Deborah F. Talkington

Development and Evaluation of Real-Time PCR-Based Fluorescence Assays for Detection of Chlamydia pneumoniae

ABSTRACT Chlamydia pneumoniae is an important respiratory pathogen recently associated with atherosclerosis and several other chronic diseases. Detection of C. pneumoniae is inconsistent, and standardized PCR assays are needed. Two real-time PCR assays specific for C. pneumoniae were developed by using the fluorescent dye-labeled TaqMan probe-based system. Oligonucleotide primers and probes were designed to target two variable domains of the ompA gene, VD2 and VD4. The limit of detection for each of the two PCR assays was 0.001 inclusion-forming unit. Thirty-nine C. pneumoniae isolates obtained from widely distributed geographical areas were amplified by the VD2 and VD4 assays, producing the expected 108- and 125-bp amplification products, respectively. None of the C. trachomatis serovars, C. psittaci strains, other organisms, or human DNAs tested were amplified. The amplification results of the newly developed assays were compared to the results of culturing and two nested PCR assays, targeting the 16S rRNA and ompA genes. The assays were compared by testing C. pneumoniae purified elementary bodies, animal tissues, 228 peripheral blood mononuclear cell (PBMC) specimens, and 179 oropharyngeal (OP) swab specimens obtained from ischemic stroke patients or matched controls. The real-time VD4 assay and one nested PCR each detected C. pneumoniae in a single, but different, PBMC specimen. Eleven of 179 OP specimens (6.1%) showed evidence of the presence of C. pneumoniae in one or more tests. The real-time VD4 assay detected the most positive results of the five assays. We believe that this real-time PCR assay offers advantages over nested PCR assays and may improve the detection of C. pneumoniae in clinical specimens.

Induction of Proinflammatory Cytokines in Human Lung Epithelial Cells during Chlamydia pneumoniae Infection

ABSTRACT Chlamydia pneumoniae is an obligate intracellular human pathogen that causes acute respiratory diseases such as pneumonia and bronchitis. Previous studies have established that C. pneumoniae can induce cytokines in mouse and/or human cells, but little information is available on the cytokine response of respiratory epithelial cells, a first line of infection. In this study, heparin treatment of C. pneumoniae significantly reduced its ability to induce interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-α) mRNA in human lung carcinoma cells, indicating that cytadherence is an important early stimulus for induction of proinflammatory mediators. Although the IL-8, gamma interferon, and TNF-α message was consistently induced by infection of A549 cells not treated with heparin, only an elevation of IL-8 protein was detected in A549 supernatants. A549 IL-β and IL-6 mRNA and supernatant protein profiles were not significantly changed by infection. Heat or UV inactivation of C. pneumoniae only partially reduced the cytokine response, and inhibition of C. pneumoniae protein or DNA synthesis did not affect its ability to induce cytokine gene expression. To prevent stress-induced cytokine release by the A549 cells, centrifugation was not utilized for infection experiments. These experiments establish the importance of cytadherence in cytokine release by cells of respiratory epithelial origin and suggest that further work in the area of cytokine mediators is warranted to gain valuable pathogenic and therapeutic insights.

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.

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.

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.

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.

Mycoplasma pulmonis V-1 surface protein variation: occurrence in vivo and association with lung lesions☆

The V-1 antigen of Mycoplasma pulmonis is exposed to the surface of the mycoplasma and has an immunoblot banding pattern that varies in vitro between and within strains. To determine if V-1 variation occurs in vivo, we infected C3H/HeNCr mice intranasally with 5 X 10(8) colony-forming units of M. pulmonis strain 5782C. We isolated M. pulmonis clones from the respiratory tracts of mice up to 28 days post-infection, then used anti-V-1 monoclonal antibody P39 to visualize their V-1 immunoblot banding patterns. By the 28th day following infection, 92% of the recovered clones had variant V-1 banding patterns. Additionally, there was a significant correlation between the severity of lung lesions and the percentage of V-1 variant clones recovered from the respiratory tracts of individual mice. These studies prove that V-1 variation does occur in vivo, and suggest that mice with more severe pulmonary lesions tend to have more V-1 variant clones as a percentage of the M. pulmonis population. Thus, variation in the V-1 protein may be a mechanism by which M. pulmonis persists in the in vivo environment, possibly by evasion of host immune surveillance or by alteration of its surface membrane to take better advantage of its environmental niche in the host.

Genetic and Phenotypic Features of Streptococcus pyogenes Strains Isolated in Brazil That Harbor New emm Sequences

ABSTRACT 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 emmtype 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 uniquesof gene sequence types, while OF-negative isolates weresof 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.

Pathogenesis and Significance of Urogenital Mycoplasmal Infections

U. urealyticum and M. hominis can no longer be considered as harmless commensals of the lower genitourinary tract. Both can produce disease in humans. Diagnosis and management of infections due to these organisms must be based upon isolation of the organisms from the affected site and preferably the number of organisms present. Due to the frequent resistance of both organisms to tetracycline, treatment must be based upon appropriate antibiotic sensitivities. For a more detailed description of the basic biology of these organisms and isolation and identification and treatment, the reader is referred to several recent reviews.

Seroepidemiologic Survey of Epidemic Cholera in Haiti to Assess Spectrum of Illness and Risk Factors for Severe Disease

To assess the spectrum of illness from toxigenic Vibrio cholerae O1 and risk factors for severe cholera in Haiti, we conducted a cross-sectional survey in a rural commune with more than 21,000 residents. During March 22–April 6, 2011, we interviewed 2,622 residents ≥ 2 years of age and tested serum specimens from 2,527 (96%) participants for vibriocidal and antibodies against cholera toxin; 18% of participants reported a cholera diagnosis, 39% had vibriocidal titers ≥ 320, and 64% had vibriocidal titers ≥ 80, suggesting widespread infection. Among seropositive participants (vibriocidal titers ≥ 320), 74.5% reported no diarrhea and 9.0% had severe cholera (reported receiving intravenous fluids and overnight hospitalization). This high burden of severe cholera is likely explained by the lack of pre-existing immunity in this population, although the virulence of the atypical El Tor strain causing the epidemic and other factors might also play a role.