Bacterial Polymerase Chain Reaction Research Articles

Clinical and epidemiological characteristics in children with community-acquired mycoplasma pneumonia in Taiwan: A nationwide surveillance.

Community-acquired pneumonia (CAP) is the leading cause of hospitalization of children. Mycoplasma pneumoniae is one of the most common pathogens. The disease severity is diverse, and the diagnosis remains a challenge to clinical pediatricians. The aims of this study are to provide a nationwide surveillance of the epidemiology and clinical manifestations of community-acquired mycoplasma pneumonia (CAMP) in children in Taiwan. The medical records of children enrolled by the Taiwan Pediatric Infectious Disease Alliance (TPIDA) project during 2010-2011 were reviewed. Hospitalized children with segmental or lobar pneumonia were included. The demographic, clinical, laboratory and radiographic data were analyzed. Nasopharyngeal swabs, pleural effusion, and serum were collected for multiplex viral and bacterial polymerase chain reaction (PCR), mycoplasma immunoglobulin M (IgM), or paired immunoglobulin G (IgG) titer. There were overall 127 children with CAMP. Among them, 16 (12.6%) children had PCR and IgM positivity, 74 (58.3%) children had a positive serologic study, 34 (27.8%) children had positive PCR detection, and three (2.4%) children had paired IgG above a four-fold increase. Enrolled patients were divided into two groups before and after the age of 5 years. Children younger than 5 years or younger had a significantly longer hospitalization, higher intensive care unit (ICU) admission rates, and more complications. They were more frequent to receive oxygen supplementation and even surgical intervention. The white blood cell counts and C-reactive protein levels were higher in children 5 years old or younger. Mycoplasma pneumoniae is an important etiology of CAP in children 5 years or younger. They had a longer length of hospitalization, higher inflammatory responses, and more complications, compared to children older than 5 years.

Mailed versus frozen transport of nasal swabs for surveillance of respiratory bacteria in remote Indigenous communities in Australia

BackgroundSurveillance programs and research for acute respiratory infections in remote Australian communities are complicated by difficulties in the storage and transport of frozen samples to urban laboratories for testing. This study assessed the sensitivity of a simple method for transporting nasal swabs from a remote setting for bacterial polymerase chain reaction (PCR) testing.MethodsWe sampled every individual who presented to a remote community clinic over a three week period in August at a time of low influenza and no respiratory syncytial virus activity. Two anterior nasal swabs were collected from each participant. The left nare specimen was mailed to the laboratory via routine postal services. The right nare specimen was transported frozen. Testing for six bacterial species was undertaken using real-time PCR.ResultsOne hundred and forty participants were enrolled who contributed 150 study visits and paired specimens for testing. Respiratory illnesses accounted for 10% of the reasons for presentation. Bacteria were identified in 117 (78%) presentations for 110 (79.4%) individuals; Streptococcus pneumoniae and Haemophilus influenzae were the most common (each identified in 58% of episodes). The overall sensitivity for any bacterium detected in mailed specimens was 82.2% (95% CI 73.6, 88.1) compared to 94.8% (95% CI 89.4, 98.1) for frozen specimens. The sensitivity of the two methods varied by species identified.ConclusionThe mailing of unfrozen nasal specimens from remote communities appears to influence the utility of the specimen for bacterial studies, with a loss in sensitivity for the detection of any species overall. Further studies are needed to confirm our finding and to investigate the possible mechanisms of effect.Clinical trial registrationAustralia and New Zealand Clinical Trials Registry Number: ACTRN12609001006235.

Diagnostic lumbar puncture in suspected acute bacterial meningitis: avoiding catastrophe

Every physician is wedded to the notion of ‘primum non nocere’. The NHS in the UK has made patient safety and optimal clinical outcomes as quality indicators of service provision.1 Consequently, it is vital to learn from adverse clinical outcomes and revisit and challenge accepted clinical practice in the light of personal experience. Recently, we saw a child with acute onset worsening headaches and neck stiffness who had a lumbar puncture for suspected bacterial meningitis, confirming the diagnosis. There were no contraindications such as signs of raised intracranial pressure (ICP). One hour later, she abruptly deteriorated. Brain magnetic resonance imaging 12 hours later demonstrated tonsillar descent through the foramen magnum with patchy ischaemic infarction of the brainstem, cerebellum, and high cervical cord. Despite emergency foramen magnum decompression, bilateral facial, bulbar, respiratory, and 4-limb flaccid paralysis with absent sensory and autonomic function below mid-pons developed, with modest recovery subsequently. The National Institute for Health and Care Excellence (NICE) advocates lumbar puncture prior to antibiotic therapy in suspected cases of bacterial meningitis, unless there are signs suggesting raised ICP; shock; extensive/spreading purpura; coagulation abnormalities; convulsions (until stabilized); respiratory insufficiency; and local superficial infection at the lumbar puncture site. In such cases, it recommends delaying the procedure, and using clinical assessment and not brain computed tomography to determine when it is safe to proceed.2 The guideline does not place any emphasis on a history suggestive of raised ICP. Among other features, acute onset worsening headaches has been recognized as a potential symptom of significant raised ICP.3 Also, it makes no recommendation to routinely measure opening pressure at lumbar puncture. The spectre of ‘coning’ leading to death or secondary brain damage in patients with raised ICP is a haunting one. Raised ICP almost always accompanies bacterial meningitis and was reported in 33 out of 35 children with pyogenic meningitis.4 Brain herniation is not common in acute bacterial meningitis, reported in 5% of cases and contributing to 30% mortality.5, 6 Brain herniation has been implicated temporally with performance of lumbar puncture for diagnosis of suspected bacterial meningitis in many reports, usually occurring within 3 to 12 hours of the procedure,5, 6 but can occur spontaneously.7 While it is ideal to aspire to microbiological diagnosis by cerebrospinal fluid studies,8 can lumbar puncture justifiably be delayed or omitted altogether in some circumstances? Given the recent establishment of rapid, reliable polymerase chain reaction (PCR) testing for common causes of meningitis (which remain sensitive for up to 96 hours after antibiotic administration),2, 9 I wonder how justifiable protocolized lumbar puncture is, given its potentially catastrophic sequelae in the face of significant raised ICP. Additionally, are we paying too much attention to clinical examination2, 8 or investigations suggesting raised ICP at the sacrifice of pertinent features in the history, which could inform the decision for lumbar puncture at an earlier stage? The sick child with suspected bacterial meningitis should be assessed by an experienced clinician.10 More attention must be paid to features in the history suggesting the presence of significant raised ICP, which is where caution and judgement should be exercised, rather than just following protocol. Best practice from patient safety and outcome perspective would be to defer lumbar puncture, if clinically considered appropriate and as the guideline recommends, obtain blood for real time PCR and cultures, and treat with appropriate antimicrobial therapy.2, 9 In addition, immediate measures to monitor and reduce ICP should be initiated.6 Bacterial PCR on cerebrospinal fluid can be performed at a later time.2, 9 A systematic national surveillance study on mortality and morbidity of ‘coning’ post lumbar puncture through either the Royal College of Paediatrics and Child Health or the British Paediatric Neurology Association would greatly assist in determining the risk–benefit of this potentially risky procedure.

Detection and Localization ofRickettsia spin Mealybug

Mealybug, Phenacoccus solenopsis Tinsley, is a sap-sucking hemipteran insect. It is an agricultural pest that is now widely distributed in India. In this study we report the presence of Rickettsia from P. solenopsis. We constructed a 16S rRNA gene library to study the bacterial diversity associated with this insect and we found that all the clones from the library were only of Candidatus Tremblaya phenacola. This study also highlights that the normal protocol adopted to study the bacterial diversity from environmental sample, by preparation of a 16S rRNA gene library, does not work when the bacterial population is highly skewed in favor of one bacteria (primary endosymbiont in this case). Hence, we used bacterial genus specific polymerase chain reaction primers to test the presence of any of the widely known secondary endosymbionts associated with insects. We tested for the presence of Cardinium, Rickettsia, Wolbachia, and Arsenophonus in P. solenopsis collected from 10 different locations across India. Only Rickettsia was detected from four locations while we were not able to find any other bacteria. We confirmed the presence of these bacteria by localizing Rickettsia and the primary endosmbiont, Candidatus Tremblaya sp. to the bacteriocyte of P. solenopsis using fluorescent in situ hybridization.

The incidence and aetiology of hospitalised community-acquired pneumonia among Vietnamese adults: a prospective surveillance in Central Vietnam

BackgroundLower respiratory tract infection (LRTI) including Community-acquired pneumonia (CAP) is a common infectious disease that is associated with significant morbidity and mortality. The patterns of aetiological pathogens differ by region and country. Special attention must be paid to CAP in Southeast Asia (SEA), a region facing rapid demographic transition. Estimates burden and aetiological patterns of CAP are essential for the clinical and public health management. The purposes of the study are to determine the incidence, aetiological pathogens, clinical pictures and risk factors of community-acquired pneumonia (CAP) in the Vietnamese adult population.MethodsA prospective surveillance for hospitalised adult CAP was conducted in Khanh Hoa Province, Central Vietnam. All adults aged ≥15 years with lower respiratory tract infections (LRTI) admitted to a provincial hospital from September 2009 to August 2010 were enrolled in the study. Patients were classified into CAP and non-pneumonic LRTI (NPLRTI) according to the radiological findings. Bacterial pathogens were identified from sputum samples by the conventional culture and polymerase chain reaction (PCR) for Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis; 13 respiratory viruses were identified from nasopharyngeal specimens by PCR.ResultsOf all 367 LRTI episodes examined, 174 (47%) were CAP. Older age, the presence of underlying respiratory conditions, and higher index score of smoking were associated with CAP. The one-year estimated incidence of hospitalised adult CAP in our study population was 0.81 per 1,000 person years. The incidence increased considerably with age and was highest among the elderly. The case fatality proportion of hospitalised CAP patients was 9.8%. Among 286 sputum samples tested for bacterial PCR, 79 (28%) were positive for H. influenzae, and 65 (23%) were positive for S. pneumoniae. Among 357 samples tested for viral PCR, 73 (21%) were positive for respiratory viruses; influenza A (n = 32, 9%) was the most common.ConclusionsThe current adult CAP incidence in Vietnam was relatively low; this result was mainly attributed to the young age of our study population.

Unique Vaginal Microbiota That Includes an Unknown Mycoplasma-Like Organism Is Associated With Trichomonas vaginalis Infection

The prevalence of Trichomonas vaginalis infection is highest in women with intermediate Nugent scores. We hypothesized that the vaginal microbiota in T. vaginalis-infected women differs from that in T. vaginalis-uninfected women. Vaginal samples from 30 T. vaginalis-infected women were matched by Nugent score to those from 30 T. vaginalis-uninfected women. Equal numbers of women with Nugent scores categorized as normal, intermediate, and bacterial vaginosis were included. The vaginal microbiota was assessed using 454 pyrosequencing analysis of polymerase chain reaction-amplified 16S ribosomal RNA gene sequences. The 16S ribosomal RNA gene sequence of an unknown organism was obtained by universal bacterial polymerase chain reaction amplification, cloning, and sequencing. Principal coordinates analysis of the pyrosequencing data showed divergence of the vaginal microbiota in T. vaginalis-infected and T. vaginalis-uninfected patients among women with normal and those with intermediate Nugent scores but not among women with bacterial vaginosis. Cluster analysis revealed 2 unique groups of T. vaginalis-infected women. One had high abundance of Mycoplasma hominis and other had high abundance of an unknown Mycoplasma species. Women in the former group had clinical evidence of enhanced vaginal inflammation. T. vaginalis may alter the vaginal microbiota in a manner that is favorable to its survival and/or transmissibility. An unknown Mycoplasma species plays a role in some of these transformations. In other cases, these changes may result in a heightened host inflammatory response.

Rare case of post-cataract-surgery Prevotella endophthalmitis diagnosed by polymerase chain reaction DNA sequencing

We report the presentation, diagnosis, and management of endophthalmitis caused by the opportunistic Prevotella species. The case was referred to us following uneventful phacoemulsification and intraocular lens implantation. Accurate identification of this rare cause of endophthalmitis was made using bacterial polymerase chain reaction DNA sequencing. Subsequent prompt modification of antibacterial therapy allowed resolution of signs and symptoms and significant visual recovery. To our knowledge, this is the first reported case of diagnosis and management of post-cataract-surgery endophthalmitis caused by the opportunistic Prevotella species.

Broad-range real-time PCR assay for detection of bacterial DNA in ocular samples from infectious endophthalmitis

To evaluate a broad-range real-time polymerase chain reaction (PCR) targeting the bacterial 16S rRNA gene for detection of bacterial DNA in infectious endophthalmitis. The bacterial 16S rRNA gene was measured by quantitative real-time PCR. For the assay, bacterial DNA was prepared from 12 Gram-positive and 4 Gram-negative strains. To determine the optimum method for DNA extraction, four extraction procedures were selected by using DNA extraction program cards with and without the use of lysozyme. To evaluate PCR sensitivity, PCR fragments were amplified from Staphylococcus aureus and Escherichia coli DNA. DNA extraction using the Bacteria card(®) without enzymes resulted in detection of all the tested strains at concentrations ≥ 10(7) copies/mL. Extraction with the Bacteria card(®) with lysozyme resulted in detection of all the tested strains at concentrations ≥ 10(6) copies/mL, indicative of no significant difference between the two procedures. DNA extraction using the Virus card(®), both with and without enzymes, resulted in reduced efficiency of detection of all strains compared with use of the Bacteria card(®). The PCR could detect as few as 1-10 colony-forming units (CFU) in diluted vitreous samples per reaction, and all tested bacterial species known to cause endophthalmitis were detected. Bacterial 16S-specific PCR can comprehensively detect the main causative bacteria of clinically suspected endophthalmitis.

Actinobaculum suisDetection Using Polymerase Chain Reaction

Actinobaculum suis is an important agent related to urinary infection in swine females. Due to its fastidious growth characteristics, the isolation of this anaerobic bacterium is difficult, thus impairing the estimation of its prevalence. The purpose of this study was to develop and test a polymerase chain reaction (PCR) for the detection and identification of A. suis and then compare these results with traditional isolation methods. Bacterial isolation and PCR were performed on one hundred and ninety-two urine samples from sows and forty-five preputial swabs from boars. The results indicate that this PCR was specific for A. suis, presenting a detection limit between 1.0 × 101 CFU/mL and 1.0 × 102 CFU/mL. A. suis frequencies, as measured by PCR, were 8.9% (17/192) in sow urine samples and 82.2% (37/45) in preputial swabs. Assessed using conventional culturing techniques, none of the urine samples were positive for A. suis; however, A. suis was detected in 31.1% (14/45) of the swabs. This PCR technique was shown to be an efficient method for the detection of A. suis in urine and preputial swabs.

Enhanced detection and serotyping ofStreptococcus pneumoniaeusing multiplex polymerase chain reaction

PurposeMethods for quick and reliable detection of Streptococcus pneumoniae are needed for the diagnosis of pneumococcal disease and vaccine studies. This study aimed to show that sequential multiplex polymerase chain reaction (PCR) is more efficient than conventional culture in achieving S. pneumoniae-positive results.MethodsNasopharyngeal (NP) secretions were obtained from 842 pediatric patients admitted with lower respiratory infections at Severance Children's Hospital in Korea between March 2009 and June 2010. For identification and serotype determination of pneumococci from the NP secretions, the secretions were evaluated via multiplex PCR technique with 35 serotype-specific primers arranged in 8 multiplex PCR sets and conventional bacteriological culture technique.ResultsAmong the results for 793 samples that underwent both bacterial culture and PCR analysis for pneumococcal detection, 153 (19.3%) results obtained by PCR and 81 (10.2%) results obtained by conventional culture technique were positive for S. pneumoniae. The predominant serotypes observed, in order of decreasing frequency, were 19A (23%), 6A/B (16%), 19F (11%), 15B/C (5%), 15A (5%), and 11A (4%); further, 26% of the isolates were non-typeable.ConclusionAs opposed to conventional bacteriological tests, PCR analysis can accurately and rapidly identify pneumococcal serotypes.

Broad‐range bacterial polymerase chain reaction in the microbiologic diagnosis of complicated pneumonia

A bacterial cause is not frequently identified in children with pneumonia complicated by parapneumonic effusion (ie, complicated pneumonia). To determine the frequency of positive blood and pleural fluid cultures in children with complicated pneumonia and to determine whether broad-range 16S rRNA polymerase chain reaction (PCR) improves identification of a microbiologic cause. This prospective cohort study included children 1-18 years of age hospitalized with complicated pneumonia. Pleural fluid drainage was performed in 64 (51.6%) of 124 children with complicated pneumonia. A microbiologic cause was identified in 11 of 64 patients (17.2%; 95% confidence interval [CI]: 8.9%-28.7%). Bacteria were isolated from pleural fluid culture in 6 of 64 patients (9.4 %; 95% CI: 3.5%-19.3%) undergoing pleural drainage; the causative bacteria were Staphylococcus aureus (n = 5) and Streptococcus pneumoniae (n = 1). Blood culture identified a bacterial cause in 3 of 44 cases (6.8%; 95% CI: 1.4%-18.7%) undergoing pleural fluid drainage; S. pneumoniae (n = 1), Haemophilus influenzae (n = 1), and S. aureus (n = 1) were isolated. Only 3 of the 19 pleural fluid samples (15.8%; 95% CI: 3.4%-39.6%) analyzed with 16S rRNA PCR were positive. S. pneumoniae was the only organism detected in all three samples; two of these three had negative pleural fluid cultures and absence of bacteria on Gram stain. S. aureus was isolated from pleural fluid culture in one patient with a negative 16S rRNA PCR test. Causative bacteria were infrequently identified in children with complicated pneumonia. Broad-range 16S rRNA PCR only modestly improved the microbiologic yield over conventional culture methods.

Cathelicidin Signaling via the Toll-like Receptor Protects Against Colitis in Mice

Cathelicidin (encoded by Camp) is an antimicrobial peptide in the innate immune system. We examined whether macrophages express cathelicidin in colons of mice with experimental colitis and patients with inflammatory bowel disease, and we investigated its signaling mechanisms. Quantitative, real-time, reverse-transcription polymerase chain reaction (PCR), bacterial 16S PCR, immunofluorescence, and small interfering RNA (siRNA) analyses were performed. Colitis was induced in mice using dextran sulfate sodium (DSS); levels of cathelicidin were measured in human primary monocytes. Expression of cathelicidin increased in the inflamed colonic mucosa of mice with DSS-induced colitis compared with controls. Cathelicidin expression localized to mucosal macrophages in inflamed colon tissues of patients and mice. Exposure of human primary monocytes to Escherichia coli DNA induced expression of Camp messenger RNA, which required signaling by extracellular signal-regulated kinase (ERK); expression was reduced by siRNAs against Toll-like receptor (TLR)9 and MyD88. Intracolonic administration of bacterial DNA to wild-type mice induced expression of cathelicidin in colons of control mice and mice with DSS-induced colitis. Colon expression of cathelicidin was significantly reduced in TLR9(-/-) mice with DSS-induced colitis. Compared with wild-type mice, Camp(-/-) mice developed a more severe form of DSS-induced colitis, particularly after intracolonic administration of E coli DNA. Expression of cathelicidin from bone marrow-derived immune cells regulated DSS induction of colitis in transplantation studies in mice. Cathelicidin protects against induction of colitis in mice. Increased expression of cathelicidin in monocytes and experimental models of colitis involves activation of TLR9-ERK signaling by bacterial DNA. This pathway might be involved in the pathogenesis of ulcerative colitis.

Bacterial Communities Associated with Chenopodium album and Stellaria media Seeds from Arable Soils

The bacterial community compositions in Chenopodium album and Stellaria media seeds recovered from soil (soil weed seedbank), from bulk soil, and from seeds harvested from plants grown in the same soils were compared. It was hypothesized that bacterial communities in soil weed seedbanks are distinct from the ones present in bulk soils. For that purpose, bacterial polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) fingerprints, made from DNA extracts of different soils and seed fractions, were analyzed by principal component analysis. Bacterial fingerprints from C. album and S. media seeds differed from each other and from soil. Further, it revealed that bacterial fingerprints from soil-recovered and plant-harvested seeds from the same species clustered together. Hence, it was concluded that microbial communities associated with seeds in soil mostly originated from the mother plant and not from soil. In addition, the results indicated that the presence of a weed seedbank in arable soils can increase soil microbial diversity. Thus, a change in species composition or size of the soil weed seedbank, for instance, as a result of a change in crop management, could affect soil microbial diversity. The consequence of increased diversity is yet unknown, but by virtue of identification of dominant bands in PCR-DGGE fingerprints as Lysobacter oryzae (among four other species), it became clear that bacteria potentially antagonizing phytopathogens dominate in C. album seeds in soil. The role of these potential antagonists on weed and crop plant growth was discussed.

Enrichment culture of CSF is of limited value in the diagnosis of neonatal meningitis

Neonatal meningitis is difficult to diagnose due to the subtle and nonspecific symptoms in neonates, and confirmation requires cerebrospinal fluid examination (CSF) [1]. Gram stain, culture of CSF directly onto agar plates, and broth enrichment culture are well established methods for diagnosing bacterial meningitis [2–5]. Other methods under evaluation include use of bacterial polymerase chain reaction combined with DNA sequencing [6]. The aim of CSF broth enrichment culture is to facilitate the isolation of damaged organisms and to recover those present in small numbers [7, 8]. The exact origin of enrichment culture is unknown [9]. Beijerinck and Winogradski are believed to be the first to recommend enrichment techniques [10]. We previously reported on the utility of various microbiology tests for the diagnosis of bacterial meningitis in the newborn [7]. We showed that enrichment cultures (inoculation of CSF into a brain-heart infusion broth incubated for 48 hrs) when performed on all lumbar puncture specimens are often falsely positive, because the prevalence of true bacterial meningitis is low in neonatal intensive care units and the test lacks specificity. We suggested that enrichment culture should be confined to settings where the prevalence of bacterial meningitis was higher, such as in samples with raised CSF white cell count (WCC), where organisms are seen on the Gram stain or where pathogens may be difficult to grow such as when babies have already received antibiotics. The aim of our current study was to assess the performance of enrichment culture when performed on CSF samples selected on the basis of a raised WCC of ≥30 /mm3.

PCR-restriction endonuclease analysis of Mycobacterium avium subsp. paratuberculosis isolates from goats, sheep, and cattle in Jordan

Paratuberculosis is an endemic disease and induces high economical losses in Jordan. There is no information available on genotypic variation of Mycobacterium avium subsp. paratuberculosis (MAP) isolated from animals in Jordan. In this study, we investigated 150 fecal samples from sheep, goats, and cattle for the presence of paratuberculosis using bacterial culture and polymerase chain reaction (PCR)-RFLP analysis of insertion sequence IS1311. Analysis of the results revealed that genotypic information from sheep, goat, and cattle could classify them into cattle or sheep strains. All culture isolates from cattle, 12.5% of the isolates from sheep, and 50% of the isolates from goats were cattle strain, while 87.5% of the isolates from sheep and 50% of the isolates from goats were sheep strain. Sequencing of the IS1311 268 bp PCR product from the three animal species confirmed the different MAP patterns.

Determination of ascitic bacterial 16S rRNA gene in the rapid diagnosis of spontaneous bacterial peritonitis

To evaluate the value of ascitic bacterial 16S rRNA gene determination in the rapid diagnosis of spontaneous bacterial peritonitis (SBP). 16S rRNA gene from bacterial DNA in ascites was determined by quantitative fluorescent polymerase chain reaction (PCR) in 76 patients with suspected SBP and 6 patients with non-infectious ascites. The results were compared with those obtained from bacterial culture. The positive rate of SBP was 22.4% among patients detected with ascitic bacterial 16S rRNA gene determination-based quantitative fluorescent PCR, which was significantly higher than that (7.9%) in patients only received bacterial culture (P<0.05). In addition,in 6 patients with non-infectious ascites,both the 16S rRNA gene determination-based quantitative fluorescent PCR and bacterial culture showed negative results. 16S rRNA gene determination-based quantitative fluorescent PCR can be an effective tool for the rapid diagnosis of SBP. It is more sensitive than the bacterial culture.

Indirect immunofluorescence test using polyclonal antibodies for the detection of Taylorella equigenitalis

Contagious equine metritis is a horse disease that causes endometrial inflammation due to Taylorella equigenitalis. Since Taylorella asinigenitalis was characterized, genital swab culture has proved to be an insufficient method for distinguishing between the two Taylorella species. Here, we developed an indirect immunofluorescence (IIF) test using polyclonal antibodies. Specificity, sensitivity, and detection limit were assessed using isolated bacteria (55 T. equigenitalis strains, 46 T. asinigenitalis strains and 18 other bacterial species), experimental and genital swabs in comparison to bacterial culture and polymerase chain reaction (PCR) testing. Our results indicated that IIF using polyclonal antibodies allows T. equigenitalis to be discriminated from T. asinigenitalis. This test constitutes a rapid, sensitive and specific tool for confirming presumptive colonies of T. equigenitalis.

Diagnostic Utility of Polymerase Chain Reaction on Intraocular Specimens to Establish the Etiology of Infectious Endophthalmitis

To evaluate the utility of polymerase chain reaction (PCR) on intraocular clinical specimens (aqueous humor [AH] and vitreous fluid [VF]) as an etiologic diagnostic tool relative to microbiological culture methods in infectious endophthalmitis. Conventional bacterial and mycologic cultures and PCR for eubacterial and panfungal genomes were applied for etiologic diagnosis on pairs of AH and VF obtained from 72 patients with clinically established infectious endophthalmitis. Based on cultures, an infectious etiology was established in 27 (37.5%) of 72 patients. PCR detected infectious etiology in all 72 patients. PCR increased the clinical sensitivity over culture by 62.5% (p<0.0001, McNemar test). The frequency of culture positivity, single infections, and polymicrobial infection varied significantly among the types of endophthalmitis (p<0.0001, chi-square test). PCR detected an infectious etiology in 48 patients and polymicrobial infection in 24 patients. An etiology was established by PCR on 56 (77.8%) AH and 65 (90.3%) VF of the 72 patients and this difference had no statistical significance. PCR on intraocular specimens as an etiologic diagnostic tool has been shown to be specific and severalfold more sensitive than cultures and clinically useful. Therefore, PCR may be considered the gold standard to establish the etiology of infectious endophthalmitis. As there is no statistically significant difference in the results of PCR on AH and VF, PCR on AH could be the method of choice considering safety and simplicity of the procedure of its collection.

Investigation and Diagnosis of Nontuberculous Mycobacteriosis in a Captive Herd of Aoudad (Ammotragus lervia)

An epizootic of nontuberculous mycobacteriosis occurred in a captive herd of aoudad (Ammotragus lervia) over a period of 18 mo. Each of the affected animals was subject to a thorough postmortem examination that included histopathology, tissue concentration and acid-fast staining, aerobic and anaerobic bacterial culture, mycobacterial culture, and real-time polymerase chain reaction specific for Mycobacterium tuberculosis DNA. Histopathologic lesions consistent with pulmonary mycobacteriosis, including the presence of acid-fast bacteria, were identified in two captive adult male aoudad. M. avium was isolated in culture from the pulmonary parenchyma, and M. parafortuitum was isolated from a mesenteric lymph node of a third animal, an adult female, euthanized subsequent to an illness characterized by progressive dyspnea and tachypnea. M. intracellulare was isolated within the bronchial lymph node of a fourth aoudad, an adult female that was euthanized due to chronic weight loss. Diagnostic testing of the 34 individuals in the herd included collection of blood for an interferon-gamma assay, intradermal tuberculin testing, and radiometric fecal culture for M. avium subsp. paratuberculosis. On the basis of this investigation, mycobacteriosis associated with M. bovis, M. tuberculosis, and/or M. avium subsp. paratuberculosis was ruled out and nontuberculous mycobacteriosis was confirmed in this herd.

Septicemia, Endocarditis, and Cerebral Infarction Due to Staphylococcus aureus in a Harp seal (phoca groenlandica)

An adult, wild-collected, male harp seal (Phoca groenlandica) was transferred from a rehabilitation center to a display facility because of unilateral phthisis bulbi and decreased use of the right forelimb, which precluded its release. In quarantine, the animal demonstrated limited use of the right forelimb, which acutely progressed to complete disuse of the limb accompanied by intermittent lethargy. One month after transfer, the animal was found dead on exhibit. Necropsy showed septic arthritis of the right scapulohumeral joint, valvular endocarditis with systemic bacterial thromboembolism, and infarction of the cerebrum and myocardium. Culture of the blood and affected joint space revealed Staphylococcus aureus. Bacterial polymerase chain reaction of formalin-fixed tissues from the heart and brain were also positive for S. aureus. Staphylococcus aureus infection should be considered as an additional cause of endocarditis and embolic encephalitis in seals.