Rapid detection of coinfections by Trichomonas vaginalis, Mycoplasma hominis, and Ureaplasma urealyticum by a new multiplex polymerase chain reaction

We developed a multiplex polymerase chain reaction (M-PCR) assay to simultaneously detect Trichomonas vaginalis, Mycoplasma hominis, and Ureaplasma urealyticum. The test is extremely specific and has a sensitivity of 10 cells for T. vaginalis and U. urealyticum and of 1 cell for M. hominis. The technique was validated on vaginal swabs from 240 women presenting symptoms of vaginitis, and results were compared with data obtained using microscopic and culture techniques on the same patients. The M-PCR revealed to be greatly more sensitive and specific than traditional techniques. It has been well demonstrated, in vitro, that T. vaginalis can establish a symbiosis with M. hominis; our data confirm in vivo this strict association: in fact, M. hominis has been detected in 78.6% of all samples positive for T. vaginalis, as compared to only 4.8% of women without trichomoniasis. The species specificity of this association has been confirmed by the absence of any significant correlation between T. vaginalis and U. urealyticum.     

Co-colonization of vanA and vanB Enterococcus faecium of clonal complex 17 in a patient with bacteremia due to vanA E. faecium

A 53-year-old Vietnamese man with liver cirrhosis was transferred from a Vietnamese hospital to our tertiary care hospital in Korea in order to undergo a liver transplantation. Bacteremia due to vanA Enterococcus faecium was diagnosed, and stool surveillance cultures for vancomycin-resistant enterococci (VRE) were positive for both vanA and vanB E. faecium. Pulsed-field gel electrophoresis analysis revealed that the 2 vanA VRE isolates from the blood and stool were clonal, but the vanB VRE was unrelated to the vanA VRE. vanA and vanB VRE were ST64 and ST18, single-allele variations of clonal complex 17, respectively. This is the first case report of vanA VRE bacteremia in a Vietnamese patient and demonstrates the reemergence of vanB VRE since a single outbreak occurred 15 years ago in Korea. The reemergence of vanB VRE emphasizes the importance of VRE genotyping to prevent the spread of new VRE strains.     

KRAS and PIK3CA but not BRAF genes are frequently mutated in Chinese cholangiocarcinoma patients

Cholangiocarcinoma (CCA) is a rare but lethal malignancy arising from the biliary tract epithelium. It has a poor prognosis largely due to the difficulties of early diagnosis and the lack of effective therapies. It is thus imperative to develop new and effective treatments for CCA, which depends heavily on the mechanistic understanding of the disease. Previous studies have suggested that somatic mutations in KRAS, BRAF, and PIK3CA genes are frequently found in several types of human cancers including colon, breast, and lung carcinomas as well as CCA. Yet, the frequency and the involvement of these oncogenic mutations in CCA in Chinese population have not been investigated. In this study, we evaluated the hotspot mutations of KRAS, BRAF, and PIK3CA genes in 34 Chinese CCA patients. Sequencing analysis revealed 13 (38.2%) and 11 (32.4%) patients bearing KRAS and PIK3CA mutations, in which two (5.9%) of them harbored both KRAS and PIK3CA mutations. Surprisingly, no BRAF mutation was detected in all 34 CCA samples. Our findings indicate that somatic mutations in KRAS and PIK3CA but not BRAF oncogenes are closely associated with the development of CCA in Chinese population and provide new potential targets for future therapeutic treatments of the disease.     

Acinetobacter pittii and Acinetobacter nosocomialis among clinical isolates of the Acinetobacter calcoaceticus-baumannii complex in Sichuan,China

Among 82 clinical isolates of the Acinetobacter calcoaceticus-baumannii complex recovered in 13 hospitals of Sichuan, China, in 2011, 13 were Acinetobacter pittii and 2 were Acinetobacter nosocomialis. Multilocus sequence typing revealed a novel sequence type (ST) of A. nosocomialis and 7 novel STs of A. pittii. Most isolates were hospital-acquired and colonized in the respiratory tract, while 6 cases with pneumonia due to A. pittii were identified. This study provided a snapshot of the local incidence of A. pittii and A. nosocomialis.     

Escherichia albertii, a newly emerging enteric pathogen with poorly defined properties

Escherichia albertii is a newly emerging enteric pathogen that has been associated with sporadic infections among humans and birds. Selected coliform isolates were screened for allelic variation in 2 housekeeping genes (lysP and mdh) specific for E. albertii. The 48 strains that were identified as E. albertii were tested for 15 virulence markers and biochemical and serogical properties. All E. albertii strains were non-motile, fermented D-glucose (with gas), D-mannitol, and D-mannose, but failed to ferment lactose and other sugars. Variable positive reactions were noted for other tests. Most strains were rough or failed to agglutinate with Shigella boydii 13 antisera and E. coli antisera with few exceptions. All strains were positive for the eaeA gene, and variable numbers were positive for the cdtB, phoE, ehxA, and stx2f genes. Results illustrate the variability extent within this lineage and highlight the importance of accurately distinguishing it within the genus Escherichia and including information within commercial databases to improve their identification.     

A case of osteitis due to Staphylococcus aureus and Arcanobacterium bernardiae coinfection

Arcanobacterium bernardiae human infections remain rare. Only 2 reports are described in the literature. We report on an immunocompetent patient with a long history of chronic osteitis who developed a polymicrobial infection of the knee. The initial isolation of Staphylococcus aureus confounded the diagnosis of A. bernardiae infection, which underlines the need for extended period of incubation and subcultures of enriched liquid culture media. A. bernardiae is a new bacterium implicated in osteoarticular infections.     

Predictive genetic risk markers for strong biofilm-forming Staphylococcus aureus: fnbB gene and SCCmec type III

To find predictive genetic risk markers for strong biofilm producers of Staphylococcus aureus, we studied the relatedness of agr and SCCmec types and fnbB and IS256 genes to biofilm-forming ability in 465 clinical isolates. fnbB and SCCmec type III are candidates as genetic risk predictors for the strong biofilm producers.     

Molecular epidemiology and antifungal susceptibility of Candida parapsilosis sensu stricto, Candida orthopsilosis, and Candida metapsilosis in Taiwan

Candida parapsilosis was recently reclassified into 3 closely related species, C. parapsilosis sensu stricto, Candida orthopsilosis, and Candida metapsilosis. Variation in susceptibility characteristics and prevalence of the 3 genomic species could have therapeutic and epidemiologic implications. The aim of this study is to characterize the genetic and antifungal susceptibility profiles of 97 C. parapsilosis isolates from 71 patients. Among the 71 nonduplicate isolates, 85.9% (61/71) were identified as C. parapsilosis sensu stricto, 5.6% (4/71) as C. metapsilosis, and 8.5% (6/71) as C. orthopsilosis species based on sequences of the internal transcribed spacer (ITS) region. The delineation of these 3 species is concordant with that achieved by pulsed-field gel electrophoresis of BssHII restriction fragments at 75% similarity. Antifungal susceptibility tests showed that most isolates were susceptible to flucytosine, azoles, amphotericin B, and echinocandins, whereas 3 C. metapsilosis isolates from 1 patient showed resistance and susceptible-dose dependence to fluconazole. The C. metapsilosis isolates exhibited significantly higher MIC values to both fluconazole and voriconazole than those of C. parapsilosis sensu stricto and C. orthopsilosis. On the other hand, the C. metapsilosis isolates showed significantly lower MIC values on 24 h to caspofungin than those of C. parapsilosis sensu stricto and C. orthopsilosis. For micafungin, the isolates of C. parapsilosis sensu stricto had significantly higher MIC values on 24 h than those of C. orthopsilosis and C. metapsilosis. Compared to Candida albicans, mutations from proline to alanine were identified on the hot spot 1 of Fks1 in all these C. parapsilosis sensu lato isolates regardless of their MIC levels. Some of the C. orthopsilosis and C. metapsilosis isolates expressed the isoleucine to valine substitution on the hot spot 2 region. However, the amino acid variations in these isolates did not correlate to their MIC values of echinocandin.     

The Utility of Wet Prep in Predicting Neisseria gonorrhoeae and Chlamydia trachomatis

Background

Diagnosing Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) cervical infections can be difficult in the Emergency Department without real-time testing, as historical and physical elements are known to be unreliable.

Objective

To evaluate the utility of the vaginal wet mount preparation (wet prep) in predicting an infection with NG or CT.

Methods

A retrospective chart review was performed on 12 months of data from September 2007 to August 2008 on patients aged 18 years and above who had a chief complaint requiring a pelvic examination and had concurrent testing for NG/CT and a wet prep. Wet preps were analyzed and reported as quantity of white cells and clue cells present (none, few, moderate, or many) as well as the presence of Trichomonas vaginalis (TV). Wet prep results were evaluated to see if there was a correlation with NG/CT.

Results

There were 2439 patient encounters reviewed. A total of 373/2439 (15.3%) patient encounters were positive for NG or CT; 272/2439 (11.2%) were positive for TV, whereas 966/2439 (39.6%) had white cells and 995/2439 (40.8%) had clue cells on wet prep. Clue cells and TV did not correlate with the presence of NG or CT. Only the presence of “moderate” and “many” white cells correlated with NG or CT (odds ratio [OR] 1.58, 95% confidence interval [CI] 1.12–2.22 and OR 2.47, 95% CI 1.86–3.27, respectively).

Conclusion

In patients who are diagnosed with NG or CT, the presence of TV or clue cells on wet prep is an unreliable marker for diagnosis. However, having moderate or many white cells present on wet prep does increase the probability of concurrent NG or CT infection and may be used in cases where the clinical suspicion is equivocal.     

Rapid identification of Salmonella enterica subsp. arizonae and S. enterica subsp. diarizonae by real-time polymerase chain reaction

Reptiles are popular as pets, leading to an increased risk of human infections due to uncommon Salmonella strains including the Arizona group (subspecies arizonae and diarizonae). We present a real-time Arizona-specific polymerase chain reaction demonstrating 100% specificity and 99.6% sensitivity, offering savings in time and labor over traditional identification methods.     

NDRG3 and NDRG4, two novel tumor-related genes

The N-myc downstream-regulated genes, NDRG3 and NDRG4, are suggested to play important roles in biological processes and pathogenesis. Expression of NDRG3 and NDRG4 has been shown to be reduced or absent in numerous cancer cell lines and tumor types, suggesting that they may exert function as a tumor suppressor gene. In this review, we will summarize the current research on NDRG3 and NDRG4, including the molecular structure, cellular and tissue distribution, biological function, and function in cancer. We tried to show their significance in studying disease and their therapeutic potential.     

Validation of an immunologic diagnostic kit for infectious vaginitis by Trichomonas vaginalis, Candida spp., and Gardnerella vaginalis

FemPure is a kit for the rapid diagnosis of vaginitis by Trichomonas vaginalis, Candida spp., and Gardnerella vaginalis, based on aggregation of latex particles joined to specific antibodies. The validation of the method involved the parameters specificity, detection limit, robustness, clinical sensitivity, and clinical specificity. Also, samples analyzed in parallel by the validated test and other recognized tests conducted by external laboratory were included. The method was specific for the 3 infectious agents, and no cross-reaction with other microorganisms usually present in vaginal exudates. The detection limit ≥1 × 10⁶ CFU/mL for Candida albicans and G. vaginalis avoids the detection of concentrations considered normal flora, whereas T. vaginalis was detected until 1 × 10⁵ cells/mL. Values of clinical sensitivity ≥80% and clinical specificity ≥90% and concordance ≥90% were found between samples evaluated in parallel by different methods. Robustness showed that the test can be used in laboratories with different management systems; its simple implementation without equipment allows the use in primary health care areas.     

Clinical significance and antimicrobial susceptibilities of Aerococcus sanguinicola and Aerococcus urinae

A retrospective chart review was performed on 92 patients from whom 118 isolates of Aerococcus sanguinicola (n = 52) or Aerococcus urinae (n = 66) were obtained from urine cultures between October 2007 and June 2008 to assess clinical presentation and antimicrobial susceptibilities. The mean patient age was 82 (range 24–101) years. The majority was female (76% and 87% for A. sanguinicola and A. urinae, respectively) and institutionalized (61% and 60%, respectively). The majority of male patients had underlying prostatic disease (55% and 63%, respectively). Thirty-one of 46 patients with A. sanguinicola and 45 of 57 patients with A. urinae isolated from the urine had a clinical diagnosis of urinary tract infection. One subject had A. sanguinicola isolated from blood cultures. A. sanguinicola and A. urinae had low ceftriaxone, penicillin, and vancomycin MICs. MICs to erythromycin and levofloxacin were ≥0.5 and >4 μg/mL in 41% and 78% of A. sanguinicola and 17% and 23% of A. urinae isolates, respectively. In conclusion, A. sanguinicola and A. urinae are not infrequent causes of urinary tract infection and most A. sanguinicola isolates have elevated MICs to levofloxacin.     

Molecular diagnosis of Arcobacter and Campylobacter in diarrhoeal samples among Portuguese patients

The present study was conducted to investigate the prevalence and diversity of Arcobacter and Campylobacter spp. in 298 stool samples of patients with diarrhoea, collected from 22 Portuguese hospitals, between September and November 2012. Detection of Arcobacter and Campylobacter spp. was performed using molecular-based detection techniques, such as real-time fluorescence resonance energy transfer PCR, species-specific PCR, and sequencing of amplified PCR products. Overall, 1.3% of the samples were positive for Arcobacter butzleri and 0.3% for Arcobacter cryaerophilus. Campylobacter spp. were found in 31.9% of diarrhoeic faeces. Campylobacter jejuni and Campylobacter concisus were the most prevalent species (13.7% and 8.0%, respectively). The prevalence of Arcobacter and Campylobacter spp. was significantly different between children and adults (39.7% versus 22.8%, P = 0.003). We underline the high prevalence of these pathogens in diarrhoeal samples among Portuguese patients, with particular relevance in the paediatric age group.     

Standardization of multilocus sequence typing scheme for Mycobacterium abscessus and Mycobacterium massiliense

This study aims to develop a multilocus sequence typing (MLST) scheme for Mycobacterium abscessus complex for the typing of stains within each species. A total of 89 clinical isolates of M. abscessus complex from 71 patients of 2 tertiary care hospitals in South Korea were included. Forty-two isolates were identified as M. abscessus, and 29, as Mycobacterium massiliense through sequencing of 8 housekeeping genes and rpoB. The MLST scheme identified 26 different sequence types(STs) and 13 different clonal complexes (CCs) in M. abscessus and 12 different STs and 6 different CCs in M. massiliense. The MLST data showed high concordance with the XbaI-macrorestriction patterns of pulsed-field gel electrophoresis in the duplicated isolates. Our MLST schemes could identify different strains of M. abscessus and M. massiliense, and the schemes also showed a reliable reproducibility. Therefore, our MLST schemes may be useful in studying the epidemiology of M. abscessus and M. massiliense infections.     

Development of a single-tube polymerase chain reaction assay for the simultaneous detection of Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus spp. directly in clinical samples

This study describes the development and evaluation of a multiplex single-tube polymerase chain reaction assay for the simultaneous detection of Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus spp. used as target species-specific or genus-specific genes. The assay enables the detection of 5 to 50 pg of bacterial DNA. The sensitivity of the assay was evaluated as 100% for P. aeruginosa, S. aureus, and Streptococcus spp., and 94.3% for H. influenzae; the specificity was 100% for all 4 microorganisms (positive predictive value, 100%; negative predictive value, 98.2%). The assay permits rapid and accurate detection of these 4 microorganisms in a wide range of clinical samples such as whole blood, cerebrospinal, ear, pleural and ophthalmic fluids, as well as bronchoalveolar lavage and bronchial secretions.     

Evaluation of colorimetric detection methods for Shigella, Salmonella, and Vibrio cholerae by loop-mediated isothermal amplification

We evaluated loop-mediated isothermal amplification end-point detection methods for Salmonella, Shigella, and Vibrio cholerae. Detection sensitivities were comparable to real-time PCR methods. The colorimetric dyes hydroxynaphthol blue and SYBR Green I showed increased sensitivity when compared to visual and automated turbidity readings. End-point colorimetric dyes promise great utility in developing settings.     

Comparative analyses of individual and multiple alterations of p53, PTEN and p16 in non-small cell lung carcinoma,glioma and breast carcinoma samples

p53, p16 and PTEN are the most commonly altered tumor suppressor genes in human cancers. In the present study, we compared the presence of individual and multiple alterations of these tumor suppressors in non-small cell lung carcinoma (NSCLC), glioma and breast carcinoma, in order to evaluate specificity of each tumor type regarding the number of altered genes, as well as their combinations. We tested the mutational status, loss of heterozygosity and methylation status of these genes. Effects of gene alterations on patients’ survival were also assessed. In NSCLC samples, single gene alterations occurred rarely, while there was considerable increase in incidence of double gene alterations. Furthermore, coexistence of aberrant p53, PTEN and p16 was the most frequent and had significant adverse effect on the survival of NSCLC patients. On the contrary, in glioma and breast cancer specimens, substantial number of cases had aberrant single gene only. Moreover, glioma and breast carcinoma also differ in genotypes that were predominant. Specifically, in glioma samples, prevalent were co-alterations of PTEN and p16, followed by aberrant only PTEN. In breast cancer samples, alterations in all three genes as well as in p53 and p16 were the most common. Moreover, PTEN was altered exclusively with aberrant p53, with statistically significant correlation among them. Overall, our results suggest that NSCLC, glioma and breast cancer need different approaches in molecular diagnosis and treatment with particular attention toward the number and combination of targeted genes.     

Accuracy of commercial systems for identification of Burkholderia pseudomallei versus Burkholderia cepacia

Infection caused by Burkholderia pseudomallei or Burkholderia cepacia may result in fatal outcome unless the causative agent is accurately identified in a short period, which is critical for treatment. We evaluated the reliability of the commonly used commercial systems, API 20NE, VITEK 2, and WalkAway 96, for comparative identification of B. pseudomallei versus B. cepacia clinical isolates. Based on biochemical and molecular tests as reference methods, API 20NE was probably the most reliable, with an accuracy of 87% and 93%, for the identification of B. pseudomallei and B. cepacia, respectively. The VITEK 2 and WalkAway 96 systems resulted in a number of misidentification and, thus, were less reliable. The performance of each system and identification guidelines for B. pseudomallei and B. cepacia are discussed. Our study emphasized that laboratories should carefully interpret the identification of B. pseudomallei and B. cepacia when using commercial systems.     

Rapid detection of vancomycin-resistant enterococci from rectal swabs by the Cepheid Xpert vanA/vanB assay

The detection of vancomycin-resistant enterococci using a novel commercial multiplex real-time polymerase chain reaction assay (Xpert™ vanA/vanB, Cepheid, Sunnyvale, CA) was evaluated on 804 rectal swab specimens. Compared to enriched culture, sensitivity and negative predictive value of this method were 100%. Many false-positive results were recovered (sensitivity, 85.4%; positive predictive value, 8.7%), especially for the vanB gene.