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BACKGROUND:Carbapenem-resistant Klebsiella pneumoniae (CRKP) is emerging as a significant pathogen causing healthcare-associated infections. Matrix-assisted laser desorption/ionisation mass spectrometry time-of-flight mass spectrometry (MALDI-TOF MS) is used by clinical microbiology laboratories to address the need for rapid, cost-effective and accurate identification of microorganisms. We evaluated application of machine learning methods for differentiation of drug resistant bacteria from susceptible ones directly using the profile spectra of whole cells MALDI-TOF MS in 46 CRKP and 49 CSKP isolates. METHODS:We developed a two-step strategy for data preprocessing consisting of peak matching and a feature selection step before supervised machine learning analysis. Subsequently, five machine learning algorithms were used for classification. RESULTS:Random forest (RF) outperformed other four algorithms. Using RF algorithm, we correctly identified 93% of the CRKP and 100% of the CSKP isolates with an overall classification accuracy rate of 97% when 80 peaks were selected as input features. CONCLUSIONS:We conclude that CRKPs can be differentiated from CSKPs through RF analysis. We used direct colony method, and only one spectrum for an isolate for analysis, without modification of current protocol. This allows the technique to be easily incorporated into clinical practice in the future.

The 14-3-3 proteins are cerebrospinal fluid (CSF) markers of neuronal damage during infectious meningitis and Creutzfeldt-Jakob disease. Little is known about dynamic changes in the individual isoforms in response to parasitic eosinophilic meningitis. The purposes of this study were to determine the 14-3-3 protein isoform patterns, examine the kinetics and correlate the severity of blood brain barrier (BBB) damage with the expressions of these markers in mice with eosinophilic meningitis. Mice were orally infected with 50 A. cantonensis L3 via an oro-gastric tube and sacrificed every week for 3 consecutive weeks after infection. The Evans blue method and BBB junctional protein expressions were used to measure changes in the BBB. Hematoxylin and eosin staining was used to analyze pathological changes in the mice brains following 1-3 weeks of infection with A. cantonensis. The levels of 14-3-3 protein isoforms in serum/CSF and brain homogenates were analyzed by Western blot, and immunohistochemistry (IHC) was used to explore the different isoform distributions of 14-3-3 proteins and changes in BBB junctional proteins in the mice brain meninges. Dexamethasone was injected intraperitoneally from the seventh day post infection (dpi) until the end of the study (21 dpi) to study the changes in BBB junctional proteins. The amounts of Evans blue, tight junction and 14-3-3 protein isoforms in the different groups of mice were compared using the nonparametric Kruskal-Wallis test. There were significant increases in 14-3-3 protein isoforms β and γ in the CSF in the second and third weeks after infection compared to the controls and first week of infection, which were correlated with the severity of BBB damage in brain histology, and Evans blue extravasation. Using IHC to assess the distribution of 14-3-3 protein isoforms and changes in BBB junctional proteins in the mice brain meninges, the expressions of isoforms β, γ, ε, and θ and junctional proteins occludin and claudin-5 in the brain meninges increased over a 3-week period after infection compared to the controls and 1 week after infection. The administration of dexamethasone decreased the expressions of BBB junctional proteins occludin and claudin-5 in the mice brain meninges. Our findings support that 14-3-3 proteins β and γ can potentially be used as a CSF marker of neuronal damage in parasitic eosinophilic meningitis caused by A. cantonensis.

Rapid identification of mycobacteria is important for timely treatment and the implementation of public health measures. The MGIT system ensures rapid detection of mycobacteria, but identification is usually delayed by days to weeks due to further subculture on solid medium. Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) was demonstrated to effectively identify mycobacteria isolates subcultured from solid or liquid media. Reports of identification directly from MGIT broths of both sterile and non-sterile clinical specimens, omitting the subculture step, were limited and not satisfactory before. Our identification method dramatically shortened delay from detection to identification of mycobacteria.We assessed the performance of the Vitek MS IVD version 3.0 for direct identification of NTM and M.tuberculosis from primary MGIT cultures, and assessed two sample preparation methods.Direct identification of NTM from positive MGIT broths, using MALDI-TOF VITEK MS with IVD v.3.0, generated high rates of acceptable results reaching 96.4% (80/83), and up to 100% (83/83) for sample preparations including a 0.1% SDS washing step. The sensitivity of VITEK MS to identify M.tuberculosis from MGIT tubes was 58/72 (80.6%), when using immunochromatography (ICA) test as gold standard. A characteristic colony clumping, wool-like appearance was observed in 48, and all 58 (100%) were correctly identified as M.tuberculosis using MALDI-TOF. The detection rate of M.tuberculosis complex was low (10/24, 41.6%) in the 24 MGIT tubes that was polymicrobial. Our method significantly reduced both the reagent cost and turnaround time.Based on a simplified protocol, we showed that MALDI-TOF MS can be used for rapid identification of NTM directly from primary MGIT cultures within the routine clinical laboratory workflow. However, we recommend an initial ICA test to screen for M.tuberculosis complex, due to a low identification rate of M. tuberculosis in the presence of polymicrobial cultures using MALDI-TOF.

BACKGROUND:Dengue fever is an important arboviral disease. The clinical manifestations vary from a mild non-specific febrile syndrome to severe life-threatening illness. The virus can usually be detected in the blood during the early stages of the disease. Dengue virus has also been found in isolated cases in the cerebrospinal fluid, urine, nasopharyngeal sections and saliva. In this report, we describe the isolation of dengue virus from the upper respiratory tract of four confirmed cases of dengue. METHODS:We reviewed all laboratory reports of the isolation of dengue virus from respiratory specimens at the clinical microbiology laboratory of the Kaohsiung Veterans General Hospital during 2007 to 2015. We then examined the medical records of the cases from whom the virus was isolated to determine their demographic characteristics, family contacts, clinical signs and symptoms, course of illness and laboratory findings. RESULTS:Dengue virus was identified in four patients from a nasopharyngeal or throat culture. Two were classified as group A dengue (dengue without warning signs), one as group B (dengue with warning signs) and one as group C (severe dengue). All had respiratory symptoms. Half had family members with similar respiratory symptoms during the period of their illnesses. All of the patients recovered uneventfully. CONCLUSIONS:The isolation of dengue virus from respiratory specimens of patients with cough, rhinorrhea and nasal congestion, although rare, raises the possibility that the virus is capable of transmission by the aerosol route among close contacts. This concept is supported by studies that show that the virus can replicate in cultures of respiratory epithelium and can be transmitted through mucocutaneous exposure to blood from infected patients. However, current evidence is insufficient to prove the hypothesis of transmission through the respiratory route. Further studies will be needed to determine the frequency of respiratory colonization, viable virus titers in respiratory secretions and molecular genetic evidence of transmission among close contacts.

Bile esculin azide with vancomycin (BEAV) medium is a sensitive, but slightly less specific method for vancomycin-resistant enterococci (VRE) screening. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a rapid method for identification of clinical pathogens. This study aimed to assess the performance of a novel combination screening test for VRE, using BEAV broth combined with MALDI-TOF MS.Clinical specimens were collected from patients at risk of VRE carriage, and tested by the novel combination method, using selective BEAV broth culture method followed by MALDI-TOF MS identification (SBEAVM). The reference method used for comparison was the ChromID VRE agar method.A total of 135 specimens were collected from 78 patients, and 63 specimens tested positive for VRE positive using the ChromID VRE method (positive rate 46.7%). The sensitivity, specificity, positive predictive value, and negative predictive value of SBEAVM method after an incubation period of 28 hours were 93.7%, 90.3%, 89.4%, and 94.2%, respectively. The SBEAVM method when compared to the ChromID VRE method had a shorter turnaround time (29 vs. 48-72 hours) and lower laboratory cost ($2.11 vs. $3.23 per test).This study demonstrates that SBEAVM is a rapid, inexpensive, and accurate method for use in VRE screening.

BACKGROUND:Predicting the risk of tuberculosis (TB) in people living with HIV (PLHIV) using a single test is currently not possible. We aimed to develop and validate a clinical algorithm, using baseline CD4 cell counts, HIV viral load (pVL), and interferon-gamma release assay (IGRA), to identify PLHIV who are at high risk for incident active TB in low-to-moderate TB burden settings where highly active antiretroviral therapy (HAART) is routinely provided. MATERIALS AND METHODS:A prospective, 5-year, cohort study of adult PLHIV was conducted from 2006 to 2012 in two hospitals in Taiwan. HAART was initiated based on contemporary guidelines (CD4 count < = 350/μL). Cox regression was used to identify the predictors of active TB and to construct the algorithm. The validation cohorts included 1455 HIV-infected individuals from previous published studies. Area under the receiver operating characteristic (ROC) curve was calculated. RESULTS:Seventeen of 772 participants developed active TB during a median follow-up period of 5.21 years. Baseline CD4 < 350/μL or pVL ≥ 100,000/mL was a predictor of active TB (adjusted HR 4.87, 95% CI 1.49-15.90, P = 0.009). A positive baseline IGRA predicted TB in patients with baseline CD4 ≥ 350/μL and pVL < 100,000/mL (adjusted HR 6.09, 95% CI 1.52-24.40, P = 0.01). Compared with an IGRA-alone strategy, the algorithm improved the sensitivity from 37.5% to 76.5%, the negative predictive value from 98.5% to 99.2%. Compared with an untargeted strategy, the algorithm spared 468 (60.6%) from unnecessary TB preventive treatment. Area under the ROC curve was 0.692 (95% CI: 0.587-0.798) for the study cohort and 0.792 (95% CI: 0.776-0.808) and 0.766 in the 2 validation cohorts. CONCLUSIONS:A validated algorithm incorporating the baseline CD4 cell count, HIV viral load, and IGRA status can be used to guide targeted TB preventive treatment in PLHIV in low-to-moderate TB burden settings where HAART is routinely provided to all PLHIV. The implementation of this algorithm will avoid unnecessary exposure of low-risk patients to drug toxicity and simultaneously, reduce the burden of universal treatment on the healthcare system.