A LAMP-based system for rapid detection of eight common pathogens causing lower respiratory tract infections.

Lower respiratory tract infections (LRTIs) are a leading cause of morbidity and mortality worldwide and lack a rapid diagnostic method. To improve the diagnosis of LRTIs, we established an available loop-mediated isothermal amplification (LAMP) assay for the detection of eight common lower respiratory pathogens, including Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli, Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis. The whole process can be achieved within 1 h (sample to results read out). We established an extraction free isothermal system. 528 sputum samples collected from patients suspected to have LRTIs were analyzed by the system (8 tests in each sample, a total of 4224 tests) and compared with the standard culture method (SCM). The samples with inconsistent results were further verified by Sanger sequencing and High-throughput sequencing (NGS). The detection limits of the LAMP assay for the 8 pathogens ranged from 103 to 104 CFU/mL. Upon testing 528 samples, the Kappa coefficients of all pathogens ranged between 0.5 and 0.7 indicated a moderate agreement between the LAMP assay and the SCM. All inconsistent samples were further verified by Sanger sequencing, we found that the developed LAMP assay had a higher consistency level with Sanger sequencing than the SCM for all pathogens. Additionally, when the NGS was set to a diagnostic gold standard, the specificity and sensitivity of the LAMP assay for LRTIs were 94.49% and 75.00%. The present study demonstrated that the developed LAMP has high consistency with the sequencing methods. Meanwhile, the LAMP assay has a higher detection rate compared to the SCM. It may be a powerful tool for rapid and reliable clinical diagnosis of LRTIs in primary hospitals.

Co-carriage of Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis among three different age categories of children in Hungary.

BACKGROUND: The nasopharynx can from time to time accommodate otherwise pathogenic bacteria. This phenomenon is called asymptomatic carriage. However, in case of decreased immunity, viral infection or any other enhancing factors, severe disease can develop. Our aim in this study was to survey the nasal carriage rates of four important respiratory pathogens in three different age groups of children attending nurseries, day-care centres and primary schools. This is the first study from Hungary about the asymptomatic carriage of H. influenzae and M. catarrhalis. METHODS: Altogether 580 asymptomatic children were screened in three Hungarian cities. Samples were collected from both nostrils with cotton swabs. The identification was based on both colony morphology and species-specific PCRs. Serotyping was performed for S. pneumoniae, H. influenzae and M. catarrhalis. Antibiotic susceptibility was determined with agar dilution, according to the EUCAST guidelines. Clonality was examined by PFGE. RESULTS AND CONCLUSIONS: Whereas the carriage rates of S. pneumoniae, H. influenzae and M. catarrhalis clearly decreased with age, that of S. aureus showed an opposite tendency. Multiple carriage was least prevalent if S. aureus was one of the participants. The negative association between this bacterium and the others was statistically significant. For pneumococcus, the overall carriage rate was lower compared to earlier years, and PCV13 serotypes were present in only 6.2% of the children. The majority of H. influenzae isolates was non-typeable and no type b was detected; serotype A was dominant among M. catarrhalis. All four bacteria were more sensitive to antibiotics compared to clinical isolates. No MRSAs were detected, but we found three mupirocin resistant strains. The positive effect of Hib- and PCV-vaccination is undoubted. Continued surveillance of these pathogens is required.

Genotypic differences in CC224, CC363, CC449 and CC446 of Moraxella catarrhalis isolates based on whole genome SNP, MLST and PFGE typing.

Understanding the evolutionary path of M. catarrhalis from macrolide-susceptible to macrolide-resistant organism, is important for hindering macrolide resistance from propagation. Multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and whole genome SNP typing (WGST), as useful and practical typing tools, have both advantages and disadvantages. We studied the utility of these 3 typing methods, including the level of agreement, consistency and drawbacks, in characterizing M. catarrhalis clones and clonal complexes. We focused on four clonal complexes [CC224, CC363, CC449 (CCN10) and CC446 (CCN08)] and found that PFGE and WGST had a high level of agreement and a proper consistency of the same clone or very closely related clones, while MLST is less discriminatory for different clones. Furthermore, we also established an evolutionary distance cut-off value for "The same clone". Moreover, we detected macrolide-resistant M. catarrhalis in CC224, which had previously been considered as a macrolide-susceptible clonal complex. A higher number of isolates belonged to ST215 compared to ST446, implying that ST215 is more likely to be the primary founder. Our study also demonstrated that all the four clonal complexes belong to the M. catarrhalis lineage 1, which is considered to be related to increased virulence potential and serum resistance. We also observed that copB II was highly related to CC449 and LOS type B was mainly confined in CC224. In conclusion, these findings provide further insight into the evolutionary characteristics of M. catarrhalis.

Molecular epidemiology study of a nosocomial Moraxella catarrhalis outbreak in a neurological rehabilitation unit.

BACKGROUND: Moraxella catarrhalis is a common agent causing upper and lower respiratory tract infections, particularly of ventilated patients. The bacteria are transmitted between humans by direct and indirect contacts. However, reports of nosocomial outbreaks by this pathogen are scarce. AIM: To analyse M. catarrhalis strains isolated during an outbreak in a medical rehabilitation centre to reveal their clonal relationship and to elucidate potential transmission routes. METHODS: Extensive environmental and medical staff sampling was performed. Phenotypic and genotypic analyses of 15 isolates were executed, including repetitive element palindromic polymerase chain reaction (repPCR) and whole-genome sequencing. Furthermore, an intensified hygiene regimen was installed. FINDINGS: The clonal nature of nine patient isolates and a simultaneous presence of separate entities including a strain isolated from a physician during staff screening was confirmed. Although neither asymptomatic carriers among the staff persons nor outbreak strain-contaminated fomites were identified for a specific intervention, the outbreak ceased due to maximum general and specific hygiene precautions. Retrospective analysis showed the increasing prevalence of M. catarrhalis strains over a period of two years before the incidence. Since then and after returning to the regular hygiene regimen, only one patient with a phenotypically diverse M. catarrhalis isolate has been documented. CONCLUSION: The first M. catarrhalis outbreak involving nine patients of a neurological and trauma rehabilitation centre was reported. Potential transmission pathways were discussed. Comprehensive outbreak analyses insinuated the extension of routine laboratory storage time for defined species.

Sputum Moraxella catarrhalis strains exhibit diversity within and between COPD subjects.

PURPOSE: Moraxella catarrhalis is implicated in the pathogenesis of some COPD exacerbations. We sought to investigate whether the M. catarrhalis strain is variable between COPD subjects; that an exacerbation is associated with acquisition of a new strain and that certain strains are more commonly associated with exacerbations. PATIENTS AND METHODS: Sputum samples were collected at stable and exacerbation visits from COPD subjects from a single center as part of the COPDMAP consortium. Samples identified as M. catarrhalis positive by qPCR were recultured in liquid cultures grown to extract genomic DNA; underwent Illumina MiSeq and bacterial genome sequences were de novo assembled and Multi Locus Sequence Type (MLST) was determined. RESULTS: Thirty-five samples were obtained from 18 subjects. These included 13 stable and 22 exacerbation samples. The diversity between samples was very large with 25 different M. catarrhalis MLSTs being identified out of the 35 samples of which 12 MSLTs have not been described previously. Change and persistence of M. catarrhalis strain were observed between stable visits, from stable to exacerbation and vice-a-versa, and between exacerbation visits. CONCLUSION: Sputum M. catarrhalis strains exhibit marked diversity within and between COPD subjects. Acquisition of a new strain is common between stable and exacerbation events such that no strain is specifically associated with an exacerbation.

Multilocus sequence typing-based analysis of Moraxella catarrhalis population structure reveals clonal spreading of drug-resistant strains isolated from childhood pneumonia.

This work revealed the drug resistance and population structure of Moraxella catarrhalis strains isolated from children less than three years old with pneumonia. Forty-four independent M. catarrhalis strains were analyzed using broth dilution antimicrobial susceptibility testing and multilocus sequence typing (MLST). The highest non-susceptibility rate was observed for amoxicillin (AMX), which reached 95.5%, followed by clindamycin (CLI) (n=33; 75.0%), azithromycin (AZM) (61.4%), cefaclor (CEC) (25.0%), trimethoprim-sulfamethoxazole (SXT) (15.9%), cefuroxime (CXM) (4.5%), tetracycline (TE) (2.3%), and doxycycline (DOX) (2.3%). There was no strain showing non-susceptibility to other six antimicrobials. Using MLST, the 44 M. catarrhalis strains were divided into 33 sequence types (STs). Based on their allelic profiles, the 33 STs were divided into one CC (CC363) and 28 singletons. CC363 contained five STs and ST363 was the founder ST. CC363 contained 63.6%, 33.3%, and 40.7% of CEC non-susceptible, CLI non-susceptible and AZM non-susceptible strains, respectively. The proportions of CEC non-susceptible, CLI non-susceptible and AZM non-susceptible strains in CC363 were higher than that of singletons; these differences were significant for CEC (p=0.002) and AZM (p=0.011). Furthermore, CC363 contained more AMX-CLI-AZM co-non-susceptible and AMX-CEC-CLI-AZM co-non-susceptible strains than the singletons (p=0.007 and p<0.001, respectively). CC363 is a drug-resistant clone of clinical M. catarrhalis strains in China. Expansion of this clone under selective pressure of antibiotics should be noted and long-term monitoring should be established.

Virulence determinants of Moraxella catarrhalis: distribution and considerations for vaccine development.

Moraxella catarrhalis is a human-restricted opportunistic bacterial pathogen of the respiratory mucosa. It frequently colonizes the nasopharynx asymptomatically, but is also an important causative agent of otitis media (OM) in children, and plays a significant role in acute exacerbations of chronic obstructive pulmonary disease (COPD) in adults. As the current treatment options for M. catarrhalis infection in OM and exacerbations of COPD are often ineffective, the development of an efficacious vaccine is warranted. However, no vaccine candidates for M. catarrhalis have progressed to clinical trials, and information regarding the distribution of M. catarrhalis virulence factors and vaccine candidates is inconsistent in the literature. It is largely unknown if virulence is associated with particular strains or subpopulations of M. catarrhalis, or if differences in clinical manifestation can be attributed to the heterogeneous expression of specific M. catarrhalis virulence factors in the circulating population. Further investigation of the distribution of M. catarrhalis virulence factors in the context of carriage and disease is required so that vaccine development may be targeted at relevant antigens that are conserved among disease-causing strains. The challenge of determining which of the proposed M. catarrhalis virulence factors are relevant to human disease is amplified by the lack of a standardized M. catarrhalis typing system to facilitate direct comparisons of worldwide isolates. Here we summarize and evaluate proposed relationships between M. catarrhalis subpopulations and specific virulence factors in the context of colonization and disease, as well as the current methods used to infer these associations.

Bead-based flow-cytometry for semi-quantitative analysis of complex membrane vesicle populations released by bacteria and host cells.

During infection, the release of nano-sized membrane vesicle is a process which is common both for bacteria and host cells. Host cell-derived membrane vesicles can be involved in innate and adaptive immunity whereas bacterial membrane vesicles can contribute to bacterial pathogenicity. To study the contribution of both membrane vesicle populations during infection is highly complicated as most vesicles fall within a similar size range of 30-300nm. Specialized techniques for purification are required and often no single technique complies on its own. Moreover, techniques for vesicle quantification are either complicated to use or do not distinguish between host cell-derived and bacterial membrane vesicle subpopulations. Here we demonstrate a bead-based platform that allows a semi-quantitatively analysis by flow-cytometry of bacterial and host-cell derived membrane vesicles. We show this method can be used to study heterogeneous and complex vesicle populations composed of bacterial and host-cell membrane vesicles. The easy accessible design of the protocol makes it also highly suitable for screening procedures to assess how intrinsic and environmental factors affect vesicle release.

Molecular characterization of clinical isolates of Moraxella catarrhalis by randomly amplified polymorphic DNA fingerprinting.

Moraxella catarrhalis, a less virulent microorganism that colonizes the upper respiratory tract, has recently been associated with lower respiratory disease, especially in HIV-positive immunocompromised individuals and children. Here, we correlated the DNA clustering pattern of 24 clinical isolates of M. catarrhalis for ß-lactamase production and drug resistance, from different disease groups using three different arbitrarily selected primers, P1 (5'-TCACGATGCA-3'), P14 (5'-GATCAAGTCC-3') and P17 (5'-GATCTGACAC-3'). M. catarrhalis revealed three distinct banding patterns with primer P1, four with P14 and P17. 71% (n = 17) of the isolates revealed pattern 2 with primer P1, which discriminated majority (12/21) of the isolates grouped under the major branch of the dendrogram. The minor branch had only three isolates. Separation of M. catarrhalis into two subpopulations (major and minor clusters) with primer P1 is suggestive of diverse genetic lineage. A high level of concordance between RAPD and antibiotic profile was observed. Clustering of M. catarrhalis recovered from different disease groups reflect the identical clinical background or the common geographical/temporal factors. The presence or absence of ß-lactamase in a cluster confirmed their single source of origin.

Comparing the anterior nare bacterial community of two discrete human populations using Illumina amplicon sequencing.

The anterior nares are an important reservoir for opportunistic pathogens and commensal microorganisms. A barcoded Illumina paired-end sequencing method targeting the 16S ribosomal RNA V1-2 hypervariable region was developed to compare the bacterial diversity of the anterior nares across distinct human populations (volunteers from Germany vs a Babongo Pygmy tribe, Africa). Of the 251 phylotypes detected, 231 could be classified to the genus level and 109 to the species level, including the unambiguous identification of the ubiquitous Staphylococcus aureus and Moraxella catarrhalis. The global bacterial community of both adult populations revealed that they shared 85% of the phylotypes, suggesting that our global bacterial communities have likely been with us for thousands of years. Of the 34 phylotypes unique to the non-westernized population, most were related to members within the suborder Micrococcineae. There was an even more overwelming distinction between children and adults of the same population, suggesting a progression of a childhood community of high-diversity comprising species of Moraxellaceae and Streptococcaceae to an adult community of lower diversity comprising species of Propionibacteriaceae, Clostridiales Incertae Sedis XI, Corynebacteriaceae and Staphylococcaceae. Thus, age was a stronger factor for accounting for differing bacterial assemblages than the origin of the human population sampled.

Comparison of restriction enzymes for pulsed-field gel electrophoresis typing of Moraxella catarrhalis.

NotI, the most prevalent restriction enzyme used for typing Moraxella catarrhalis, failed to digest genomic DNA from respiratory samples. An improved pulsed-field gel electrophoresis (PFGE) methodology determined SpeI as the best choice for typing this bacterial species, with a good restriction of clinical samples and a good clustering correlation with NotI.

Usefulness of gram-stained sputum obtained just after administration of antimicrobial agents as the earliest therapeutic indicator for evaluating the effectiveness of empiric therapy in community-acquired pneumonia caused by pneumococcus or Moraxella catarrhalis.

We present here three cases in which morphological changes and/or a decreased number of Streptococcus pneumoniae or Moraxella catarrhalis could be observed in gram-stained sputum obtained just after the first administration of an antimicrobial agent. Case 1 was a 53-year-old man with pneumonia caused by gram-positive diplococcus, identified as S. pneumoniae, who was administered 2 g of ampicillin over a period of 1 h. Gram-stained sputum showed smaller or gram-negative pneumococci at the completion of administration of the agent, a decreased number of cocci at 1 h after administration, and almost no cocci at 12 h after the completion of administration. Case 2 was a 72-year-old woman with pneumonia caused by diplococcus, identified as S. pneumoniae, who was administered 2 g of ampicillin over a period of 1 h. Gram-stained sputum showed weakly stained, small cocci at the completion of administration of the agent and few cocci at 1 h after the completion of administration. Case 3 was a 58-year-old woman with pneumonia caused by a gram-negative diplococcus, identified as Moraxella catarrhalis, who was administered 1 g of cefotaxime over a period of 30 min. Gram-stained sputum showed few extracellular cocci and some intracellular cocci inside neutrophils 1 h after administration and no cocci 2 h after the completion of administration. These three cases showed that gram-stained sputum obtained just after and/or 1 h after administration of the first antimicrobial agent were suitable as the quickest therapeutic indicator of the effectiveness of empiric therapy, with the effectiveness of the agent being shown much earlier than with markers such as the white blood cell count and C-reactive protein level.

Nutrient agar with sodium chloride supplementation for presumptive detection of Moraxella catarrhalis in clinical specimens.

We previously reported that Nissui nutrient agar (N medium) promoted the growth of Moraxella catarrhalis but not commensal Neisseria spp. In the present study, we examined which constituent of N medium was responsible for the selective growth of M. catarrhalis using 209 M. catarrhalis and 100 commensal Neisseria spp. clinical strains. We found that peptone, but not meat extract or agar of N medium, had growth-promoting or growth-inhibiting ability with respect to M. catarrhalis and commensal Neisseria spp. Thus, we investigated the amino acid content of N peptone and found it had higher concentrations of amino acids than other commercial peptone products. On varying the sodium chloride concentration of reconstituted N medium, we noted that the concentration was an important factor in bacterial growth differences. Varying the sodium chloride concentration of other commercial nutrient agars achieved similar results to those for N medium. This is, to our knowledge, the first study observing that sodium chloride concentration is responsible for difference in growth between the two organisms. We also successfully isolated colonies of M. catarrhalis from respiratory specimens on N medium, whereas the growth of commensal Neisseria spp. was inhibited, and by adding bovine hematin and ß-NAD we were able to isolate Haemophilus influenzae colonies as efficiently as with a chocolate agar. In conclusion, nutrient agar can be used as a medium for the preferential isolation of M. catarrhalis from upper respiratory tract specimens.

Antimicrobial resistance of Moraxella catarrhalis isolates in Taiwan.

BACKGROUND/PURPOSE: The prevalence of ampicillin-resistant Moraxella catarrhalis has been higher in Taiwan than in other countries, with reports of 97.7% in the 1990s. The aims of this study were to assess resistance trends for M. catarrhalis, which causes respiratory tract infections, against several classes of oral antibiotics and to compare the minimum inhibitory concentration (MIC) of antimicrobial agents against M. catarrhalis isolates between 1993-1994 and 2001-2004. METHODS: Clinical isolates of M. catarrhalis (n = 314) were collected from 11 large medical centers in Taiwan between 2001 and 2004. ß-Lactamase production tests were performed. The MICs for 13 different oral antibiotics were calculated using the agar dilution method. Pulsed-field gel electrophoresis (PFGE) was performed for 18 randomly selected high-level ampicillin-resistant (BRO-1 ß-lactamase-positive, MIC ≥ 32 µg/mL) isolates to investigate their genetic relatedness. RESULTS: The overall rate of ß-lactamase-producing isolates was 97.8% (307/314). All isolates were susceptible to amoxicillin + clavulanate, chloramphenicol, cefixime, ciprofloxacin, erythromycin, levofloxacin, moxifloxacin, and roxithromycin. The rate of resistance to cefaclor and cefuroxime was 8.3% and 1.3%, respectively, while no resistance was found in 1993-1994. Resistance to trimethoprim-sulfamethoxazole (SXT) and tetracycline was 18.5% and 19.8%, respectively. Comparison of 1993-1994 and 2001-2004 isolates revealed that the zone diameter for amoxicillin + clavulanate disks decreased from 43 mm in 1993-1994 to 32 mm in 2001-2004 (p < 0.001). However, MIC(50) (0.25 µg/mL in both 1993-1994 and 2001-2004) and MIC(90) (0.5 µg/mL in both 1993-1994 and 2001-2004) for amoxicillin + clavulanate did not differ between the study periods. The PFGE typing results demonstrate that at least two closely related BRO-1 clones are spreading in Taiwan. CONCLUSION: The rates of resistance to cefaclor, cefuroxime, tetracycline and SXT are now increasing in Taiwan. Molecular typing showed that at least two closely related BRO-1 clones are circulating. Although amoxicillin + clavulanate remains the antimicrobial therapy of choice for M. catarrhalis infections, continued surveillance of antimicrobial susceptibility and application of control measures against further transmission are required to inhibit the emergence of the resistant strains.

A prospective study of intrafamilial transmission and antimicrobial susceptibility of Moraxella catarrhalis.

Moraxella catarrhalis has been recognized as a particularly threatening respiratory tract pathogen in humans. A prospective study was performed to investigate which strains of M. catarrhalis can be transmitted within families; the study also addressed features of antimicrobial susceptibility. Seventy-five strains were isolated from six participants between July 2002 and February 2004, including 73 that were verified as beta-lactamase-producing strains. Antimicrobial susceptibility was tested for six types of antibiotics and no treatment issues were found. Pulsed-field gel electrophoresis (PFGE) was performed on all strains and 25 independent PFGE patterns were detected. The dominant pattern L (defined in the present study) was found in 21 (28%) of strains that were continuously recovered from children from the same family over an 8-month period. Strains with the patterns G, J, L, M, R, S, U, and W seemed to spread among the children, but there was no evidence of child-parent transmission. In the present study, the characteristics of M. catarrhalis within families have been documented, and PFGE profiles found to reveal alternating colonization and intrafamilial transmission.

Comparative analyses of the Moraxella catarrhalis type-IV pilus structural subunit PilA.

Moraxella catarrhalis is a Gram-negative aerobic diplococcus that is a mucosal pathogen of the upper and lower respiratory tracts in humans. In order to colonize the human host and establish an infection, M. catarrhalis must be able to effectively attach to the respiratory mucosal epithelia. Although little is known about M. catarrhalis pathogenesis, our laboratory has previously shown that expression of type IV pili (TFP) contributes to mucosal colonization. TFP are filamentous surface appendages primarily composed of a single protein subunit termed pilin, which is encoded by pilA in M. catarrhalis. These surface structures play a crucial role in the initiation of disease by a wide range of pathogenic bacteria. Our studies also indicate that unlike the pilin of the pathogenic Neisseria species, which exhibit both phase and antigenic variation, the pilin subunit of M. catarrhalis appears to be more highly conserved as there are no major pilin variants produced by a single strain and only two major PilA antigenic variants, termed clade 1 and clade 2, have been observed between strains. Moreover, we have determined that these highly conserved bacterial surface structures are expressed by all M. catarrhalis clinical isolates evaluated. Therapeutic or vaccine-based interventions that prevent or diminish nasopharyngeal colonization will likely decrease acute and recurrent M. catarrhalis infections in prone populations. Thus, our data indicate that additional studies aimed at elucidating the role of PilA in the pathogenesis and host response to M. catarrhalis infections are warranted.

Colonization of healthy children by Moraxella catarrhalis is characterized by genotype heterogeneity, virulence gene diversity and co-colonization with Haemophilus influenzae.

The colonization dynamics of Moraxella catarrhalis were studied in a population comprising 1079 healthy children living in Rotterdam, The Netherlands (the Generation R Focus cohort). A total of 2751 nasal swabs were obtained during four clinic visits timed to take place at 1.5, 6, 14 and 24 months of age, yielding a total of 709 M. catarrhalis and 621 Haemophilus influenzae isolates. Between January 2004 and December 2006, approximate but regular 6-monthly cycles of colonization were observed, with peak colonization incidences occurring in the autumn/winter for M. catarrhalis, and winter/spring for H. influenzae. Co-colonization was significantly more likely than single-species colonization with either M. catarrhalis or H. influenzae, with genotypic analysis revealing no clonality for co-colonizing or single colonizers of either bacterial species. This finding is especially relevant considering the recent discovery of the importance of H. influenzae-M. catarrhalis quorum sensing in biofilm formation and host clearance. Bacterial genotype heterogeneity was maintained over the 3-year period of the study, even within this relatively localized geographical region, and there was no association of genotypes with either season or year of isolation. Furthermore, chronological and genotypic diversity in three immunologically important M. catarrhalis virulence genes (uspA1, uspA2 and hag/mid) was also observed. This study indicates that genotypic variation is a key factor contributing to the success of M. catarrhalis colonization of healthy children in the first years of life. Furthermore, variation in immunologically relevant virulence genes within colonizing populations, and even within genotypically identical M. catarrhalis isolates, may be a result of immune evasion by this pathogen. Finally, the factors facilitating M. catarrhalis and H. influenzae co-colonization need to be further investigated.

The evaluation of putative endogenous control housekeeping genes for real-time polymerase chain reaction expression studies in Moraxella catarrhalis.

Comparisons of endogenous control genes in real-time polymerase chain reaction gene expression studies involving Moraxella catarrhalis are rare. This study shows that a combination of the iron sequestering gene copB and 16S rRNA genes would be useful for lineage 1 (16S rRNA type 1) isolates, but not lineage 2 (16S rRNA types 2 and 3) isolates.

Efficacy of nasopharyngeal culture in identification of pathogen in middle ear fluid in chronic otitis media with effusion.

PURPOSE: Chronic otitis media with effusion (OME) is the leading cause of hearing loss during childhood. In bacterial etiology of OME, the most frequent pathogens responsible are Haemophilus influenzae followed by Streptococcus pneumoniae and Moraxella catarrhalis. This study aimed at evaluating the accuracy of nasopharyngeal (NP) specimens in the identification of pathogens in the middle ear fluid (MEF) in patients with OME. MATERIALS AND METHODS: In this cross sectional, case-control study, 95 MEFs and 53 NP secretion specimens were obtained from 53 children. As a control group, 102 NP specimens were taken from children having an operation other than an otological disease. Conventional culture methods and multiplex-PCR method have been used to determine the etiology of OME; NP carriage between cases and control groups were compared using conventional culture methods. Pearson Chi-Square and Fisher's Exact tests were used in statistical analysis. RESULTS: Bacteria were isolated by culture in 37.9% of MEF specimens, 14.7% of which belonged to the group H. influenzae, S. pneumoniae and M. catarrhalis. PCR was positive in 30.5% specimens targeting the same pathogens. There was a two-fold increase in carriage rate of S. pneumoniae and H. influenzae in patients than controls for each pathogen. CONCLUSION: PCR is a more reliable method to detect middle ear pathogens in MEF in comparison with the conventional culture methods. The NP colonization wasn't found to be an indicator of the pathogen in MEF although middle ear pathogens colonize more in nasopharynx of diseased children.

Molecular epidemiological study of Moraxella catarrhalis isolated from nosocomial respiratory infection patients in a community hospital in Japan.

BACKGROUND: Moraxella catarrhalis, occasionally, plays the essential role in nosocomial respiratory infection (NRI). Few studies have reported the route by which this organism spreads in a nosocomial infection outbreak. We identified characteristics of the strains isolated from NRI and attempted to reveal the potential nosocomial transmission routes. METHODS: A follow-up study has been performed in a Japanese community hospital between July 2002 and January 2003. M. catarrhalis clinical isolates were identified and beta-lactamase production test as well as the minimal inhibitory concentrations (MICs) have been examined. Pulsed-field gel electrophoresis (PFGE) and the multi locus sequence typing method (MLST) have been introduced as the effective "fingerprinting" methods. RESULTS: A total of 29 strains were isolated from 17 participants; 7 independent DNA fragment patterns were detected by PFGE. Pattern B (defined in this study) was dominant, and was detected both in strains from a health care worker (HCW) and inpatients. In the 9 selected strains analyzed by MLST, 7 unique MLST types were identified, which showed the congruence with the results of PFGE results. CONCLUSION: Epidemiological analysis proved the transmission route from patient to patient, and suggested that more studies should be focused on identifying the possible transmission route between HCWs and inpatients.