An Application of Outer Membrane Protein P6-Specific Enzyme-Linked Immunosorbent Assay for Detection of Haemophilus influenzae in Middle Ear Fluids and Nasopharyngeal Secretions

Hotomi, Muneki; Togawa, Akihisa; Kono, Masamitsu; Sugita, Gen; Sugita, Rinya; Fujimaki, Yutaka; Kamide, Yosuke; Uchizono, Akihiro; Kanesada, Keiko; Sawada, Shoichi; Okitsu, Naohiro; Masuda, Hisayo; Tanaka, Hideaki; Tanaka, Yumi; Yamanaka, Noboru

doi:10.1371/journal.pone.0071774

Cited by 5 publications

(4 citation statements)

References 37 publications

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“…In our study, for the identification of H influenzae, we amplified the P6 gene of this organism. This gene is encoded by both typable strains and non‐typable strains of H influenzae , and hence, we could not differentiate between these strains. H influenzae type b is the most aggressive type of this organism.…”

Section: Discussionmentioning

confidence: 97%

Detection of the major bacterial pathogens among children suffering from empyema in Ahvaz city, Iran

Amin

pour

Navidifar

2019

Clinical Laboratory Analysis

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IntroductionEmpyema is one of the important causes of pediatric hospital admissions.AimIn this study, we had investigated the frequency rates of S aureus, S pneumoniae, H influenzae, and P aeruginosa using PCR and bacterial culture among children suffering from empyema in Ahvaz city, Iran.MethodsThis was a descriptive study conducted on the patients hospitalized in ICUs of two teaching Hospitals of Ahvaz, Iran, between March and September 2018 on 105 pleural fluid (PF) samples of the children less than 16 years of age with the diagnosis of empyema thoracis. These specimens were inoculated on the bacterial culture media and identified using biochemical characteristics. Then, the existence of the four pathogens mentioned above was evaluated using PCR method.ResultIn this study, these bacteria agents were identified in 81 (77.14%) and 30 (28.57%) cases using the PCR assay and bacterial culture, respectively. Moreover, the PCR assay identified the infectious agents in 51 (68%) of PFs where the culture method failed. S pneumoniae (63 cases) was recognized as the most common pathogen, followed by P aeruginosa(19 cases), S aureus(15 cases), and H influenzae (9 cases) using the bacterial culture and PCR. Co‐infections were detected in 21 samples (20%) using PCR and one sample using the bacterial culture (P aeruginosa and S pneumoniae).ConclusionIn this study, we found the higher frequencies of these microorganisms using PCR than culture. In addition, we showed that PCR was a sensitive and accurate method that unaffected by antibiotic therapy and could detect well co‐infections.

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Section: Discussionmentioning

confidence: 97%

Detection of the major bacterial pathogens among children suffering from empyema in Ahvaz city, Iran

Amin

pour

Navidifar

2019

Clinical Laboratory Analysis

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“…Several OMPs of NTHi have been proposed as potential vaccine antigens on the basis of their sequence conservation, immunogenicity, and demonstration of significant protection in animal models following immunization (27). Two highly conserved proteins among NTHi strains have shown significant potential as vaccine candidates: P6 and protein D (13,14,22,(28)(29)(30). It was reported in a chinchilla model that immunization with P6 provides protection against AOM due to NTHi (31), and intranasal immunization with P6 has been shown to confer antigen-specific mucosal immunity and enhance mucosal clearance of NTHi (32).…”

Section: Discussionmentioning

confidence: 99%

“…Finally, the color development reaction was terminated by adding 100 l of 2 M H 2 SO 4 , and the absorbance at 450 nm was read. To provide quantitative results on the antibody concentrations, the level of the specific antibody present in the unknown sample was determined by comparison to an internal reference serum (pool of recovered NTHi patient serum with high anti-P6 or protein D titers) (21,22).…”

Section: Methodsmentioning

confidence: 99%

Serum Concentrations of Antibodies against Outer Membrane Protein P6, Protein D, and T- and B-Cell Combined Antigenic Epitopes of Nontypeable Haemophilus influenzae in Children and Adults of Different Ages

Chen

Shang

et al. 2016

Clin Vaccine Immunol

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e Nontypeable Haemophilus influenzae (NTHi) is one of the most common etiologies of acute otitis media, rhinosinusitis, and pneumonia. Outer membrane proteins (OMPs) are the main focus in new vaccine development against NTHi, as the H. influenzae type b (Hib) vaccine does not cover noncapsulated NTHi. The OMPs P6 and protein D are the most promising candidate antigens for an NTHi vaccine, and low antibody levels against them in serum may be correlated with infection caused by NTHi. In the current study, we measured the antibody titers against P6, protein D, and their T-and B-cell combined peptide epitopes in healthy individuals of different ages. We found that children <1 month old had the lowest antibody levels against NTHi P6, protein D, and their T-and B-cell combined antigenic epitopes. Antibody titers increased at ages 1 to 6 months, peaked at 7 months to 3 years, and remained high at 4 to 6 years. The antibody titers started to decrease after 6 years and were the lowest in the 21-to 30-year group. The geometric mean titers (GMTs) of T-and B-cell combined antigenic epitopes in P6 and protein D were positively correlated with those of the protein antigens. Among 12 peptides tested, P6-61, P6-123, and protein D-167 epitopes were better recognized than others in human serum. These findings might contribute to the development of an effective serotypeindependent vaccine for H. influenzae. Haemophilus influenzae is one of the normal inhabitants of the human nasopharynx and is responsible for pneumonia, acute otitis media (AOM), and acute rhinosinusitis (1-3). The presence or absence of a polysaccharide capsule segregates this bacterial species into two well-defined groups: one group of encapsulated strains and another group of noncapsulated strains, commonly referred to as nontypeable H. influenzae (NTHi) (3). Common infections caused by NTHi include otitis media in children and lower airway infections of chronic obstructive pulmonary disease in adults (4, 5). Vaccines composed of polysaccharide capsule conjugated to protein carriers have virtually eliminated infections caused by encapsulated H. influenzae type b, including meningitis and other systemic infections, in regions where the vaccines are widely administered (6, 7). However, these conjugate vaccines have no effect on infections caused by NTHi, and in regions with H. influenzae type b vaccination programs, nontypeable strains are now the most common cause of noninvasive H. influenzae infection, so that the development of the vaccine against NTHi is an urgent and challenging task (8-10).Since NTHi organisms are noncapsulated bacteria, the outer membrane proteins (OMPs) are the main targets for vaccine designers. Several research groups have identified conserved surface proteins and tested them as putative vaccines, and the conserved NTHi antigens with demonstrated preclinical protective capacity have been identified, among which P6 and protein D are the most widely studied (11)(12)(13)(14).Experimental data derived from humans and animal models indicate t...

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“…The BLAST analysis of the longest 1298 bp sequence encoding for partial the aerobic respiration control sensor protein ArcB gene after specific H. influenzae read extraction using Kraken Tools yielded a non-b serotype, non-typeable H. influenzae strain P641-4342 with 97% sequence identity (GenBank accession number CP031687.1) belonging to H. influenzae lineage 22.1-21 ( ) [ 5 ]. This finding has been routinely validated by positive specific H. influenzae real-time PCR at 30 Ct using a LightCycler ® 480 thermal-cycler (Roche, Wilmington, NC, USA) in a 20 µL final reaction volume containing 5 µL DNA and Takyon No Roxe Probe MasterMix (Eurogentec), targeting a 167 bp fragment length of the ompP1 gene as previously described [ 6 ]. While the patient had been vaccinated for H. influenzae serotype B, whole genome sequencing identified a non-typeable H. influenzae serotype (NTHi), against which the patient was not immunized.…”

Section: Resultsmentioning

confidence: 99%

Haemophilus influenzae Meningitis Direct Diagnosis by Metagenomic Next-Generation Sequencing: A Case Report

et al. 2021

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Current routine real-time PCR methods used for the point-of-care diagnosis of infectious meningitis do not allow for one-shot genotyping of the pathogen, as in the case of deadly Haemophilus influenzae meningitis. Real-time PCR diagnosed H. influenzae meningitis in a 22-year-old male patient, during his hospitalisation following a more than six-metre fall. Using an Oxford Nanopore Technologies real-time sequencing run in parallel to real-time PCR, we detected the H. influenzae genome directly from the cerebrospinal fluid sample in six hours. Furthermore, BLAST analysis of the sequence encoding for a partial DUF417 domain-containing protein diagnosed a non-b serotype, non-typeable H.influenzae belonging to lineage H. influenzae 22.1-21. The Oxford Nanopore metagenomic next-generation sequencing approach could be considered for the point-of-care diagnosis of infectious meningitis, by direct identification of pathogenic genomes and their genotypes/serotypes.

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An Application of Outer Membrane Protein P6-Specific Enzyme-Linked Immunosorbent Assay for Detection of Haemophilus influenzae in Middle Ear Fluids and Nasopharyngeal Secretions

Cited by 5 publications

References 37 publications

Detection of the major bacterial pathogens among children suffering from empyema in Ahvaz city, Iran

Detection of the major bacterial pathogens among children suffering from empyema in Ahvaz city, Iran

Serum Concentrations of Antibodies against Outer Membrane Protein P6, Protein D, and T- and B-Cell Combined Antigenic Epitopes of Nontypeable Haemophilus influenzae in Children and Adults of Different Ages

Haemophilus influenzae Meningitis Direct Diagnosis by Metagenomic Next-Generation Sequencing: A Case Report

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