High-Integrity Sequencing of Spike Gene for SARS-CoV-2 Variant Determination.

For tiling of the SARS-CoV-2 genome, the ARTIC Network provided a V4 protocol using 99 pairs of primers for amplicon production and is currently the widely used amplicon-based approach. However, this technique has regions of low sequence coverage and is labour-, time-, and cost-intensive. Moreover, it requires 14 pairs of primers in two separate PCRs to obtain spike gene sequences. To overcome these disadvantages, we proposed a single PCR to efficiently detect spike gene mutations. We proposed a bioinformatic protocol that can process FASTQ reads into spike gene consensus sequences to accurately call spike protein variants from sequenced samples or to fairly express the cases of missing amplicons. We evaluated the in silico detection rate of primer sets that yield amplicon sizes of 400, 1200, and 2500 bp for spike gene sequencing of SARS-CoV-2 to be 59.49, 76.19, and 92.20%, respectively. The in silico detection rate of our proposed single PCR primers was 97.07%. We demonstrated the robustness of our analytical protocol against 3000 Oxford Nanopore sequencing runs of distinct datasets, thus ensuring high-integrity sequencing of spike genes for variant SARS-CoV-2 determination. Our protocol works well with the data yielded from versatile primer designs, making it easy to determine spike protein variants.

Synergy of ß-Lactams with Vancomycin against Methicillin-Resistant Staphylococcus aureus: Correlation of Disk Diffusion and Checkerboard Methods.

Modified disk diffusion (MDD) and checkerboard tests were employed to assess the synergy of combinations of vancomycin and ß-lactam antibiotics for 59 clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and Mu50 (ATCC 700699). Bacterial inocula equivalent to 0.5 and 2.0 McFarland standard were inoculated on agar plates containing 0, 0.5, 1, and 2 µg/ml of vancomycin. Oxacillin-, cefazolin-, and cefoxitin-impregnated disks were applied to the surface, and the zones of inhibition were measured at 24 h. The CLSI-recommended checkerboard method was used as a reference to detect synergy. The MICs for vancomycin were determined using the Etest method, broth microdilution, and the Vitek 2 automated system. Synergy was observed with the checkerboard method in 51% to 60% of the isolates when vancomycin was combined with any ß-lactam. The fractional inhibitory concentration indices were significantly lower in MRSA isolates with higher vancomycin MIC combinations (P < 0.05). The overall agreement between the MDD and checkerboard methods to detect synergy in MRSA isolates with bacterial inocula equivalent to McFarland standard 0.5 were 33.0% and 62.5% for oxacillin, 45.1% and 52.4% for cefazolin, and 43.1% and 52.4% for cefoxitin when combined with 0.5 and 2 µg/ml of vancomycin, respectively. Based on our study, the simple MDD method is not recommended as a replacement for the checkerboard method to detect synergy. However, it may serve as an initial screening method for the detection of potential synergy when it is not feasible to perform other labor-intensive synergy tests.

Phylodynamic reconstruction of the spatiotemporal transmission and demographic history of coxsackievirus B2.

BACKGROUND: Studies regarding coxsackievirus (CV) tend to focus on epidemic outbreaks, an imbalanced topology is considered to be an indication of acute infection with partial cross-immunity. In enteroviruses, a clear understanding of the characteristics of tree topology, transmission, and its demographic dynamics in viral succession and circulation are essential for identifying prevalence trends in endemic pathogens such as coxsackievirus B2 (CV-B2). This study applied a novel Bayesian evolutionary approach to elucidate the phylodynamic characteristics of CV-B2. A dataset containing 51 VP1 sequences and a dataset containing 34 partial 3D(pol) sequencing were analyzed, where each dataset included Taiwan sequences isolated during 1988-2013. RESULTS: Four and five genotypes were determined based on the 846-nucleotide VP1 and 441-nucleotide 3D(pol) (6641-7087) regions, respectively, with spatiotemporally structured topologies in both trees. Some strains with tree discordance indicated the occurrence of recombination in the region between the VP1 and 3D(pol) genes. The similarities of VP1 and 3D(pol) gene were 80.0%-96.8% and 74.7%-91.9%, respectively. Analyses of population dynamics using VP1 dataset indicated that the endemic CV-B2 has a small effective population size. The balance indices, high similarity, and low evolutionary rate in the VP1 region indicated mild herd immunity selection in the major capsid region. CONCLUSIONS: Phylodynamic analysis can reveal demographic trends and herd immunity in endemic pathogens.

Detection of Aichi virus with antibody targeting of conserved viral protein 1 epitope.

Aichi virus (AiV) is an emerging single-stranded, positive-sense, non-enveloped RNA virus in the Picornaviridae that causes acute gastroenteritis in humans. The first case of AiV infection in Taiwan was diagnosed in a human neonate with enterovirus-associated symptoms; the virus was successfully isolated and propagated. To establish a method to detect AiV, we analyzed the antigen epitope and generated a polyclonal antibody against AiV viral protein 1 (VP1). This peptide-purified anti-AiV VP1 antibody showed high specificity against AiV VP1 without cross-reaction to nine other tested strains of Picornaviruses. The anti-AiV VP1 antibody was used in immunofluorescence analysis, immunoblotting, and enzyme-linked immunosorbent assay to elucidate the cell tropism and replication kinetics of AiV. Use of the anti-AiV VP1 antibody also revealed AiV infection restriction with interferon type I and polyI/C antiviral treatment. The AiV infection and detection system may provide an in vitro platform for AiV virology study.

Inhibition of the Mycobacterium tuberculosis reserpine-sensitive efflux pump augments intracellular concentrations of ciprofloxacin and enhances susceptibility of some clinical isolates.

BACKGROUND/PURPOSE: Active efflux is known to play a major role in the resistance of many bacteria to antibiotics. To evaluate the possibility of overcoming resistance by suppressing the efflux, we determined the effect of reserpine, an efflux pump inhibitor. METHODS: Intracellular accumulations and the minimal inhibitory concentrations (MICs) of ciprofloxacin in M. tuberculosis H37Rv and 16 clinical isolates were determined, compared, and analyzed. Nine of the clinical isolates were resistant to isoniazid and rifampin (multiple-drug resistant MDR). Five of these were resistant to ciprofloxacin. RESULTS: A reserpine-inhibited efflux system was identified in the H37Rv control and 10:1 (90.9%) of ciprofloxacin-susceptible and 4:1 (80%) of ciprofloxacin-resistant clinical isolates. The MIC of ciprofloxacin decreased in the presence of reserpine in 3/10 (30%) of the ciprofloxacin-susceptible and 2/4 (50%) of the MDR ciprofloxacin-resistant strains that expressed efflux pumps. Two of the efflux-positive, ciprofloxacin-resistant strains in which the MIC of ciprofloxacin was not decreased by reserpine were found to carry a D94A gyrA mutation. In contrast, two strains with the D94G gyrA mutation were susceptible to ciprofloxacin in the presence of reserpine. An efflux-negative strain, highly resistant to multiple antibiotics, was found to have a novel G247S mutation that differs from known mutations in the QRDR region of the gyrA gene. CONCLUSION: These findings indicate t hat reserpine can increase intracellular concentrations of ciprofloxacin, but is unable to overcome other mechanisms of resistance in clinical isolates.

Functional ginger extracts from supercritical fluid carbon dioxide extraction via in vitro and in vivo assays: antioxidation, antimicroorganism, and mice xenografts models.

Supercritical fluid carbon dioxide extraction technology was developed to gain the active components from a Taiwan native plant, Zingiber officinale (ginger). We studied the biological effects of ginger extracts via multiple assays and demonstrated the biofunctions in each platform. Investigations of ginger extracts indicated antioxidative properties in dose-dependant manners on radical scavenging activities, reducing powers and metal chelating powers. We found that ginger extracts processed moderate scavenging values, middle metal chelating levels, and slight ferric reducing powers. The antibacterial susceptibility of ginger extracts on Staphylococcus aureus, Streptococcus sobrinus, S. mutans, and Escherichia coli was determined with the broth microdilution method technique. The ginger extracts had operative antimicroorganism potentials against both Gram-positive and Gram-negative bacteria. We further discovered the strong inhibitions of ginger extracts on lethal carcinogenic melanoma through in vivo xenograft model. To sum up, the data confirmed the possible applications as medical cosmetology agents, pharmaceutical antibiotics, and food supplements.

Attention-Guided Transfer Learning for Identification of Filamentous Fungi Encountered in the Clinical Laboratory.

This study addresses the challenge of accurately identifying filamentous fungi in medical laboratories using transfer learning with convolutional neural networks (CNNs). The study uses microscopic images from touch-tape slides with lactophenol cotton blue staining, the most common method in clinical settings, to classify fungal genera and identify Aspergillus species. The training and test data sets included 4,108 images with representative microscopic morphology for each genus, and a soft attention mechanism was incorporated to enhance classification accuracy. As a result, the study achieved an overall classification accuracy of 94.9% for four frequently encountered genera and 84.5% for Aspergillus species. One of the distinct features is the involvement of medical technologists in developing a model that seamlessly integrates into routine workflows. In addition, the study highlights the potential of merging advanced technology with medical laboratory practices to diagnose filamentous fungi accurately and efficiently. IMPORTANCE This study utilizes transfer learning with CNNs to classify fungal genera and identify Aspergillus species using microscopic images from touch-tape preparation and lactophenol cotton blue staining. The training and test data sets included 4,108 images with representative microscopic morphology for each genus, and a soft attention mechanism was incorporated to enhance classification accuracy. As a result, the study achieved an overall classification accuracy of 94.9% for four frequently encountered genera and 84.5% for Aspergillus species. One of the distinct features is the involvement of medical technologists in developing a model that seamlessly integrates into routine workflows. In addition, the study highlights the potential of merging advanced technology with medical laboratory practices to diagnose filamentous fungi accurately and efficiently.

Susceptibility of Mycobacterium tuberculosis to sulfamethoxazole, trimethoprim and their combination over a 12 year period in Taiwan.

OBJECTIVES: This study was designed to determine the susceptibility of clinical isolates of multidrug-resistant (MDR) and non-MDR Mycobacterium tuberculosis to sulfamethoxazole, trimethoprim and trimethoprim/sulfamethoxazole over a 12 year period in Taiwan. PATIENTS AND METHODS: We examined a total of 117 clinical isolates of M. tuberculosis collected from Southern Taiwan, 116 from 1995 to 2006 and an extensively drug-resistant (XDR) isolate in 2009. These included 28 isolates susceptible to all four first-line agents, 52 MDR isolates and 36 isolates with a mixed combination of drug resistance patterns other than MDR and 1 XDR isolate. RESULTS: Sulfamethoxazole inhibited 80% growth of all 117 isolates regardless of their susceptibility to the first-line agents at an MIC(90) of 9.5 mg/L. The concentration required to inhibit 99% growth was 38 mg/L. There were no significant changes in the MIC(50) or MIC(90) of sulfamethoxazole over a 12 year period. All 117 isolates were resistant to trimethoprim at >8 mg/L. The combination of trimethoprim/sulfamethoxazole at a ratio of 1:19 had no additive or synergistic effects. CONCLUSIONS: Sulfamethoxazole inhibited the growth of clinical isolates of M. tuberculosis at achievable concentrations in plasma after oral administration. Susceptibility to sulfamethoxazole remained constant over a 12 year period. Trimethoprim was inactive against M. tuberculosis and trimethoprim/sulfamethoxazole provided no additional activity. Although the current and prior studies demonstrate that sulfamethoxazole is active against M. tuberculosis the search needs to continue for more active, lipid-soluble sulphonamides that are better absorbed into tissues and have improved therapeutic efficacy.

Epidemiology of group B Streptococcus ST-17 clone in pregnant women of South Taiwan.

OBJECTIVES: We aimed to utilize a simple molecular assay to simultaneously detect both group B Streptococcus (GBS) and virulent ST-17 rectovaginal colonization. We also attempted to estimate the prevalence of maternal GBS and ST-17 carriers and to evaluate their seasonal association. SUBJECTS AND METHODS: We used an optimized multiplex PCR method employing scp-B and ST-17 primers to analyze DNA extracted from rectovaginal swabs of 3,064 cases collected over 3 years. The incidence trends, seasonal variations, and temperature preference were analyzed. RESULTS: The overall prevalence of maternal colonization for GBS and ST-17 clone were 13.25 and 2.48%, respectively. The ST-17 to GBS ratio was 18.72%. The occurrence of ST-17 colonization was significantly associated with seasonal variations with a preference for lower temperatures. CONCLUSIONS: We developed a novel multiplex PCR method suitable for the simultaneous detection of GBS and ST-17 clone. The phenomenon of lower temperature preference for ST-17 clone necessitates further investigation. The epidemiological data for GBS and ST-17 incidence are especially important to establish a public policy for universal GBS screening in the future.

Biological properties of acidic cosmetic water from seawater.

This current work was to investigate the biological effects of acidic cosmetic water (ACW) on various biological assays. ACW was isolated from seawater and demonstrated several bio-functions at various concentration ranges. ACW showed a satisfactory effect against Staphylococcus aureus, which reduced 90% of bacterial growth after a 5-second exposure. We used cultured human peripheral blood mononuclear cells (PBMCs) to test the properties of ACW in inflammatory cytokine release, and it did not induce inflammatory cytokine release from un-stimulated, normal PBMCs. However, ACW was able to inhibit bacterial lipopolysaccharide (LPS)-induced inflammatory cytokine TNF-α released from PBMCs, showing an anti-inflammation potential. Furthermore, ACW did not stimulate the rat basophilic leukemia cell (RBL-2H3) related allergy response on de-granulation. Our data presented ACW with a strong anti-oxidative ability in a superoxide anion radical scavenging assay. In mass spectrometry information, magnesium and zinc ions demonstrated bio-functional detections for anti-inflammation as well as other metal ions such as potassium and calcium were observed. ACW also had minor tyrosinase and melanin decreasing activities in human epidermal melanocytes (HEMn-MP) without apparent cytotoxicity. In addition, the cell proliferation assay illustrated anti-growth and anti-migration effects of ACW on human skin melanoma cells (A375.S2) indicating that it exerted the anti-cancer potential against skin cancer. The results obtained from biological assays showed that ACW possessed multiple bioactivities, including anti-microorganism, anti-inflammation, allergy-free, antioxidant, anti-melanin and anticancer properties. To our knowledge, this was the first report presenting these bioactivities on ACW.

Using a Knowledge-Based Clinical Decision Support System to Reduce the Time to Appropriate Antimicrobial Therapy in Hospitalized Patients With Bloodstream Infections: A Single-Center Observational Study.

Background: Inappropriate antimicrobial use is a crucial determinant of mortality in hospitalized patients with bloodstream infections. Current literature reporting on the impact of clinical decision support systems on optimizing antimicrobial prescription and reducing the time to appropriate antimicrobial therapy is limited. Methods: Kaohsiung Veterans General Hospital implemented a hospital-wide, knowledge-based, active-delivery clinical decision support system, named RAPID (Real-time Alert for antimicrobial Prescription from virtual Infectious Diseases experts), to detect whether there was an antimicrobial agent-pathogen mismatch when a blood culture result was positive. Once RAPID determines the current antimicrobials as inappropriate, an alert text message is immediately sent to the clinicians in charge. This study evaluated how RAPID impacted the time to appropriate antimicrobial therapy among patients with bloodstream infections. Results: During the study period, 633 of 11 297 recorded observations (5.6%) were determined as inappropriate antimicrobial prescriptions. The time to appropriate antimicrobial therapy was significantly shortened after the implementation of RAPID (1.65 vs 2.45 hours, P < .001), especially outside working hours (1.24 vs 6.43 hours, P < .001), in the medical wards (1.40 vs 2.14 hours, P < .001), in participants with candidemia (0.74 vs 5.36 hours, P < .001), and for bacteremia due to non-multidrug-resistant organisms (1.66 vs 2.49 hours, P < .001). Conclusions: Using a knowledge-based clinical decision support system to reduce the time to appropriate antimicrobial therapy in a real-world scenario is feasible and effective. Our results support the continued use of RAPID.

Low Seroprevalence of Aichi Virus Infection in Taiwan.

Aichi virus (AiV) belongs to the genus Kobuvirus of the family Picornaviridae; it is a single-stranded positive-sense RNA virus without an envelope. AiV causes acute gastroenteritis, abdominal pain, nausea, vomiting, and fever. Low incidence and high seroprevalence of AiV infections have been reported in several regions of the world; however, little was known on the prevalence of AiV infections in Taiwan. This study described the first two cases of AiV infection and analyzed AiV seroprevalence in Taiwan. A total of 700 sera were collected from a single hospital in southern Taiwan. The neutralization assay was employed to assess AiV neutralization antibodies in the serum. The test identified 48 positive cases, with a seroprevalence of 6.86%. Results also showed a gradual increase in AiV seroprevalence rate with age. Compared with other countries, Taiwan had a relatively low AiV seroprevalence, suggesting a low incidence of or sporadic AiV infections.

Clinical significance of isolation of Mycobacterium avium complex from respiratory specimens.

BACKGROUND/PURPOSE: Mycobacterium avium complex (MAC) is frequently considered to be a contaminant or transient colonizer. To the best of our knowledge, there have been very few reports regarding the clinical significance of MAC isolates in respiratory specimens, and the associated disease spectrum in Taiwan. The purpose of this study was to investigate the clinical significance of MAC isolates in respiratory specimens. METHODS: We retrospectively reviewed the medical records of patients in a medical center in Southern Taiwan from whom MAC isolates were recovered from respiratory specimens, and analyzed their clinical features, chest imaging findings, treatment and prognosis. We also performed an antibiotic susceptibility test on our MAC isolates. RESULTS: The 64 isolates used in this study were recovered from April to October 2001 from respiratory specimens in 54 patients admitted to Kaohsiung Veterans General Hospital, Taiwan. According to the 2007 criteria of the American Thoracic Society, a total of 12 patients (22.2%) had clinically significant MAC pulmonary disease. CONCLUSION: Despite the increased frequency of recovering MAC from respiratory specimens, most cases did not meet the criteria of American Thoracic Society for clinically significant nontuberculous pulmonary disease. The minimum inhibitory concentrations of drugs against these MAC isolates might help to guide treatment, but further studies should be done.

Detection of carbapenem-resistant Klebsiella pneumoniae on the basis of matrix-assisted laser desorption ionization time-of-flight mass spectrometry by using supervised machine learning approach.

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.

In vitro activities of linezolid against clinical isolates of Mycobacterium tuberculosis complex isolated in Taiwan over 10 years.

Significant increases in the MIC(90)s of linezolid in multidrug-resistant Mycobacterium tuberculosis isolates were seen between the baseline period of 2001 to 2003 (0.5 microg/ml) and 2004 (2 microg/ml). The MICs were 4 microg/ml in three strains. Both fluoroquinolones (except levofloxacin) and kanamycin were found to have statistically significant degrees of concordance with linezolid.

Clinical application of reverse-transcription polymerase chain reaction and intravenous immunoglobulin for enterovirus encephalitis.

Although polymerase chain reaction (PCR) is a highly sensitive procedure for the diagnosis of enteroviruses, it has never been systemically applied to the treatment of enteroviral encephalitis using intravenous immunoglobulin (IVIg). We conducted a 2-year randomized, controlled comparison of reverse transcription (RT)-PCR of cerebrospinal fluid (CSF) with traditional viral isolation to guide IVIg treatment. Seventy-five patients were enrolled and classified into three groups: one group with clinical manifestations of enteroviral infections and two without. The latter two groups were separated on the basis of whether IVIg treatment was guided by RT-PCR or virus culture assay. CSF specimens from the 18 confirmed cases of enteroviral encephalitis were RT-PCR positive for enterovirus in all but one case. Of the remaining 57 cases of nonenteroviral encephalitis, only 4 were positive for enterovirus RT-PCR. One patient in the group of IVIg treatment guided by viral isolation subsequently displayed a sequel of epilepsy. No patients in the IVIg treatment groups guided by RT-PCR had any neurological sequelae. In conclusion, the use of RT-PCR allowed rapid, sensitive, and specific detection of enteroviral RNA in CSF. When used to guide IVIg treatment, RT-PCR may shorten hospitalization and improve outcomes of patients with enteroviral encephalitis.

Antimicrobial resistance of rapidly growing mycobacteria in western Taiwan: SMART program 2002.

BACKGROUND/PURPOSE: To understand the resistance patterns of rapidly growing mycobacteria (RGM) in Taiwan, antimicrobial resistance of clinical isolates was determined as part of the SMART (Surveillance from Multicenter Antimicrobial Resistance in Taiwan) program. METHODS: During the period from January 2002 to December 2003, clinical isolates were collected from eight hospitals located on the west side of Taiwan and one reference laboratory. Broth microdilution minimum inhibitory concentrations of 11 antimicrobial agents were determined for 312 clinical isolates of RGM, including the Mycobacterium fortuitum group (110 isolates), Mycobacterium abscessus group (168 isolates), and Mycobacterium chelonae group (34 isolates). RESULTS: Nearly all of the RGM were susceptible to amikacin and ofloxacin (= 90%) and resistant to doxycycline (less than 3% susceptible). Tobramycin showed similar in vitro activity against the M. fortuitum and M. chelonae (77%) groups, but was less active against the M. abscessus group (58%). Ciprofloxacin was active mainly against M. fortuitum (95%). Nearly all RGM were resistant to erythromycin and doxycycline. However, around half of the RGM isolates remained susceptible to minocycline (50-54%). Clarithromycin was active against the M. abscessus group (53% susceptible), with a high rate of resistance in the M. chelonae (38% susceptible) and M. fortuitum (15% susceptible) group. Cefoxitin was more active against the M. fortuitum group (65%) than the other two RGM (40-44%), and les than 40% of the RGM isolates remained susceptible to imipenem (21-38%). CONCLUSION: The resistance of RGM in Taiwan is not as high as previously reported (notably for tobramycin, ciprofloxacin and cefoxitin), but reduction in the susceptibility rates of clarithromycin and imipenem for the M. fortuitum and M. abscessus groups demonstrates the importance of in vitro susceptibility testing of clinically important isolates, as susceptibility may differ in different geographical areas, even regionally, and over time.

Rapid identification of mycobacteria from positive MGIT broths of primary cultures by MALDI-TOF mass spectrometry.

BACKGROUND: 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. METHODOLOGY: 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. RESULTS: 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. CONCLUSIONS: 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.

Human Parechovirus Infection in Children in Taiwan: a Retrospective, Single-Hospital Study.

To understand human parechovirus (HPeV) infections in Taiwanese children, we analyzed data for 112 children (age≤10 years) with HPeV infection diagnosed between July 2007 and June 2016 in a medical center in Kaohsiung, southern Taiwan. The patients were infected with HPeV1 (n=94), HPeV3 (n=3), HPeV4 (n=3), HPeV6 (n=1) and non-typeable HPeV (n=11). We compared the clinical implications for children younger than 3 months (n=56) and 3 months and older (n=31), excluding 25 children with concomitant infections. Fever was noted in almost half of the children younger than 3 months but was more frequent in older than in younger children (83.9% vs 46.4%). As compared with older children, children younger than 3 months had a lower incidence of respiratory symptoms (30.1% vs 83.9%), more frequently required intensive care unit admission (28.6% vs 3.2%), and had longer hospital stays (mean 10.95 vs 5.13 days). Importantly, about one-third of the children were suspected to have hospital-acquired or cluster infections in the environment of medical institutions, with a significantly high proportion of 42.9% (24/56) in younger infants. Hospital-acquired infections might play a key role in the spread of HPeV, especially in children younger than 3 months.

Parechovirus A Detection by a Comprehensive Approach in a Clinical Laboratory.

Parechovirus A (Human parechovirus, HPeV) causes symptoms ranging from severe neonatal infection to mild gastrointestinal and respiratory disease. Use of molecular approaches with RT-PCR and genotyping has improved the detection rate of HPeV. Conventional methods, such as viral culture and immunofluorescence assay, together with molecular methods facilitate comprehensive viral diagnosis. To establish the HPeV immunofluorescence assay, an antibody against HPeV capsid protein VP0 was generated by using antigenic epitope prediction data. The specificity of the anti-HPeV VP0 antibody was demonstrated on immunofluorescence assay, showing that this antibody was specific for HPeV but not enteroviruses. A total of 74 HPeV isolates, 7 non⁻polio-enteroviruses and 12 HPeV negative cell culture supernatant were used for evaluating the efficiency of the anti-HPeV VP0 antibody. The sensitivity of HPeV detection by the anti-HPeV VP0 antibody was consistent with 5'untranslated region (UTR) RT-PCR analysis. This study established comprehensive methods for HPeV detection that include viral culture and observation of cytopathic effect, immunofluorescence assay, RT-PCR and genotyping. The methods were incorporated into our routine clinical practice for viral diagnosis. In conclusion, this study established a protocol for enterovirus and HPeV virus identification that combines conventional and molecular methods and would be beneficial for HPeV diagnosis.