level of oral mutans streptococci of preschoolers: block-randomised trial.

OBJECTIVES: To assess the influence of xylitol chewing gum consumption on mutans streptococci level of 3-4 years old Japanese preschoolers. METHODS: 248 participants were examined regarding caries-related factors at baseline and were followed up at 6, 9, and 12 months after the baseline: assessors were blinded, subjects were open labelled and blocked parallel randomised; 142 were selected to use xylitol gum for 3 months (from months 6 to 9) and 106 were controls. RESULTS: 161 participants were analysed (xylitol n = 76, control n = 85). Nineteen caries-related variables, including xylitol gum consumption, were analysed for any association with the main outcome, plaque mutans streptococci scores development within the intervention period, by logistic regression. Six showed statistically significant associations by univariate analysis (P < 0.05). However, only xylitol gum consumption remained a significant negative association (P < 0.05) by multiple analyses. Interestingly, over 10% xylitol group children experienced diarrhoea, which was larger than previous investigations. CONCLUSION: Xylitol gum is effective in avoiding increased plaque mutans streptococci in young children.

Tuberculosis: A persistent unpleasant neighbour of humans.

Mycobacterium tuberculosis, the bacterium that causes tuberculosis, has long been an unpleasant neighbour of humans. Following transmission of the bacterium from patients with active infection, new hosts do not immediately develop symptoms, as M. tuberculosis initially remains quiescent. However, it is eventually triggered, leading to the infection of other individuals. Humans are the exclusive host, and the rapid proliferation of the human population worldwide along with increasing globalisation have contributed to the pathogen's persistence, as have the survival strategies employed by M. tuberculosis, especially its resistance to several antimicrobials. Defeating this enemy will require novel approaches.

23-valent polysaccharide vaccine (PPSV23)-targeted serotype-specific identification of Streptococcus pneumoniae using the loop-mediated isothermal amplification (LAMP) method.

Reports of invasive disease due to Streptococcus pneumoniae have declined since the introduction of pneumococcal conjugate vaccines (PCV7 and PCV13). The incidence of invasive diseases due to S. pneumoniae that are not addressed by the vaccines, however, has increased in children and adults, creating a global public health problem. Previously, we established the loop-mediated isothermal amplification (LAMP) method for a PCV13 serotype-specific assay. In the current study, we developed a rapid, simple, and cost-effective assay to detect serotypes in the 23-valent pneumococcal polysaccharide vaccine (PPSV23) using the LAMP method. In this study, LAMP primer sets for serotypes 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, and 33F of S. pneumoniae were developed. The reactivity, specificity, and sensitivity of LAMP assays were determined and compared to those of conventional PCR. The feasibility of LAMP assays in clinical application in patients with invasive pneumococcal diseases was validated by defining the detection limit of the LAMP assay with bacterial genomic DNA-spiked blood specimens. The specificity of each LAMP assay was determined using 44 serotypes of pneumococcal strains. Their sensitivity was 100 copies per reaction versus 103 to 106 copies per reaction for PCR assays. Using DNA-spiked blood specimens, excluding the LAMP assay that targeted serotype 22F (103 copies per reaction), the limit of detection of the LAMP assay was similar to that with purified DNA as the template (102 copies per reaction), compared with 103 to >106 copies per reaction for PCR assays. In conclusion, a rapid and simple LAMP-based PPSV23-targeted serotype detection assay was developed for use in many countries. This study is the first report of a LAMP-based assay for identification of PPSV23 serotypes. Further evaluation of this assay is needed through surveillance and vaccine efficacy studies.

Rapid and Accurate Species Identification of Mitis Group Streptococci Using the MinION Nanopore Sequencer.

Differentiation between mitis group streptococci (MGS) bacteria in routine laboratory tests has become important for obtaining accurate epidemiological information on the characteristics of MGS and understanding their clinical significance. The most reliable method of MGS species identification is multilocus sequence analysis (MLSA) with seven house-keeping genes; however, because this method is time-consuming, it is deemed unsuitable for use in most clinical laboratories. In this study, we established a scheme for identifying 12 species of MGS (S. pneumoniae, S. pseudopneumoniae, S. mitis, S. oralis, S. peroris, S. infantis, S. australis, S. parasanguinis, S. sinensis, S. sanguinis, S. gordonii, and S. cristatus) using the MinION nanopore sequencer (Oxford Nanopore Technologies, Oxford, UK) with the taxonomic aligner "What's in My Pot?" (WIMP; Oxford Nanopore's cloud-based analysis platform) and Kraken2 pipeline with the custom database adjusted for MGS species identification. The identities of the species in reference genomes (n = 514), clinical isolates (n = 31), and reference strains (n = 4) were confirmed via MLSA. The nanopore simulation reads were generated from reference genomes, and the optimal cut-off values for MGS species identification were determined. For 31 clinical isolates (S. pneumoniae = 8, S. mitis = 17 and S. oralis = 6) and 4 reference strains (S. pneumoniae = 1, S. mitis = 1, S. oralis = 1, and S. pseudopneumoniae = 1), a sequence library was constructed via a Rapid Barcoding Sequencing Kit for multiplex and real-time MinION sequencing. The optimal cut-off values for the identification of MGS species for analysis by WIMP and Kraken2 pipeline were determined. The workflow using Kraken2 pipeline with a custom database identified all 12 species of MGS, and WIMP identified 8 MGS bacteria except S. infantis, S. australis, S. peroris, and S. sinensis. The results obtained by MinION with WIMP and Kraken2 pipeline were consistent with the MGS species identified by MLSA analysis. The practical advantage of whole genome analysis using the MinION nanopore sequencer is that it can aid in MGS surveillance. We concluded that MinION sequencing with the taxonomic aligner enables accurate MGS species identification and could contribute to further epidemiological surveys.

Development of a Novel Loop-Mediated Isothermal Amplification Method to Detect Guiana Extended-Spectrum (GES) ß-Lactamase Genes in Pseudomonas aeruginosa.

Infections caused by multidrug-resistant Pseudomonas aeruginosa in hospitalized patients are often fatal, and nosocomial infections caused by Guiana extended-spectrum (GES) ß-lactamase-producing strains are of growing concern. Several genotypes of the GES ß-lactamase gene (bla GES) include a single missense mutation, a change from G to A at nucleotide position 493 (G493A) that changes glycine to serine; the mutant enzyme exhibits carbapenemase activity. Rapid and reliable identification of drug-resistance is important in clinical settings; however, culture methods remain the gold standard. Conventional and real-time PCR cannot identify carbapenemase-producing genotypes, and direct DNA sequencing is essential. We established a novel loop-mediated isothermal amplification (LAMP) method to detect various genotypes of bla GES and another LAMP method to discriminate carbapenemase genotypes of bla GES. We evaluated the two assays using clinical P. aeruginosa strains. Two primer sets targeting bla GES (GES-LAMP) and the point mutation (Carba-GES-LAMP) were designed and evaluated for specificity and sensitivity. The detection limit of the GES-LAMP method was assessed using purified DNA and DNA-spiked clinical samples (urine, sputum, and blood). To determine the clinical usefulness of the methods, we used different (genotypically and phenotypically) P. aeruginosa clinical isolates, collected from diverse geographical locations between 2003 and 2012. The novel LAMP assay targeting bla GES was highly specific. The detection limit was 10 DNA copies per reaction; the assay was 10-fold more sensitive than conventional PCR. The LAMP assay detected bla GES with high sensitivity in all DNA-spiked samples; PCR did not detect bla GES in blood samples. The GES-LAMP method correctly detected the 5 isolates containing bla GES among the 14 isolates tested. Using these isolates, we confirmed that our Carba-GES-LAMP method of detecting point mutations correctly identified the two bla GES positive organisms with carbapenemase activity. To the best of our knowledge, this is the first report of the GES ß-lactamase gene detection assay using the LAMP method. Our new assays effectively detect bla GES and critical unique mutations.

Molecular serotype-specific identification of Streptococcus pneumoniae using loop-mediated isothermal amplification.

In children, the incidence of pneumococcal meningitis has decreased since the introduction of pneumococcal conjugate vaccine (PCV7 and PCV13). However, since the introduction of the vaccine, developed countries have seen the emergence of non-PCV13 serotypes. However, invasive pneumococcal disease (IPD) caused by PCV13-targeted serotypes still represents an important public health problem in resource-limited countries. To develop a rapid, simple, and cost-effective assay to detect serotypes of Streptococcus pneumoniae, we developed a novel loop-mediated isothermal amplification (LAMP) assay based on the sequences available for the 13 capsular types that are included in PCV13: 1, 3, 4, 5, 6 A, 6B, 7 F, 9 V, 14, 18 C, 19 A, 19 F, and 23 F. We evaluated test reactivity, specificity, sensitivity and performance, and compared the results between established LAMP and conventional PCR assays. To support its clinical use, the detection limits of the LAMP assay were evaluated using bacterial genomic DNA-spiked cerebrospinal fluid (CSF) and blood specimens. We confirmed the specificity of the LAMP assay using 41 serotypes of pneumococcal strains. The sensitivity of the LAMP assay was 10 to 100 copies per reaction, compared to 10 to 104 copies per reaction for PCR assays. The detection limits of the LAMP assay were comparable when using DNA-spiked CSF and blood specimens, as compared to using purified DNA as the template. In conclusion, a rapid and simple LAMP-based pneumococcal serotyping method has been developed. This is the first report of a LAMP method for a PCV13 serotype-specific identification assay, which could be a promising step to facilitate epidemiological studies of pneumococcal serotyping.

Novel loop-mediated isothermal amplification method for detection of the JP2 clone of Aggregatibacter actinomycetemcomitans in subgingival plaque.

We developed a loop-mediated isothermal amplification method that detects the JP2 clone of Aggregatibacter actinomycetemcomitans, which induces aggressive periodontitis in adolescents of North and West African descents. Being independent of special equipment, this specific and sensitive method offers significant advantages for screening of patients on a population basis and in clinical settings.

Rapid and simple detection of Ureaplasma species from vaginal swab samples using a loop-mediated isothermal amplification method.

PROBLEM: Ureaplasma species occasionally cause chorioamnionitis and premature labor. We developed a novel assay employing a loop-mediated isothermal amplification (LAMP) method to detect Ureaplasma parvum and Ureaplasma urealyticum. METHOD OF STUDY: Loop-mediated isothermal amplification primers were designed to amplify Ureaplasma-specific ureaseB genes. Four U. parvum strains, 5 U. urealyticum strains and 14 reference bacterial species were evaluated. Forty-six vaginal swab samples were analyzed by LAMP, culture, and PCR. RESULTS: Our LAMP primers were specific to each species and had no cross-reaction. Of 46 clinical specimens, the sensitivity, specificity, and positive and negative predictive values of the LAMP method were 100% (12/12), 100% (34/34), 100% (12/12), and 100% (34/34), respectively, whereas those of PCR were 66.7% (8/12), 100% (34/34), 100% (8/8), and 89.5% (34/38), respectively, compared to culture-based detection. CONCLUSION: The LAMP detection method outperformed the culture and PCR methods. Early detection enables appropriate antibiotic selection for improved prenatal outcomes.

Loop-Mediated Isothermal Amplification Methods for Diagnosis of Bacterial Meningitis.

The rapid, accurate, and efficient identification of an infectious disease is critical to ensure timely clinical treatment and prevention in public health settings. In 2015, meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis was responsible for 379,200 (range: 322,700-444,700) deaths. Clinical features alone cannot determine whether bacterial meningitis is present; an analysis of cerebrospinal fluid (CSF) is essential. Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method offering an alternative to polymerase chain reaction (PCR). LAMP-based assays for detection of three leading bacteria in CSF for diagnosis of meningitis have been established. The typing assays using LAMP for detection of meningococcal serogroups A, B, C, W, X, and Y as well as H. influenzae serotypes a, b, c, d, e, and f were launched. In comparative analysis of the meningitis pathogen assays, LAMP assays did not yield false negative results, and the detection rate of LAMP assays was superior compared with PCR or conventional culture methods. LAMP assays provide accurate and rapid test results to detect major bacterial meningitis pathogens. Accumulating evidence suggests that LAMP assays have the potential to provide urgently needed diagnostics for bacterial meningitis in resource-limited settings of both developed and developing countries.

Detection of Haemophilus influenzae by loop-mediated isothermal amplification (LAMP) of the outer membrane protein P6 gene.

It is difficult and time-consuming to distinguish Haemophilus influenzae from the genotypically similar Haemophilus parainfluenzae, which is a commensal of the human oral cavity. The novel nucleic acid amplification technique of loop-mediated isothermal amplification (LAMP), which amplifies DNA under isothermal conditions (63 degrees C) with high specificity, efficiency, and rapidity, was evaluated for H. influenzae detection. A H. influenzae-specific LAMP primer set was designed for the outer membrane protein P6 gene. Primer set specificity was validated using 4 Haemophilus spp. and 13 other species. Within 60 min, LAMP detected 100 or more copies of purified DNA with a sensitivity that was 10-fold higher than that of conventional PCR. This method can be used to differentiate H. influenzae from H. parainfluenzae strains. Thus, LAMP may represent a sensitive and reliable means of diagnosing H. influenzae infection.