Genetic and phenotypic features of Streptococcus pyogenes strains isolated in Brazil that harbor new emm sequences.

In the present study, 37 group A Streptococcus (GAS) strains belonging to 13 new emm sequence types identified among GAS strains randomly isolated in Brazil were characterized by using phenotypic and genotypic methods. The new types were designated st204, st211, st213, st809, st833, st854, st2904, st2911, st2917, st2926, st3757, st3765, and st6735. All isolates were susceptible to the antimicrobial agents tested, except to tetracycline. They all carried the speB gene, and 94.6% produced detectable SpeB. Most strains belonging to a given emm type had similar or highly related pulsed-field gel electrophoresis profiles that were distinct from profiles of strains of another type. The other characteristics were variable from isolate to isolate, although some associations were consistently found within some emm types. Unlike the other isolates, all type st213 isolates were speA positive and produced SpeA. Strains belonging to st3765 were T6 and opacity factor (OF) negative. Individual isolates within OF-positive emm types were associated with unique sof gene sequence types, while OF-negative isolates were sof negative by PCR. This report provides information on new emm sequence types first detected in GAS isolates from a geographic area not extensively surveyed. Such data can contribute to a better understanding of the local and global dynamics of GAS populations and of the epidemiological aspects of GAS infections occurring in tropical regions.

Standardizing Chlamydia pneumoniae assays: recommendations from the Centers for Disease Control and Prevention (USA) and the Laboratory Centre for Disease Control (Canada).

Chlamydia pneumoniae has been associated with atherosclerosis and several other chronic diseases, but reports from different laboratories are highly variable and "gold standards" are lacking, which has led to calls for more standardized approaches to diagnostic testing. Using leading researchers in the field, we reviewed the available approaches to serological testing, culture, DNA amplification, and tissue diagnostics to make specific recommendations. With regard to serological testing, only use of microimmunofluorescence is recommended, standardized definitions for "acute infection" and "past exposure" are proposed, and the use of single immunoglobulin (Ig) G titers for determining acute infection and IgA for determining chronic infection are discouraged. Confirmation of a positive culture result requires propagation of the isolate or confirmation by use of polymerase chain reaction (PCR). Four of 18 PCR assays described in published reports met the proposed validation criteria. More consistent use of control antibodies and tissues and improvement in skill at identifying staining artifacts are necessary to avoid false-positive results of immunohistochemical staining. These standards should be applied in future investigations and periodically modified as indicated.

Mycoplasma microti sp. nov., isolated from the respiratory tract of prairie voles (Microtus ochrogaster).

Mycoplasmas were isolated from the respiratory tracts of prairie voles (Microtus ochrogaster). This paper presents biochemical, serological and molecular genetic characterizations of those organisms and proposes a new species, Mycoplasma microti sp. nov. The type strain of Mycoplasma microti is strain IL371T (ATCC 700935T).

Azithromycin prophylaxis during a hospital outbreak of Mycoplasma pneumoniae pneumonia.

Outbreaks of Mycoplasma pneumoniae (MP) in closed communities can have a high attack rate and can last several months. Azithromycin chemoprophylaxis has not been evaluated as a means of limiting transmission. This randomized, double-blinded placebo-controlled trial of azithromycin was conducted among asymptomatic hospital employees during an MP outbreak. Oropharyngeal swabs were obtained for detection of MP by polymerase chain reaction, and questionnaires were administered to assess clinical illness. Of the 147 employees who were enrolled, 73 received azithromycin and 74 received placebo. Carriage was similar within and between groups at weeks 1 and 6 (9.6% vs. 6.7% and 10.3% vs. 13.2%, respectively). Four episodes of clinically significant respiratory illness occurred in the azithromycin group versus 16 episodes in the placebo group (protective efficacy, 75%; 95% confidence interval, 28%-91%). Use of azithromycin prophylaxis in asymptomatic persons during an MP outbreak in a closed setting may be of value in reducing clinical illness.

Analysis of complement fixation and commercial enzyme immunoassays for detection of antibodies to Mycoplasma pneumoniae in human serum.

The Meridian ImmunoCard (IC), GenBio ImmunoWELL-IgM, and Remel EIA commercial antibody tests are qualitative enzyme immunoassays that detect antibodies to Mycoplasma pneumoniae in serum. These tests were compared to an M. pneumoniae complement fixation (CF) assay, which uses a commercially available antigen component. The Meridian IC and the ImmunoWELL-IgM detect immunoglobulin M (IgM) only; the Remel EIA and the CF test detect both IgM and IgG antibodies. Detection of specific IgM antibody, which appears early in infection, can be, but is not always, indicative of a recent or current infection. Paired serum samples from 64 adult patients with probable M. pneumoniae infection were examined with each of the four tests. Thirty (47%) of the 64 acute-phase sera were IgM positive by Meridian IC, 26 (41%) were positive by Remel EIA, 24 (38%) were positive by CF, and 15 (23%) were positive by ImmunoWELL-IgM. When both the acute- and convalescent-phase serum samples from each patient were examined, 61 (95%) of the 64 patients were positive by CF, 60 patients (94%) were positive by Remel EIA, 52 patients (81%) were IgM positive by the Meridian IC, and 29 patients (45%) were IgM positive by the ImmunoWELL-IgM assay. The Meridian IC was more sensitive than the other tests for early detection of IgM antibodies. However, after examining paired serum samples, we concluded that the detection of IgM alone may not be useful for all cases of mycoplasma infection, especially in an adult population.

An outbreak of acute respiratory disease caused by Mycoplasma pneumoniae and adenovirus at a federal service training academy: new implications from an old scenario.

Outbreaks of Mycoplasma pneumoniae and adenovirus have been reported in military institutions for several decades. During a recent outbreak in a federal service training academy, we performed an epidemiological and laboratory investigation to better characterize and control the outbreak. Of 586 students responding to a questionnaire, 317 (54%) reported having a respiratory illness during the outbreak period. Among 42 students who underwent complete laboratory testing, 24 (57%) had evidence of M. pneumoniae infection, 8 (19%) had evidence of adenovirus infection, and 4 (10%) had evidence of both. Polymerase chain reaction testing of oropharyngeal swabs revealed more acute M. pneumoniae infections (57% positive) than did serology or culture. Multivariate analysis revealed that visiting the campus health clinic >3 times for a nonrespiratory condition, such as injury, was a significant risk factor for illness among freshmen early in the course of the outbreak, whereas having an ill roommate was a risk factor throughout the duration of the outbreak.

Outbreak of pneumonia in a long-term care facility: antecedent human parainfluenza virus 1 infection may predispose to bacterial pneumonia.

OBJECTIVES: To determine the causes of an outbreak of lobar pneumonia. DESIGN: Matched (1:2) case-control study. SETTING: A 70-bed chronic care facility for older people. PARTICIPANTS: Residents of the facility. RESULTS: Ten residents developed pneumonia over a 10-day period. Two residents died. One case-patient had Streptococcus pneumoniae bacteremia; another had polymerase chain reaction evidence of S. pneumoniae infection. No other etiologic agent was identified. Only four of 10 case-patients had received routine diagnostic cultures of blood or sputum before the administration of antibiotics. Symptoms of upper respiratory illness (URI) among residents before the pneumonia outbreak corresponded with elevation of antibodies to human parainfluenza virus 1 (HPIV1). In a matched case-control study, six of 10 case-patients, compared with five of 20 controls, had symptoms of URI during the preceding month (matched odds ratio (MOR) = 4.5, 95% CI = 0.8-33). Nine case-patients had serum available, and five of these had both serologic evidence of recent HPIV1 infection and recent URI, compared with two of 18 controls (MOR = 9.0, 95% CI = 1.2-208). Only three residents had documentation of pneumococcal vaccination. CONCLUSIONS: Noninfluenza viral infections may play a role in the pathogenesis of some bacterial pneumonias. S. pneumoniae was the cause of at least two pneumonias; lack of preantibiotic cultures may have interfered with isolation of S. pneumoniae in others. Recent HPIV1 infection was epidemiologically linked to subsequently developing pneumonia. Spread of HPIV1 in the facility may have contributed to increased susceptibility to S. pneumoniae and, potentially, to other bacterial pathogens.

Diagnosis of Mycoplasma pneumoniae infection in autopsy and open-lung biopsy tissues by nested PCR.

A nested PCR specific for the Mycoplasma pneumoniae P1 gene was used to diagnose mycoplasma infection in two cohort patients with severe pneumonia within 24 h of tissue receipt. A postmortem diagnosis of M. pneumoniae infection was obtained for the first patient, who died without the collection of appropriate paired samples for serodiagnosis. An open-lung biopsy obtained from the second patient allowed a quick, definitive diagnosis and proper selection of therapy.

Pathogenicity of Mycoplasma volis in mice and rats.

A new species of Mycoplasma, M. volis, was isolated from the respiratory tract of clinically normal field-trapped prairie voles (Microtus ochrogaster) that were to be housed in close proximity to other rodents. To determine the pathogenic potential of the new mycoplasmal isolate, three groups of rodents (Sprague Dawley rats, BALB/c mice, and severe combined immunodeficient [SCID] mice) were intranasally inoculated with 2 x 10(8) color-changing units (CCU) of M. volis and were observed for 4 to 6 weeks. Experimental animals did not manifest clinical signs of disease; however, one experimental SCID mouse was euthanized 5 days after inoculation because of a severe circling disorder. Lung lesions in experimental SD rats ranged from mild to severe bronchial-associated lymphoid tissue (BALT) hyperplasia. Lung lesions in BALB/c and SCID mice ranged from no lesions to mild pneumonia. We were able to isolate M. volis from some control mice, none of which had lung lesions. All mice were seronegative for Sendai virus, mouse hepatitis virus, and M. pulmonis. All immunocompetent experimental animals (BALB/c mice and Sprague Dawley rats) were seropositive for M. volis. All immunocompetent control animals and SCID mice were seronegative for M. volis. Our data suggest that M. volis is capable of causing microscopic lesions and seroconversion in rats and mice, and therefore these rodents should not be housed in close proximity to voles.

Enhanced control of an outbreak of Mycoplasma pneumoniae pneumonia with azithromycin prophylaxis.

There are currently no recommended epidemic-control measures for Mycoplasma pneumoniae pneumonia outbreaks in closed communities. Previous studies have suggested the usefulness of chemoprophylaxis administered to close contacts of case-patients. To evaluate the effectiveness of various epidemic-control measures during an institutional outbreak, an observational study was undertaken during a very large outbreak of M. pneumoniae pneumonia at a facility for developmentally disabled residents (n = 142 cases). Control measures evaluated included no control, standard epidemic-control measures, and targeted azithromycin prophylaxis (500 mg on day 1, 250 mg/day on days 2-5) plus standard epidemic-control measures. The combined use of azithromycin prophylaxis and standard epidemic-control measures was associated with a significant reduction in the secondary attack rate. This study suggests that the addition of antibiotic prophylaxis to standard epidemic-control measures can be useful during institutional outbreaks of M. pneumoniae pneumonia.

Increase in endocervical CD4 lymphocytes among women with nonulcerative sexually transmitted diseases.

To assess associations of nonulcerative sexually transmitted diseases (STDs) with human immunodeficiency virus (HIV)-susceptible leukocytes on female genital mucosa, cervicovaginal specimens from 32 HIV-negative STD clinic patients with gonorrhea, chlamydial infection, or trichomoniasis were compared with specimens from 32 clinic patients without these infections. Twenty-eight patients had single infections (15 gonorrhea, 10 chlamydial infection, 3 trichomoniasis), and 4 had dual infections. A saline vaginal wash and saline suspensions of vaginal wall scrapings, ectocervical scrapings, and endocervical brushings were analyzed by flow cytometry. Specimens from the endocervix had the highest proportions of lymphocytes, monocytes, and Langerhans' cells. The median number of endocervical CD4 lymphocytes/10,000 cells was greater among patients with STDs than among those without (476 vs. 245; P < .001). These data suggest that the endocervix may have a particularly important role in heterosexual HIV transmission and that nonulcerative STDs may facilitate HIV transmission by increasing the presence of CD4 lymphocytes at this site.

Protection of mice against fatal pneumococcal challenge by immunization with pneumococcal surface adhesin A (PsaA).

Pneumococcal surface adhesin A (PsaA) is a 37-kDa surface protein present on Streptococcus pneumoniae having significant homology with fimbrial adhesion proteins. Immunization of CBA/CaHNJ Xid mice with PsaA using either complete Freund's or TiterMax adjuvants significantly protected mice against heterologous intravenous challenge with type 3 pneumococcal strain WU2 at doses up to 45 times the LD50. The results indicate that PsaA warrants further evaluation as a vaccine candidate for human pneumococcal disease.

Isolation of mycoplasmas from prairie voles (Microtus ochrogaster).

A new species of mycoplasmas was isolated from the lungs and nasopharyngeal washings of prairie voles (Microtus ochrogaster). Clinical signs of disease and microscopic lesions were not observed at the time of this isolation. The organism was cultured in SP4 medium; it grew aerobically, anaerobically, and in 5% CO2 in 5 to 7 days, and fermented glucose. Transmission electron microscopy revealed the organism to lack a cell wall and to have typical mycoplasmal ultrastructural morphology. The complete nucleotide sequence of the 16S rRNA gene from an isolate was determined by amplification with polymerase chain reaction and by sequencing with the dideoxynucleotide chain termination method. The sequence did not match any known sequences in the GenBank of the National Institutes of Health. The 16S rRNA sequence of the organism, Mycoplasma volis (proposed species novum), is unique and most closely resembles that of M. muris and M. iowae. Because this vole colony will be housed in rooms with other rodents, pathogenicity studies of this new species of mycoplasmas in mice and rats are underway.

Characterization of ear fluid isolates of Alloiococcus otitidis from patients with recurrent otitis media.

Nineteen isolates of Alloiococcus otitidis from ear fluid samples collected by tympanostomy from patients at four geographic locations were identified by phenotypic characterization and genetic relatedness. Initial growth of A. otitidis isolates occurred after 3 days at 37 degrees C on brain heart infusion (BHI) agar with 5% rabbit blood. Heavy growth occurred in BHI broth supplemented with 0.07% lecithin and 0.5% Tween 80 after 4 days of incubation. The isolates were gram-positive cocci that divided on an irregular plane and produced metabolic lactic acid, pyrrolidonyl arylamidase, and leucine aminopeptidase. These cocci grew sparsely in 6.5% NaCl-BHI broth, were asaccharolytic on both fermentative and oxidative bases, and were cytochrome negative by the iron-porphyrin test. The cellular fatty acid profile of A. otitidis was distinguished from those of related genera and characterized by major amounts ( > or = 14%) of 16:0, 18:2, 18:1 omega 9c, and 18:0 and smaller amounts of 14:0, 16:1 omega 7c, 17:0, and 18:1 omega 7c. Fifteen isolates demonstrated > 69% relatedness by DNA-DNA hybridization. Four isolates plus the original 15 were confirmed as A. otitidis by dot blot hybridization with a digoxigenin-labeled nucleotide probe specific for this species. The intergenic space between the genes coding for the 16S and 23S rRNAs of alloiococci was amplified by PCR, analyzed by restriction fragment length polymorphism, and determined to consist of three different genetic types. Although beta-lactamase negative, A. otitidis demonstrated intermediate levels of resistance to beta-lactams, including expanded-spectrum cephalosporins, and were resistant to trimethoprim-sulfamethoxazole and erythromycin.

Group A streptococcal necrotizing fasciitis following varicella in children: case reports and review.

We report four cases of necrotizing fasciitis that occurred following varicella in children ranging in age from 2 to 8 years. The only organism isolated from each of these patients was Streptococcus pyogenes or group A beta-hemolytic Streptococcus (GABHS). Each child recovered; however, three required repeated surgical debridements in addition to therapy with antibiotics. An interesting finding in these patients was the development of hyponatremia and/or hypocalcemia. M-typing and T-typing of the isolates demonstrated that the GABHS strain in two children who attended the same school was M5; M1 and M3 strains were identified in the other two children. In addition to the children described in this series, eleven other cases of children with necrotizing fasciitis following varicella have been reported in the English-language literature since 1970. We believe that these cases provide further evidence that varicella is an important risk factor for necrotizing fasciitis that is caused by more-virulent strains of GABHS.

Comparison of two rapid commercial tests with complement fixation for serologic diagnosis of Mycoplasma pneumoniae infections.

The complement fixation (CF) test is the current reference serologic test for the diagnosis of Mycoplasma pneumoniae infection. However, it is reported to be insensitive and nonspecific, and it is labor intensive. To determine if a faster and more sensitive diagnosis of M. pneumoniae could be obtained, we examined 50 paired serum samples from patients with suspected M. pneumoniae infection by the CF test and two commercial rapid antibody detection kits, the Remel M. pneumoniae immunoglobulin G (IgG)-IgM antibody test system (Remel, Lenexa, Kans.) and the Seradyn Color Vue M. pneumoniae IgG-IgM kit (Seradyn, Indianapolis, Ind.). The Remel test, a 5-min qualitative immunobinding assay, detected antibodies in three patient serum samples with CF titers of 32 and in all but one sample with titers of > or = 64. The Seradyn test, a 40-min qualitative agglutination test, was less sensitive than CF or Remel. The Seradyn test was positive in 68% of cases, compared with 94 and 96% of cases tested by CF or Remel, respectively. Both commercial tests are faster and less technically demanding to perform than is the CF test.

Detection of streptococcal pyrogenic exotoxin genes by a nested polymerase chain reaction.

Severe invasive disease associated with group A Streptococcus (GAS) has recently increased in frequency. Isolates of GAS from normally sterile sites were examined for the streptococcal pyrogenic exotoxin genes spe A, spe B and spe C to determine if they play a role in this disease. Four primers for each gene were used in a nested polymerase chain reaction (PCR) configuration. The first PCR generated fragments of 818, 1106, and 801 bp, respectively, for the extotoxin genes. The second PCR generated fragments of 500, 912 and 654 bp for the spe A, spe B and spe C genes using the fragments from the first PCR as template. Of 62 strains tested, 35 (56%) contained the spe A gene, and 17 (27%) contained the spe C gene. All GAS strains studied, regardless of disease association, contained the spe B gene. These data corroborate accumulating evidence that the genes encoding pyrogenic exotoxin types B and C are not associated with severe invasive streptococcal illness including streptococcal toxic shock-like syndrome. This PCR-based gene detection system has clinical and epidemiologic applications because of its ease of performance, non-isotope labelling, high specificity and sensitivity, and lack of requirement for purified DNA.

Association of phenotypic and genotypic characteristics of invasive Streptococcus pyogenes isolates with clinical components of streptococcal toxic shock syndrome.

Sixty-two invasive Streptococcus pyogenes strains, including 32 strains isolated from patients with streptococcal toxic shock syndrome (STSS), were analyzed for the following phenotypic and genotypic characteristics: M-protein type, serum opacity factor production, protease production, the presence of streptococcal pyrogenic exotoxin (Spe) genes A, B, and C, and in vitro production of SpeA and SpeB. These characteristics were analyzed for possible associations with each other as well as with clinical components of STSS. M-type 1, the most commonly isolated M-type, was significantly associated with protease production. Protease activity was significantly associated with the clinical sign of soft tissue necrosis. M-type 1 and 3 strains from STSS patients were significantly associated with the clinical signs of shock and organ involvement as well as with SpeA production in vitro. Finally, the production of SpeA was significantly associated with the clinical component of shock and organ involvement as well as with rash. These data suggest that STSS does not make up a single syndrome but, rather, that the multiple STSS clinical criteria probably reflect different phenotypic characteristics of individual S. pyogenes isolates.

The changing epidemiology of invasive group A streptococcal infections and the emergence of streptococcal toxic shock-like syndrome. A retrospective population-based study.

OBJECTIVE: To determine disease incidence and changes in the epidemiology of invasive group A streptococcal infections in a community in Arizona. DESIGN AND SETTING: We retrospectively surveyed microbiology records from all 10 hospitals in Pima County, Arizona, to identify patients who had Streptococcus pyogenes isolated from blood, sterile body fluid, or tissue biopsy specimens between April 1985 and March 1990. Demographic and clinical information was abstracted from the medical records of these patients. PATIENTS: A total of 128 patients with a median age of 53.5 years (range, 6 months to 96 years). OUTCOME MEASURES: Racial/ethnic differences in disease incidence; mortality and changes in the clinical spectrum of disease over the study period. RESULTS: The annual age-adjusted incidence was 4.3 per 100,000 but was 46.0 per 100,000 among Native Americans. Advanced age, age less than 5 years, hypotension, and multi-organ system involvement were significantly associated with increased mortality. From 1985 to 1990, the proportion of infections with hypotension, rash, desquamation, renal impairment, and gastrointestinal involvement increased significantly (chi 2 for trend P < or = .02 for each feature). A toxic shock-like syndrome occurred in 8% of infections since 1988, compared with none of the infections between 1985 and 1987 (P = .04). Patients with the syndrome were younger than patients with other invasive infections (median age 15 vs 54 years, P = .02), and were less likely to have underlying medical conditions (P = .008). CONCLUSIONS: Significant changes occurred in the spectrum of invasive group A streptococcal infections in Pima County, Arizona, between 1985 and 1990. Native Americans were at increased risk of acquiring these infections. Patients with the streptococcal toxic shock-like syndrome had epidemiologic features that distinguished them from patients with other invasive infections, including younger age and less underlying illness.

Analysis of pneumococcal PspA microheterogeneity in SDS polyacrylamide gels and the association of PspA with the cell membrane.

Pneumococcal surface protein A (PspA) is a protection-eliciting surface protein found on all pneumococci. Although highly cross-reactive, it displays interstrain variation in its size and in the expression of individual antibody reactive epitopes. PspA was not released in significant amounts from pneumococcal membranes treated with sodium carbonate, but was solubilized with SDS. Thus, PspA is either an integral membrane protein or is attached to an integral membrane component. By SDS-PAGE and immunoblot analysis, we found two predominant molecular sizes of PspA in each strain examined. The smaller band was about the size expected from the inferred amino acid sequence of PspA and the larger band appeared to be a dimer of the monomer PspA. When higher concentrations of lysate were run on SDS gels, it was also possible to detect many additional high molecular weight components that reacted with antibodies to PspA. These multiple high molecular weight PspA bands were not due to the attachment of PspA to peptidoglycan or teichoic acids, did not appear to be composed of degraded PspA and most likely resulted from non-covalent polymerization or aggregation of PspA.