Value of a novel Neisseria meningitidis--specific polymerase chain reaction assay in skin biopsy specimens as a diagnostic tool in chronic meningococcemia

BACKGROUND: Chronic meningococcemia (CM) is a diagnostic challenge. Skin lesions are frequent but in most cases nonspecific. Polymerase chain reaction (PCR)-based diagnosis has been validated in blood and cerebrospinal fluid for acute Neisseria meningitidis infection, in patients in whom routine microbiologic tests have failed to isolate the bacteria. In 2 patients with CM, we established the diagnosis by a newly developed PCR-based approach performed on skin biopsy specimens. OBSERVATIONS: Two patients presented with fever together with systemic and cutaneous manifestations suggestive of CM. Although findings from blood cultures remained negative, we were able to identify N meningitidis in the skin lesions by a newly developed PCR assay. In 1 patient, an N meningitidis strain of the same serogroup was also isolated from a throat swab specimen. Both patients rapidly improved after appropriate antibiotherapy. CONCLUSIONS: To our knowledge, we report the first cases of CM diagnosed by PCR testing on skin biopsy specimens. It is noteworthy that, although N meningitidis-specific PCR is highly sensitive in blood and cerebrospinal fluid in acute infections, our observations underscore the usefulness of PCR performed on skin lesions for the diagnosis of chronic N meningitidis infections. Whenever possible, this approach should be systematically employed in patients for whom N meningitidis infection cannot be confirmed by routine microbiologic investigations.

Real-time quantitative PCR to determine chlamydial load in men and women in a community setting

We used a PCR method to quantify the loads of Chlamydia trachomatis organisms in self-collected urine and vulvovaginal swab (VVS) samples from 93 women and 30 men participating in the Chlamydia Screening Studies Project, a community-based study of individuals not seeking health care. For women, self-collected VVS had a higher mean chlamydial load (10,405 organisms/ml; 95% confidence interval [95% CI], 5,167 to 21,163 organisms/ml) than did first-void urines (FVU) (503 organisms/ml; 95% CI, 250 to 1,022 organisms/ml; P < 0.001). Chlamydial loads in female and male self-collected FVU specimens were similar (P = 0.634). The mean chlamydial load in FVU specimens decreased with increasing age in females and males. There was no strong statistical evidence of differences in chlamydial load in repeat male and female FVU specimens taken when patients attended for treatment a median of 23.5 (range, 14 to 62) and 28 (range, 13 to 132) days later, respectively, or in VVS taken a median of 35 (range, 14 to 217) days later. In this study, chlamydial load values for infected persons in the community who were not seeking treatment were lower than those published in other studies involving symptomatic patients attending clinical settings. This might have implications for estimates of the infectiousness of chlamydia. The results of this study provide a scientific rationale for preferring VVS to FVU specimens from women.

Assessment of a real-time PCR test to diagnose syphilis from diverse biological samples

OBJECTIVES: To investigate the contribution of a real-time PCR assay for the detection of Treponema pallidum in various biological specimens with the secondary objective of comparing its value according to HIV status. METHODS: Prospective cohort of incident syphilis cases from three Swiss hospitals (Geneva and Bern University Hospitals, Outpatient Clinic for Dermatology of Triemli, Zurich) diagnosed between January 2006 and September 2008. A case-control study was nested into the cohort. Biological specimens (blood, lesion swab or urine) were taken at diagnosis (as clinical information) and analysed by real-time PCR using the T pallidum 47 kDa gene. RESULTS: 126 specimens were collected from 74 patients with primary (n = 26), secondary (n = 40) and latent (n = 8) syphilis. Among primary syphilis, sensitivity was 80% in lesion swabs, 28% in whole blood, 55% in serum and 29% in urine, whereas among secondary syphilis, it was 20%, 36%, 47% and 44%, respectively. Among secondary syphilis, plasma and cerebrospinal fluid were also tested and provided a sensitivity of 100% and 50%, respectively. The global sensitivity of T pallidum by PCR (irrespective of the compartment tested) was 65% during primary, 53% during secondary and null during latent syphilis. No difference regarding serology or PCR results was observed among HIV-infected patients. Specificity was 100%. CONCLUSIONS: Syphilis PCR provides better sensitivity in lesion swabs from primary syphilis and displays only moderate sensitivity in blood from primary and secondary syphilis. HIV status did not modify the internal validity of PCR for the diagnosis of primary or secondary syphilis.

Hormographiella aspergillata keratomycosis in a dog

A 4-year-old, female, Border Collie was presented to the University of Bern Veterinary Teaching Hospital, because of a corneal lesion of 10 days duration. The axial cornea presented a whitish fluorescein-positive plaque with irregular margins. A diagnosis of keratomycosis was made based on cytology. Medical therapy with local broad-spectrum antibiotic and fluconazole was instituted. After 1 week of treatment, the improvement was deemed unsatisfactory. Therefore, a lamellar keratectomy and conjunctival pedicle flap were performed. After surgery, the cornea healed uneventfully. Histology confirmed the diagnosis of keratomycosis. The fungus could not be grown in culture and a precise etiological diagnosis could only be obtained with genetic identification of the fungus. A PCR technique was used to amplify the fungal genome from the cornea. Hormographiella aspergillata, the asexual reproductive form of the basidiomycete Coprinopsis cinerea, was identified. As advised in human medicine, we encourage the use of this molecular technique to obtain an early species diagnosis, allowing targeted medical therapy.

[Field validation of a real-time PCR test for the detection of Mycoplasma hyopneumoniae in nasal swabs of live pigs]

Enzootic pneumonia (EP) of pigs, caused by Mycoplasma hyopneumoniae has been a notifiable disease in Switzerland since May 2003. The diagnosis of EP has been based on multiple methods, including clinical, bacteriological and epidemiological findings as well as pathological examination of lungs (mosaic diagnosis). With the recent development of a real-time PCR (rtPCR) assay with 2 target sequences a new detection method for M. hyopneumoniae became available. This assay was tested for its applicability to nasal swab material from live animals. Pigs from 74 herds (average 10 pigs per herd) were tested. Using the mosaic diagnosis, 22 herds were classified as EP positive and 52 as EP negative. From the 730 collected swab samples we were able to demonstrate that the rtPCR test was 100% specific. In cases of cough the sensitivity on herd level of the rtPCR is 100%. On single animal level and in herds without cough the sensitivity was lower. In such cases, only a positive result would be proof for an infection with M. hyopneumoniae. Our study shows that the rtPCR on nasal swabs from live pigs allows a fast and accurate diagnosis in cases of suspected EP.

Identification and detection of Actinobacillus pleuropneumoniae by PCR based on the gene apxIVA

The apxIVA gene, a recently discovered RTX determinant of Actinobacillus pleuropneumoniae, was shown to be species-specific. DNA hybridization experiments using probes for various regions of apxIVA revealed that the 3'-terminus of this gene was present in all 14 serotypes of A. pleuropneumoniae but absent from phylogenetically related species. A primer pair spanning this region specifically amplified a 422bp fragment in PCR experiments with DNA from the reference strains of the 14 serotypes and 194 field strains isolated from various geographic locations worldwide. DNA sequence analysis of PCR products derived from all serotypes were identical except in serotypes 3, 8, and 10, which showed minor differences. The PCR did not amplify any product when DNA from 17 different bacterial species closely related to A. pleuropneumoniae was used as template. In addition, the PCR was negative with DNA of several Actinobacillus sp. which were initially characterized as A. pleuropneumoniae using routine phenotypic and serological analyses but which were subsequently shown by 16S rRNA sequence analysis to belong to yet undefined Actinobacillus species. The sensitivity of the PCR was determined to be 10pg of A. pleuropneumoniae DNA. A set of nested primers amplified a 377bp fragment specifically with A. pleuropneumoniae DNA. DNA titration experiments using the flanking and nested primer pairs showed an improved level of sensitivity to approximately 10fg of genomic DNA. The nested PCR was used to monitor the spread of A. pleuropneumoniae in pigs experimentally infected with a virulent serotype 1 strain and housed in a controlled environment facility. A. pleuropneumoniae DNA could be detected by nested PCR in nasal swab samples of infected pigs receiving either a high dose (5x10(5)) or a low dose (1x10(4)) challenge and in unchallenged cohorts that were contact-infected by the inoculated animals. Furthermore, PCR confirmed the presence of A. pleuropneumoniae in 16/17 homogenates from necrotic lung lesions, while the bacterium was successfully recovered from 13 of these lesions by culture.

Microcirculation in hypertensive patients

Regardless of the mechanisms that initiate the increase in blood pressure, functional and structural changes in the systemic vasculature are the final result of long-standing hypertension. These changes can occur in the macro- but also in the microvasculature. The supply of the tissues with oxygen, nutrients, and metabolites occurs almost exclusively in the microcirculation (which comprises resistance arterioles, capillaries and venules), and an adequate perfusion via the microcirculatory network is essential for the integrity of tissue and organ function. This review focuses on results from clinical studies in hypertensive patients, which have been performed in close cooperation with different clinical groups over the last three decades. Intravital microscopy was used to study skin microcirculation, microcatheters for the analysis of skeletal muscle microcirculation, the slit lamp for conjunctival microcirculation and the laser scanning ophthalmoscope for the measurement of the retinal capillary network. The first changes of the normal microcirculation can be found in about 93% of patients with essential hypertension, long before organ dysfunctions become clinically manifest. The earliest disorders were found in skin capillaries and thereafter in the retina and the skeletal muscle. In general, the disorders in the different areas were clearly correlated. While capillary rarefaction occurred mainly in the retina and the conjunctiva bulbi, in skin capillaries morphological changes were rare. A significant decrease of capillary erythrocyte velocities under resting conditions together with a marked damping of the postischemic hyperemia was found, both correlating with the duration of hypertension or WHO stage or the fundus hypertonicus stage. Also the mean oxygen tension in the skeletal muscle was correlated with the state of the disease. These data show that the microcirculatory disorders in hypertension are systemic and are hallmarks of the long-term complications of hypertension. There is now a large body of evidence that microvascular changes occur very early and may be important in their pathogenesis and progression.

Campylobacter spp., Yersinia enterocolitica, and Salmonella enterica and their simultaneous occurrence in German fattening pig herds and their environment.

Campylobacter spp., Salmonella enterica, and Yersinia enterocolitica are common causes of foodborne infections in humans with pork as a potential source. Monitoring programs at farm level are, to date, only implemented for S. enterica, while epidemiological knowledge of the other two pathogens is still lacking. This study aimed to assess the pathogen load (in the pigs' environment) in fattening pig herds, their simultaneous occurrence, and the occurrence of Campylobacter spp. and Y. enterocolitica in herds in different Salmonella risk categories. In 50 fattening pig herds in northern Germany, four pooled fecal samples and 10 swab samples from the pigs' direct environment (pen walls, nipple drinkers), indirect environment (hallways, drive boards), and flies and rodent droppings were collected from each herd and submitted for cultural examination. Campylobacter spp. were detected in 38.1% of fecal, 32.7% of direct environment, 5.3% of indirect environment, and 4.6% of flies/pests samples collected, and Y. enterocolitica in 17.1, 8.1, 1.2, and 3.1% and S. enterica in 11.2, 7.7, 4.1, and 1.5%, respectively. For Campylobacter spp., Y. enterocolitica, and S. enterica, 80, 48, and 32% of herds were positive, respectively; 22 herds were positive for both Campylobacter spp. and Y. enterocolitica, 12 for Campylobacter spp. and S. enterica, and 7 for Y. enterocolitica and S. enterica. There was no significant association between the pathogens at herd level. Campylobacter spp. and Y. enterocolitica were found more often in samples from the low Salmonella risk category (odds ratio, 0.51; confidence interval, 0.36 to 0.73, and 0.3, 0.17 to 0.57), and this was also the case for Y. enterocolitica at herd level (odds ratio, 0.08; confidence interval, 0.02 to 0.3). This study provides evidence that the pigs' environment should be accounted for when implementing control measures on farms against Campylobacter spp. and Y. enterocolitica. An extrapolation from the current Salmonella monitoring to the other two pathogens does not seem feasible.

A longitudinal study investigating the prevalence of Staphylococcus aureus genotype B in seasonally communal dairy herds

Abstract Staphylococcus aureus is a major mastitis-causing pathogen. Various genotypes have been recently identified in Switzerland but Staph. aureus genotype B (GTB) was the only genotype associated with high within-herd prevalence. The risk of introducing this Staph. aureus genotype into a herd may be increased by frequent animal movements. This may also be the case when cows from different herds of origin are commingled and share their milking equipment for a limited period of time. The aim of the present study was to determine the prevalence of Staph. aureus GTB in seasonally communal dairy herds before and after a summer period when dairy farming is characterized by mixing cows from different herds of origin in 1 communal operation. In addition, the environment was investigated to identify potential Staph. aureus GTB reservoirs relevant for transmission of the disease. A total of 829 cows from 110 herds of origin in 9 communal operations were included in the study. Composite milk samples were collected from all cows during the first or second milking after arrival at the communal operation and again shortly before the end of the season. Swab samples from the environment, involved personnel, and herding dogs present were collected before the cows arrived. At the end of the season, sampling of personnel was repeated. All samples were analyzed for the presence of Staph. aureus GTB using an established quantitative PCR. At the beginning of the season, Staph. aureus GTB-positive cows were identified in 7 out of 9 communal operations and the within-communal operation prevalence ranged from 2.2 to 38.9%. At the second sampling, all communal operations were Staph. aureus GTB positive, showing within-communal operation prevalence from 1 to 72.1%. The between-herd of origin prevalence increased from 27.3 to 56.6% and the cow-level prevalence increased from 11.2% at the beginning of the season to 29.6% at the end of the season. On 3 different communal operations, Staph. aureus GTB-positive swabs from seasonally employed personnel were identified at the end of the season. The results indicate that Staph. aureus GTB can easily spread in communal operations when cows from different herds of origin are mixed during the summer season. Effective management measures need to be designed to prevent the spread of Staph. aureus GTB in seasonally communal herds. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved. KEYWORDS: Staphylococcus aureus; biosecurity; communal herd; epidemiology

Emergence of Klebsiella pneumoniae co-producing NDM-1, OXA-48, CTX-M-15, CMY-16, QnrA and ArmA in Switzerland

Extensively drug-resistant (XDR) Klebsiella pneumoniae isolates usually carry a single carbapenemase (e.g. KPC, NDM, OXA-48-like). Here we describe an XDR K. pneumoniae of sequence type 101 that was detected in the screening rectal swab of a patient transferred from the intensive care unit of a hospital located in Belgrade (Serbia) to Bern University Hospital (Switzerland). The isolate was resistant to all antibiotics with the exception of colistin [minimum inhibitory concentration] (MIC≤0.125μg/mL), tigecycline (MIC=0.5μg/mL) and fosfomycin (MIC=2μg/mL). The isolate co-possessed class B (NDM-1) and class D (OXA-48) carbapenemases, class A extended-spectrum β-lactamase (CTX-M-15), class C cephalosporinase (CMY-16), ArmA 16S rRNA methyltransferase, substitutions in GyrA and ParC, loss of OmpK35 porin, as well as other genes conferring resistance to quinolones (qnrA), tetracyclines [tet(A)], sulfonamides (sul1, sul2), trimethoprim (dfrA12, dfrA14), rifampicin (arr-1), chloramphenicol (cmlA1, floR) and streptomycin (aadA1). The patient was placed under contact isolation precautions preventing the spread of this nearly untreatable pathogen.

Dacryocystography using cone beam CT in patients with lacrimal drainage system obstruction.

PURPOSE To assess the usefulness of cone beam CT (CBCT) for dacryocystography (DCG) using either direct syringing or passive application of contrast medium. METHODS Ten consecutive patients with epiphora who had CBCT-DCG in a sitting position were retrospectively analyzed. CBCT-DCGs were performed using 2 techniques: direct syringing with contrast medium or using the passive technique, where patients received 3 drops of contrast medium into the conjunctival sac before CBCT-DCG. Clinical and radiologic diagnoses were compared for both groups. RESULTS The 10 patients (men = 3) had a mean age of 63.2 years. Both techniques proved to be simple procedures with good delineation of the bone, soft tissue, and the contrast medium in the lacrimal system. No side effects were noted. CONCLUSIONS CBCT-DCG is a useful alternative to determine the localization of stenosis in patients with chronic epiphora.

Comparison of three different brushing techniques to isolate and culture primary nasal epithelial cells from human subjects.

PURPOSE Primary nasal epithelial cells are used for diagnostic purposes in clinical routine and have been shown to be good surrogate models for bronchial epithelial cells in studies of airway inflammation and remodeling. We aimed at comparing different instruments allowing isolation of nasal epithelial cells. METHODS Primary airway epithelial cell cultures were established using cells acquired from the inferior surface of the middle turbinate of both nostrils. Three different instruments to isolate nasal cells were used: homemade cytology brush, nasal swab, and curette. Cell count, viability, time until a confluent cell layer was reached, and success rate in establishing cell cultures were evaluated. A standard numeric pain intensity scale was used to assess the acceptability of each instrument. RESULTS Sixty healthy adults (median with interquartile range [IQR] age of 31 [26-37] years) participated in the study. Higher number of cells (×10(5) cells/ml) was obtained using brushes (9.8 [5.9-33.5]) compared to swabs (2.4 [1.5-3.9], p < 0.0001) and curettes (5.5 [4.4-6.9], p < 0.01). Cell viability was similar between groups. Cells obtained by brushes had the fastest growth rate, and the success rate in establishing primary cell cultures was highest with brushes (90% vs. 65% for swabs and 70% for curettes). Pain was highest with curettes (VAS score 4.0 [3.0-5.0] out of 10). The epithelial phenotype of the cultures was confirmed through cytokeratin and E-cadherin staining. CONCLUSIONS All three types of instruments allow collection and growth of human nasal epithelial cells with good acceptability to study participants. The most efficient instrument is the nasal brush.

Low Yield of Methicillin-Resistant Staphylococcus aureus Screening in Hemodialysis Patients: 10 Years' Experience

OBJECTIVE To determine the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) nasal colonization in hemodialysis patients and to analyze the cost-effectiveness of our screening approach compared with an alternative strategy. DESIGN Screening study and cost-effectiveness analysis. METHODS Analysis of twice-yearly MRSA prevalence studies conducted in the hemodialysis unit of a 950-bed tertiary care hospital from January 1, 2004, through December 31, 2013. For this purpose, nasal swab samples were cultured on MRSA screening agar (mannitol-oxacillin biplate). RESULTS There were 20 mass screenings during the 10-year study period. We identified 415 patients participating in at least 1 screening, with an average of 4.5 screenings per patient. Of 415 screened patients, 15 (3.6%) were found to be MRSA carriers. The first mass screening in 2004 yielded the highest percentage of MRSA (6/101 [6%]). Only 7 subsequent screenings revealed new MRSA carriers, whereas 4 screenings confirmed previously known carriers, and 8 remained negative. None of the carriers developed MRSA bacteremia during the study period. The total cost of our screening approach, that is, screening and isolation costs, was US $93,930. The total cost of an alternative strategy (ie, no mass screening administered) would be equivalent to costs of isolation of index cases and contact tracing was estimated to be US $5,382 (difference, US $88,548). CONCLUSIONS In an area of low MRSA endemicity (<5%), regular nasal screenings of a high-risk population yielded a low rate of MRSA carriers. Twice-yearly MRSA screening of dialysis patients is unlikely to be cost-effective if MRSA prevalence is low. Infect. Control Hosp. Epidemiol. 2015;00(0):1-4.

In vivo Evolution of CMY-2 to CMY-33 β-Lactamase in Escherichia coli ST131: Characterization of an Acquired Extended-Spectrum AmpC (ESAC) Conferring Resistance to Cefepime.

Cefepime is frequently prescribed to treat infections caused by AmpC-producing Gram-negative bacteria. CMY-2 is the most common plasmid-mediated AmpC (pAmpC) β-lactamase. Unfortunately, CMY variants conferring enhanced cefepime resistance are reported. Here, we describe the evolution of CMY-2 to an extended-spectrum AmpC (ESAC) in clonally identical E. coli isolates obtained from a patient. The CMY-2-producing E. coli (CMY-2-Ec) was isolated from a wound. Thirty days later, one CMY-33-producing E. coli (CMY-33-Ec) was detected in bronchoalveolar lavage. Two weeks before the isolation of CMY-33-Ec, the patient received cefepime.CMY-33-Ec and CMY-2-Ec were identical by rep-PCR, being of hyperepidemic ST131, but showed different β-lactam MICs (e.g., cefepime 16 vs. ≤0.5 μg/ml). Identical CMY-2-Ec isolates were also found in a rectal swab. CMY-33 differs from CMY-2 by a Leu293-Ala294 deletion. Expressed in E. coli DH10B, both CMYs conferred resistance to ceftazidime (≥256 μg/ml), but cefepime MICs were higher for CMY-33 than CMY-2 (8 vs. 0.25 μg/ml). The kcat/Km or kinact/KI (μM(-1) s(-1)) indicated that CMY-33 possesses an ESBL-like spectrum compared to CMY-2 (cefoxitin: 0.2 vs. 0.4; ceftazidime: 0.2 vs. not measurable; cefepime: 0.2 vs. not measurable; tazobactam 0.0018 vs. 0.0009). Using molecular modeling, we show that a widened active site (∼4 Å shift) may play a significant role in enhancing cefepime hydrolysis. This is the first in vivo demonstration of a pAmpC that under cephalosporin treatment expands its substrate spectrum resembling an ESBL. The prevalence of CMY-2-Ec isolates is rapidly increasing worldwide, therefore awareness that cefepime treatment may select for resistant isolates is critical.

Multispecies Epidemiologic Surveillance Study after an Outbreak of Yersiniosis at an African Green Monkey Research Facility.

After an outbreak of Yersinia enterocolitica at a NHP research facility, we performed a multispecies investigation of the prevalence of Yersinia spp. in various mammals that resided or foraged on the grounds of the facility, to better understand the epizootiology of yersiniosis. Blood samples and fecal and rectal swabs were obtained from 105 captive African green monkeys (AGM), 12 feral cats, 2 dogs, 20 mice, 12 rats, and 3 mongooses. Total DNA extracted from swab suspensions served as template for the detection of Y. enterocolitica DNA by real-time PCR. Neither Y. enterocolitica organisms nor their DNA were detected from any of these samples. However, Western blotting revealed the presence of Yersinia antibodies in plasma. The AGM samples revealed a seroprevalence of 91% for Yersinia spp. and of 61% for Y. enterocolitica specifically. The AGM that were housed in cages where at least one fatality occurred during the outbreak (clinical group) had similar seroprevalence to that of AGM housed in unaffected cages (nonclinical group). However, the nonclinical group was older than the clinical group. In addition, 25%, 100%, 33%, 10%, and 10% of the sampled local cats, dogs, mongooses, rats, and mice, respectively, were seropositive. The high seroprevalence after this outbreak suggests that Y. enterocolitica was transmitted effectively through the captive AGM population and that age was an important risk factor for disease. Knowledge regarding local environmental sources of Y. enterocolitica and the possible role of wildlife in the maintenance of yersiniosis is necessary to prevent and manage this disease.