Viral-bacterial interactions and risk of acute otitis media complicating upper respiratory tract infection

Acute otitis media (AOM) is a common complication of upper respiratory tract infection whose pathogenesis involves both viruses and bacteria. We examined risks of acute otitis media associated with specific combinations of respiratory viruses and acute otitis media bacterial pathogens. Data were from a prospective study of children ages 6 to 36 months and included viral and bacterial culture and quantitative PCR for respiratory syncytial virus (RSV), human bocavirus, and human metapneumovirus. Repeated-measure logistic regression was used to assess the relationship between specific viruses, bacteria, and the risk of acute otitis media complicating upper respiratory tract infection. In unadjusted analyses of data from 194 children, adenovirus, bocavirus, Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis were significantly associated with AOM (P < 0.05 by χ(2) test). Children with high respiratory syncytial virus loads (≥3.16 × 10(7) copies/ml) experienced increased acute otitis media risk. Higher viral loads of bocavirus and metapneumovirus were not significantly associated with acute otitis media. In adjusted models controlling for the presence of key viruses, bacteria, and acute otitis media risk factors, acute otitis media risk was independently associated with high RSV viral load with Streptococcus pneumoniae (odds ratio [OR], 4.40; 95% confidence interval [CI], 1.90 and 10.19) and Haemophilus influenzae (OR, 2.04; 95% CI, 1.38 and 3.02). The risk was higher for the presence of bocavirus and H. influenzae together (OR, 3.61; 95% CI, 1.90 and 6.86). Acute otitis media risk differs by the specific viruses and bacteria involved. Acute otitis media prevention efforts should consider methods for reducing infections caused by respiratory syncytial virus, bocavirus, and adenovirus in addition to acute otitis media bacterial pathogens.     

Test-retest reliability of the nasopharyngeal culture in children

Aspects of test-retest reliability of the nasopharyngeal culture were evaluated in children with otitis media and in ear, nose and throat (ENT)-healthy children, in all 174 cases. The nasopharyngeal colonization of Streptococcus pneumoniae, Haemophilus influenzae, Branhamella catarrhalis and beta-haemolytic streptococci was determined for a group of children with well-defined otitis media effusion (OME). The valitity of the results was then tested in a new group of children with OME. Despite seasonal differences and different bacteriologists analysing the specimens, high conformity was found between the two groups regarding distribution and recovery rates of the potential pathogens studied. The test-retest reliability of the culture was also analysed by duplicate specimens in children with acute otitis media (AOM), OME and in ENT-healthy children. The reproducibility of the findings of pathogens, whether quantitative aspects were considered or not, was found to be between 70 and 80% for children with AOM and OME.     

Experimental otitis media after nasal inoculation of Streptococcus pneumoniae and influenza A virus in chinchillas.

Otitis media developed in 67% of chinchillas inoculated intranasally with type 7 Streptococcus pneumoniae and influenza A virus. Only 4% of chinchillas inoculated with influenza alone and 21% of chinchillas inoculated with S. pneumoniae alone developed otitis media. Among the chinchillas that developed otitis media after inoculation with both pneumococcus and influenza, 73% of the affected ears contained effusion, and 27% of the affected ears showed tympanic membrane inflammation without middle ear effusion obtained on paracentesis. Although a majority of the ears with effusion yielded S. pneumoniae on culture, one-third of the effusions were sterile for aerobic bacteria. This model resembles conditions accompanying otitis media in humans and suggests that respiratory viral infection contributes significantly to the pathogenesis of acute otitis media.     

Moraxella catarrhalis-induced purulent otitis media in the rat middle ear. Structure, protection, and serum antibodies.

To study the effects of viable and heat-killed Moraxella catarrhalis bacteria on the middle ear mucosa and to evaluate the protection after whole-cell immunizations, Sprague-Dawley rats were challenged and rechallenged with four different M. catarrhalis strains. The animals were monitored by clinical observations, bacterial and histological samples from middle ears, and serum IgG levels. Only viable bacteria at a high concentration induced purulent otitis media, which was culture positive in 58% of the cases on day 4. The infection was characterized by a mild acute reaction lasting otomicroscopically about 8 days, together with quantitative and qualitative changes of the goblet cells. Structurally the mucosal effects of the heat-killed bacteria were less pronounced in the early phase compared to the viable bacteria, but similar at the end of the experiment at 6 months. The intrabullar and subcutaneous immunizations evoked an IgG antibody response in all animals, and the protection rate after immunization was 50% or more. The induced protection was not strain-specific. The study showed the rat to be a possible alternative for the study of different aspects of M. catarrhalis otitis media, an infection that is clinically and structurally different from that elicited by Streptococcus pneumoniae and Haemophilus influenzae in the rat.     

Serum intercellular adhesion molecule 1 variations in young children with acute otitis media

Acute otitis media (AOM) is an inflammatory reaction in the middle ear, most often occurring in young children. Streptococcus pneumoniae, nontypeable Haemophilus influenzae, and Moraxella catarrhalis are the most common bacteria isolated. Intercellular adhesion molecule 1 (ICAM-1) is involved in the innate immune response to infection by microorganisms, in effective antigen presentation, and in subsequent T-cell activation. Here we prospectively studied levels of serum soluble ICAM-1 (sICAM-1) before, at the time of, and after antimicrobial treatment of AOM in a group of 138 children ages 6 to 30 months. Middle ear fluids were collected by tympanocentesis to identify otopathogens. We found that (i) serum levels of sICAM-1 were significantly higher in S. pneumoniae-, nontypeable H. influenzae-, and M. catarrhalis-infected children than in well children (P < 0.001), confirming that a systemic inflammatory response occurs during AOM; (ii) sICAM-1 levels varied from no elevation (110 ng/ml) to elevation to high levels (maximum, 1,470 ng/ml) among children with AOM; (iii) in paired samples, sICAM-1 levels increased 4- to 20-fold when children developed AOM compared to their sICAM-1 levels before infection; and (iv) the level of sICAM-1 returned to the pre-AOM level at the convalescent stage of AOM after successful antimicrobial therapy. We conclude that AOM often causes a systemic inflammatory reaction, as measured by elevation of the serum sICAM-1 level, and that a high variability in sICAM-1 responses occurs with the presence of otopathogens during AOM.     

Quantitative detection of Moraxella catarrhalis in nasopharyngeal secretions by real-time PCR

The recognition of Moraxella catarrhalis as an important cause of respiratory tract infections has been protracted, mainly because it is a frequent commensal organism of the upper respiratory tract and the diagnostic sensitivity of blood or pleural fluid culture is low. Given that the amount of M. catarrhalis bacteria in the upper respiratory tract may change during infection, quantification of these bacteria in nasopharyngeal secretions (NPSs) by real-time PCR may offer a suitable diagnostic approach. Using primers and a fluorescent probe specific for the copB outer membrane protein gene, we detected DNA from serial dilutions of M. catarrhalis cells corresponding to 1 to 10(6) cells. Importantly, there was no difference in the amplification efficiency when the same DNA was mixed with DNA from NPSs devoid of M. catarrhalis. The specificity of the reaction was further confirmed by the lack of amplification of DNAs from other Moraxella species, nontypeable Haemophilus influenzae, H. influenzae type b, Streptococcus pneumoniae, Streptococcus oralis, Streptococcus pyogenes, Bordetella pertussis, Corynebacterium diphtheriae, and various Neisseria species. The assay applied to NPSs from 184 patients with respiratory tract infections performed with a sensitivity of 100% and a specificity of up to 98% compared to the culture results. The numbers of M. catarrhalis organisms detected by real-time PCR correlated with the numbers detected by semiquantitative culture. This real-time PCR assay targeting the copB outer membrane protein gene provided a sensitive and reliable means for the rapid detection and quantification of M. catarrhalis in NPSs; may serve as a tool to study changes in the amounts of M. catarrhalis during lower respiratory tract infections or following vaccination against S. pneumoniae, H. influenzae, or N. meningitidis; and may be applied to other clinical samples.     

Detection rates of bacteria in chronic otitis media with effusion in children

This study was performed to investigate polymerase chain reaction-based detection of bacterial DNA in middle ear fluid and assess the correlation between the PCR-positive rate with several factors associated with middle ear effusion. The purpose was to gain a further understanding of bacterial infection as a major cause of otitis media with effusion. Of the 278 specimens of middle ear fluid, 39 (14%) tested positive by ordinary culture. The overall detection rate of bacterial DNA using the PCR method was 36.7% for middle ear effusion, and bacterial DNA detection rates of Hemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis in the middle ear effusion were 29.1%, 4.7% and 10.8%, respectively. The bacterial DNA detection rate was higher in ears with a history of acute otitis media than those without the history. High detection rates were observed in patients younger than 48 months who have had a higher tendency to present with acute otitis media. We concluded that PCR is a more sensitive method for the detection of bacteria in middle ear effusion than ordinary culture, and acute otitis media is a major contributor to the pathogenesis of otitis media with effusion.     

Activity of cefuroxime against bacterial strains isolated from acute otitis media

The acute otitis media is a frequent infantile disease and, in 80% of cases, a bacterial strain can be isolated from the otorrhoea. Haemophilus influenzae and Streptococcus pneumoniae are the two major species isolated from auricular exudate, and represent two thirds of all isolated strains, with the others comprising Staphylococcus aureus, Branhamella catarrhalis, Pseudomonas aeruginosa, Enterobacteriaceae and corynebacteria. The treatment of this disease is based principally on beta-lactams (aminopenicillins, cephalosporins) administered by the oral route. Cefuroxime is a cephalosporin which is absorbed via the digestive tract in the form of cefuroxim-axetil. The activity of this compound was studied against 210 strains isolated from otorrhoea, collected from children who presented an acute otitis media during the first half of 1989. These strains were: 112 strains of H. influenzae, of which 23 produced a beta-lactamase; 21 strains of Streptococcus pneumoniae; 3 strains of Streptococcus pyogenes; 10 strains of Branhamella catarrhalis of which 9 produced a beta-lactamase; 18 strains of S. aureus; 14 strains of Enterobacteriaceae, and 32 strains of corynebacteria. The minimal inhibitory concentration (MIC) of cefuroxime-axetil was measured by dilution in agar. The MICs of cefuroxime against H. influenzae were low and similar (MIC 50 = 1 mg/l; MIC 90 = 1 mg/l) regardless of whether the strain secreted a beta-lactamase. Overall, 90% and 98% of the 210 strains tested here were inhibited by 1 and 4 mg/l of cefuroxime respectively. These results show that the antibacterial spectrum of cefuroxime-axetil appears to be ideally suited to the bacterial strains isolated from acute otitis media.     

National surveillance programme on susceptibility patterns of respiratory pathogens in South Africa: moxifloxacin compared with eight other antimicrobial agents

AIMS: The susceptibility patterns of Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Klebsiella pneumoniae, and Streptococcus pyogenes isolated from specimens submitted to 12 private laboratories in South Africa were determined. METHODS: Minimum inhibitory concentration (MIC) determinations were performed on the isolates in the microbiology laboratory at Tygerberg Hospital according to the recommendations of the National Committee for Clinical Laboratory Standards (NCCLS). RESULTS: According to the NCCLS breakpoints, 24% of 729 S pneumoniae isolates were sensitive, 30% intermediate, and 46% resistant to penicillin. Rates of macrolide resistance were high, with 61% of the pneumococci being resistant to clarithromycin and azithromycin. Co-trimoxazole resistance was also high, with 28% of pneumococcal strains being sensitive, 21% intermediate, and 51% resistant. beta Lactamase was produced by 7% of 736 H influenzae isolates and 91% of 256 M catarrhalis isolates. The quinolones, moxifloxacin and levofloxacin, were universally active against all isolates tested, which included S pneumoniae, H influenzae, M catarrhalis, K pneumoniae, and S pyogenes. CONCLUSIONS: Haemophilus influenzae and S pneumoniae were the most commonly isolated organisms. Resistance to penicillin was one of the highest reported in the world (76%) in S pneumoniae, as was macrolide resistance in pneumonocci, although surprisingly, only 14% of S pyogenes were resistant. The quinolones, moxifloxacin and levofloxacin, were active against all organisms tested, including the penicillin and macrolide resistant strains and moxifloxacin was more active than levofloxacin against pneumococci.     

Clinically applicable multiplex PCR for four middle ear pathogens

The multiplex PCR method for the detection of Alloiococcus otitidis, Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae (P. H. Hendolin, A. Markkanen, J. Ylikoski, and J. J. Wahlfors, J. Clin. Microbiol. 35:2854-2858, 1997) in middle ear effusions (MEEs) was modified to be better suited for clinical use. To detect false-negative results, an internal amplification was added to the reaction, and to prevent carryover contamination, the dUTP-uracil-N-glycosidase system was incorporated into the procedure. Labor was minimized by using the heat-activatable AmpliTaq Gold polymerase in order to circumvent manual hot start and by detecting the amplification products on an automated sequencer. The performance of the improved protocol was verified with MEEs from patients with otitis media with effusion. In addition, a ligase detection reaction (LDR) was developed for confirmation of the PCR products. The modifications increased the reliability of the protocol and the hands-off time significantly. However, when two DNA extraction protocols were compared, gram-negative bacteria were detected more often in phenol-treated MEEs (94 versus 46%; P < 0.001), and gram-positive bacteria were detected more often in MEEs dissolved in sodium dodecyl sulfate-NaOH-chaotropic salt (83 versus 27%; P < 0.001). The LDR was found to be 100% specific. In all, the results demonstrate the feasibility of the rapid (7-h) multiplex PCR method for routine laboratory use.     

Acute otitis media with spontaneous tympanic membrane perforation

The principal aim of this review is to present the current knowledge regarding acute otitis media (AOM) with spontaneous tympanic membrane perforation (STMP) and to address the question of whether AOM with STMP is a disease with specific characteristics or a severe case of AOM. PubMed was used to search for all studies published over the past 15 years using the key words “acute otitis media” and “othorrea” or “spontaneous tympanic membrane perforation”. More than 250 articles were found, but only those published in English and providing data on aspects related to perforation of infectious origin were considered. Early Streptococcus pneumoniae infection due to invasive pneumococcal strains, in addition to coinfections and biofilm production due mainly to non-typeable Haemophilus influenzae, seem to be precursors of STMP. However, it is unclear why some children have several STMP episodes during the first years of life that resolve without complications in adulthood, whereas other children develop chronic suppurative otitis media. Although specific aetiological agents appear to be associated with an increased risk of AOM with STMP, further studies are needed to determine whether AOM with STMP is a distinct disease with specific aetiological, clinical and prognostic characteristics or a more severe case of AOM than the cases that occur without STMP. Finally, it is important to identify preventive methods that are useful not only in otitis-prone children with uncomplicated AOM, but also in children with recurrent AOM and those who experience several episodes with STMP.     

A mouse model for acute otitis media

To induce acute otitis media in the mouse and to describe the clinical and bacteriological course of the infection, middle ears of BALB/c, Swiss-Webster and C57BL/6 mice were inoculated with Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis. Systemic and local changes were monitored by clinical observations, otomicroscopy, and analysis of bacterial samples from blood and middle ears. Agglutination of mouse erythrocytes by M. catarrhalis was also tested. Depending on bacterial strain, bacterial dose, and mouse strain three responses were identified: acute otitis media, otitis media with serous effusion, or no reaction. BALB/c mice were the most susceptible animals. On day 3, 76% of the BALB/c mice had developed middle ear infection, 50% had a positive middle ear culture, 56% were bacteremic, and 10% had succumbed to a disseminated infection. The local infections lasted approximately a week. Animals which survived recovered without permanent deterioration or otomicroscopically discernible changes. In no case did M. catarrhalis induce a culture-positive middle ear infection, possibly due to an inability to agglutinate the mouse erythrocytes. The mouse model can become a useful tool in studies of pneumococcal and H. influenzae-induced otitis media, but the bacterial dose has to be carefully titrated and adjusted to the chosen mouse strain.     

Role of Nasopharyngeal Culture in Antibiotic Prescription for Patients with Common Cold or Acute Sinusitis

The aim of the present study was to assess the hypothesis that, when present in nasopharyngeal secretions, Streptococcus pneumoniae. Haemophilus influenzae, and Moraxella catarrhalis play a pathogenic role early in the course of an upper respiratory tract infection. Adults with a clinical diagnosis of acute sinusitis or common cold were enrolled. Participants were randomly assigned in a double-blind manner to receive azithromycin 500 mg daily or placebo for 3 days. The effect of treatment on symptom evolution in the predefined subset of patients with Streptococcus pneumoniae, Haemophilus influenzae, or Moraxella catarrhalis in their nasopharyngeal secretions was assessed. Of 265 patients enrolled, 132 received placebo and 133 azithromycin. Streptococcus pneumoniae, Haemophilus influenzae, or Moraxella catarrhalis was identified in nasopharyngeal secretions of 77 patients (29%). In this predefined subgroup of patients with Streptococcus pneumoniae, Haemophilus influenzae, or Moraxella catarrhalis, resolution of symptoms by day 7 occurred in 73% of those treated with azithromycin compared with 47% of those who received placebo (P=0.007). The median time before resolution of symptoms was 5 days in the azithromycin group compared to 7 days in the placebo group. Respiratory complications requiring antibiotic treatment occurred in 19% of patients in the placebo group and in 3% of the azithromycin group (P=0.025). In the remaining 188 patients without Streptococcus pneumoniae, Haemophilus influenzae, or Moraxella catarrhalis, resolution of symptoms by day 7 was similar in both groups (69% in the placebo group vs. 64% in the azithromycin group [P=0.75]). Antibiotic treatment is of clinical benefit for patients with acute sinusitis or common cold when Streptococcus pneumoniae, Haemophilus influenzae, or Moraxella catarrhalis is present in nasopharyngeal secretions. This observation provides new insights into the pathogenic role of these bacteria in the early stage of the common cold.     

A New Rat Model of Otitis Media Caused by Streptococcus pneumoniae: Conditions and Application in Immunization Protocols

Streptococcus pneumoniae (pneumococcus [Pn]) can be cultured from up to 50% of acute otitis media (AOM) effusions, and these bacteria are the most common cause of AOM-related complications. With the recent advent of antibiotic-resistant Pn strains, treatment of Pn infections may meet with serious difficulties. Prevention through vaccination, notably for the four most common occurring Pn serotypes in humans (i.e., Pn 6B, Pn 14, Pn 19F, and Pn 23F), is a helpful alternative. Testing of vaccine efficacy should occur in an appropriate animal AOM model, which is presented here. The four involved Pn serotypes are not pathogenic to the rat, which was chosen as the experimental animal for practical reasons. To induce a natural infection (i.e., ascending through the eustachian tube), the mucociliary clearance of the eustachian tube was impaired by infusing histamine into the tympanic cavity on 2 consecutive days before intranasal inoculation of the bacteria. With this simple protocol, high and reproducible infection rates, as determined with bacterial cultures, of Pn-induced AOM (approximately 70%) with the two major Pn serotypes 14 and 19F (Pn 14 and Pn 19F) were obtained, whereas lower infection rates (25 to 50%) with Pn 6B and Pn 23F were obtained. In this model, intranasal priming with pneumococci, as well as subcutaneous vaccination with Pn 14 tetanus toxoid-conjugated polysaccharide, induced a protective effect against the induction of otitis media with these bacteria. This shows that immunity to Pn 14 AOM can be induced by both mucosal and systemic presentations of antigen. In conclusion, we have developed an animal model for Pn-induced AOM, which is suitable for the evaluation of the protecting effect of immunization.     

Evaluation of the Hyplex BloodScreen Multiplex PCR-Enzyme-linked immunosorbent assay system for direct identification of gram-positive cocci and gram-negative bacilli from positive blood cultures

We evaluated the Hyplex BloodScreen PCR-enzyme-linked immunosorbent assay (ELISA) system (BAG, Lich, Germany), a new diagnostic test for the direct identification of gram-negative bacilli and gram-positive cocci from positive blood cultures, with 482 positive BACTEC 9240 blood culture bottles. The test involves amplification of the bacterial DNA by multiplex PCR and subsequent hybridization of the PCR product to specific oligonucleotide probes in an ELISA-based format. The available probes allow the separate detection of Escherichia coli, Pseudomonas aeruginosa, Enterobacter aerogenes, Klebsiella spp., Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis/Enterococcus faecium, Streptococcus pyogenes, and Streptococcus pneumoniae and the staphylococcal mecA gene. The Hyplex BloodScreen test showed an overall sensitivity of 100% for the identification of gram-negative bacilli and 96.6 to 100% for the identification of gram-positive cocci (S. aureus, 100%; S. epidermidis, 97.2%; Enterococcus faecalis/Enterococcus faecium, 96.6%; and Streptococcus pneumoniae, 100%). The specificities of the test modules ranged from 92.5 to 100% for gram-negative bacilli and 97.7 to 100% for gram-positive cocci (Escherichia coli, 92.5%; Pseudomonas aeruginosa, 98.5%; Klebsiella spp., 100%; Enterobacter aerogenes, 100%; S. aureus, 100%, S. epidermidis, 97.7%; Enterococcus faecalis/Enterococcus faecium, 99.6%; Streptococcus pyogenes, 100%; and Streptococcus pneumoniae, 99.3%). The result of the mecA gene detection module correlated with the result of the phenotypic oxacillin resistance testing in all 38 isolates of Staphylococcus aureus investigated. In conclusion, the Hyplex BloodScreen PCR-ELISA system is well suited for the direct and specific identification of the most common pathogenic bacteria and the direct detection of the mecA gene of Staphylococcus aureus in positive blood cultures.     

Pulsed-field gel electrophoresis analysis of nasopharyngeal flora in children attending a day care center

To investigate how bacterial pathogens spread from child to child in a day care center, we monitored six children, two boys and four girls, born between August 1995 and November 1997, attending a day care center and analyzed nasopharyngeal samples from them using pulsed-field gel electrophoresis (PFGE). We obtained nasopharyngeal cultures from all of the affected children and almost all of the unaffected children between September 1998 and March 1999 after some children presented simultaneously with purulent rhinorrhea. Moreover, when a child was found to have acute otitis media, nasopharyngeal secretions from the child were independently cultured during treatment. During this period, 28 isolates of Moraxella catarrhalis, 13 of Streptococcus pneumoniae, and 4 of Haemophilus influenzae were recovered. PFGE gave 8 patterns for M. catarrhalis, 10 for S. pneumoniae, and 1 for H. influenzae. PFGE patterns demonstrated spread of M. catarrhalis between children. However, each occurrence of clusters of infection with M. catarrhalis lasted 2 to 6 weeks, with a change in PFGE pattern between occurrences of clusters. The M. catarrhalis strain infecting each child also changed. Similarly, the S. pneumoniae strain in each child also changed. In contrast, infection with H. influenzae persisted for about 3 months in an affected child.     

Comparison of the nasal bacterial floras in two groups of healthy subjects and in patients with acute maxillary sinusitis.

The nasal bacterial flora was studied in 183 healthy men entering military service (entry group), 103 healthy recruits in service (service group), and 185 recruits with acute maxillary sinusitis. The 267 nasal and ipsilateral sinus aspirate findings in the same patients with acute maxillary sinusitis were compared pairwise. In the entry group presumed sinus pathogens were only rarely isolated from the nasal cavities: Haemophilus influenzae in 4%, Streptococcus pneumoniae in 1%, Branhamella catarrhalis in 3%, and Streptococcus pyogenes in 0%. The corresponding isolation frequencies in the service group were 19, 13, 3, and less than 1%, respectively, and those in the group with acute maxillary sinusitis were 61, 25, 7, and 6%, respectively. Suppression of the major components of the normal nasal flora, Corynebacterium sp., coagulase-negative staphylococci, Propionibacterium acnes, and Staphylococcus aureus, was seen in the group with acute maxillary sinusitis and also occasionally in the service group. When a sinus aspirate culture yielded a presumed sinus pathogen, the same pathogen was found in the nasal samples in 91% of the cases. The predictive value of a pathogen-positive nasal finding was highest (93.8%) for S. pyogenes, followed by 77.7% for H. influenzae and 68.7% for S. pneumoniae, and lowest (20%) for B. catarrhalis.     

Validation and reproducibility assessment of tigecycline MIC determinations by Etest

A multicenter study was conducted to validate Etest tigecycline compared to the Clinical Laboratory Standards Institute reference broth microdilution and agar dilution methodologies. A large collection of gram-negative (n = 266) and gram-positive (n = 162) aerobic bacteria, a collection of anaerobes (n = 385), and selected collections of nonpneumococcal streptococci (n = 369), Streptococcus pneumoniae (n = 372), and Haemophilus influenzae (n = 372) were tested. Strains with reduced susceptibility to tigecycline were used with all test methods. The Etest showed excellent inter- and intralaboratory reproducibility for all organism groups tested regardless of the test methodology. The essential agreement values with the reference method (+/-1 dilution) were >99% for the collection of gram-negative and gram-positive aerobes; >98% for the S. pneumoniae, H. influenzae, and anaerobe collections; and 100% for the group of nonpneumococcal streptococci. These results validate the performance accuracy and utility of Etest tigecycline and verify the reproducibility of this convenient predefined gradient methodology for tigecycline susceptibility determination.