Medical tourism and medical tourists: providing a sustainable course to integrating health treatments with tourism.

Medical tourism (MT) is an expanding multidisciplinary economic activity that combines the healthcare and tourist industries, with patients increasingly travelling worldwide for medical treatments. MT provides economic benefits to destinations while raising ethical, quality, informed and risk concerns for medical tourists. Greater cross-disciplinary studies and collaboration across sectors are advocated.

Facile and Unplugged Surface Plasmon Resonance Biosensor with NIR-Emitting Perovskite Nanocomposites for Fast Detection of SARS-CoV-2.

The emergence of the coronavirus disease 2019 (COVID-19) pandemic prompted researchers to develop portable biosensing platforms, anticipating to detect the analyte in a label-free, direct, and simple manner, for deploying on site to prevent the spread of the infectious disease. Herein, we developed a facile wavelength-based SPR sensor built with the aid of a 3D printing technology and synthesized air-stable NIR-emitting perovskite nanocomposites as the light source. The simple synthesis processes for the perovskite quantum dots enabled low-cost and large-area production and good emission stability. The integration of the two technologies enabled the proposed SPR sensor to exhibit the characteristics of lightweight, compactness, and being without a plug, just fitting the requirements of on-site detection. Experimentally, the detection limit of the proposed NIR SPR biosensor for refractive index change reached the 10-6 RIU level, comparable with that of state-of-the-art portable SPR sensors. In addition, the bio-applicability of the platform was validated by incorporating a homemade high-affinity polyclonal antibody toward the SARS-CoV-2 spike protein. The results demonstrated that the proposed system was capable of discriminating between clinical swab samples collected from COVID-19 patients and healthy subjects because the used polyclonal antibody exhibited high specificity against SARS-CoV-2. Most importantly, the whole measurement process not only took less than 15 min but also needed no complex procedures or multiple reagents. We believe that the findings disclosed in this work can open an avenue in the field of on-site detection for highly pathogenic viruses.

Clinical Impact of the Revised 2019 CLSI Levofloxacin Breakpoints in Patients with Enterobacterales Bacteremia.

The Clinical and Laboratory Standards Institute (CLSI) revised the fluoroquinolone MIC breakpoints for Enterobacterales in 2019, based on pharmacokinetic/pharmacodynamic analyses. However, clinical evidence supporting these breakpoint revisions is limited. A retrospective study was conducted at 3 hospitals in Taiwan between January 2017 and March 2019. Patients treated with levofloxacin for bacteremia caused by members of the Enterobacterales with high MICs (1 or 2 µg/ml; levofloxacin susceptible by pre-2019 CLSI breakpoints) were compared with those with low-MIC bacteremia (≤0.5 µg/ml; levofloxacin susceptible by 2019 CLSI breakpoints) to assess therapeutic effectiveness by multivariable logistic regression. The primary outcome was 30-day mortality, and the secondary outcome was the emergence of levofloxacin-resistant isolates within 90 days after levofloxacin initiation. A total of 308 patients were eligible for the study. Kaplan-Meier analysis showed that patients infected with high-MIC isolates (n = 63) had a significantly lower survival rate than those infected with low-MIC isolates (n = 245) (P = 0.001). Multivariable logistic regression revealed that high levofloxacin MIC was a predictor of 30-day mortality (odds ratio [OR], 6.05; 95% confidence interval [CI], 1.51 to 24.18; P = 0.011). We consistently found similar results in a propensity score-matched cohort (OR, 5.38; 95% CI, 1.06 to 27.39; P = 0.043). The emergence of levofloxacin-resistant isolates was more common in the high-MIC group than the low-MIC group (25.0% versus 7.5%; P = 0.065). An estimated area under the concentration-time curve/MIC ratio of ≥87 was significantly associated with better survival (P = 0.002). In conclusion, patients infected with isolates with levofloxacin MICs within the pre-2019 CLSI susceptible range of 1 or 2 µg/ml exhibited higher mortality than those infected with isolates with MICs of ≤0.5 µg/ml.

Identification of Important N-Linked Glycosylation Sites in the Hemagglutinin Protein and Their Functional Impact on DC-SIGN Mediated Avian Influenza H5N1 Infection.

DC-SIGN, a C-type lectin mainly expressed in dendritic cells (DCs), has been reported to mediate several viral infections. We previously reported that DC-SIGN mediated H5N1 influenza A virus (AIVs) infection, however, the important DC-SIGN interaction with N-glycosylation sites remain unknown. This study aims to identify the optimal DC-SIGN interacting N-glycosylation sites in HA proteins of H5N1-AIVs. Results from NetNGlyc program analyzed the H5 hemagglutinin sequences of isolates during 2004-2020, revealing that seven and two conserved N-glycosylation sites were detected in HA1 and HA2 domain, respectively. A lentivirus pseudotyped A/Vietnam/1203/04 H5N1 envelope (H5N1-PVs) was generated which displayed an abundance of HA5 proteins on the virions via immuno-electron microscope observation. Further, H5N1-PVs or reverse-genetics (H5N1-RG) strains carrying a serial N-glycosylated mutation was generated by site-directed mutagenesis assay. Human recombinant DC-SIGN (rDC-SIGN) coated ELISA showed that H5N1-PVs bound to DC-SIGN, however, mutation on the N27Q, N39Q, and N181Q significantly reduced this binding (p < 0.05). Infectivity and capture assay demonstrated that N27Q and N39Q mutations significantly ameliorated DC-SIGN mediated H5N1 infection. Furthermore, combined mutations (N27Q&N39Q) significantly waned the interaction on either H5N1-PVs or -RG infection in cis and in trans (p < 0.01). This study concludes that N27 and N39 are two essential N-glycosylation contributing to DC-SIGN mediating H5N1 infection.

Detection of Cytopathic Effects Induced by Influenza, Parainfluenza, and Enterovirus Using Deep Convolution Neural Network.

The isolation of a virus using cell culture to observe its cytopathic effects (CPEs) is the main method for identifying the viruses in clinical specimens. However, the observation of CPEs requires experienced inspectors and excessive time to inspect the cell morphology changes. In this study, we utilized artificial intelligence (AI) to improve the efficiency of virus identification. After some comparisons, we used ResNet-50 as a backbone with single and multi-task learning models to perform deep learning on the CPEs induced by influenza, enterovirus, and parainfluenza. The accuracies of the single and multi-task learning models were 97.78% and 98.25%, respectively. In addition, the multi-task learning model increased the accuracy of the single model from 95.79% to 97.13% when only a few data of the CPEs induced by parainfluenza were provided. We modified both models by inserting a multiplexer and de-multiplexer layer, respectively, to increase the correct rates for known cell lines. In conclusion, we provide a deep learning structure with ResNet-50 and the multi-task learning model and show an excellent performance in identifying virus-induced CPEs.

The Combination of CA125 and NSE Is Useful for Predicting Liver Metastasis of Lung Cancer.

PURPOSE: Liver metastasis is the final stage of cancer progression and is associated with poor prognosis. Although numerous indicators have been identified as having prognostic value for lung cancer and liver metastasis, liver metastases are still not diagnosed by imaging in many patients. To provide a more accurate method for clinical prediction of liver metastasis, we analyzed multiple factors to identify potential predictive factors for liver metastasis of lung cancer. METHODS: Patients first diagnosed with lung cancer between 2002 and 2016 (n = 1746) were divided into two groups, with and without liver metastasis. Serum concentrations of calcium, carcinoembryonic antigen (CEA), cancer antigen-125 (CA125), cancer antigen-153 (CA153), carbohydrate antigen-199 (CA199), cytokeratin fraction 21-1 (CYFRA21-1), total prostate-specific antigen (TPSA), and neuron-specific enolase (NSE) were analyzed in both patient groups. RESULTS: There was no significant difference in age or sex between the two groups. CA125 and NSE were significantly associated with liver metastasis. Compared with CA125, NSE was more specific, while it was less sensitive (P < 0.001). Further analysis of NSE concentrations was conducted in patients with non-small-cell lung cancer and indicated that NSE concentration differed significantly between those with and without liver metastasis (P = 0.023). We conducted analysis with NSE and CA125 combined, resulting in acceptable sensitivity (51.2%), specificity (72.6%), and area under the curve (0.64) values; sensitivity and area under the curve values were higher than those for individual factors, while specificity was higher than that for CA125. CONCLUSIONS: The combination of CA125 and NSE can assist prediction of liver metastasis of lung cancer, providing improved diagnostic accuracy.

CYFRA21-1/TG ratio as an accurate risk factor to predict eye metastasis in nasopharyngeal carcinoma: A STROBE-compliant article.

Nasopharyngeal carcinoma (NPC) has a distinctive geographical distribution in China, especially southern China. There are several risk factors for NPC, such as Epstein-Barr virus, genetics, and environmental exposures. Although the incidence of eye metastasis (EM) is lower than metastasis in other body parts, it often indicates poor prognosis.We assessed several serum biomarkers for their ability to predict EM in NPC. Patients with NPC were selected (n = 963), and were separated into two groups, EM and no eye metastasis. Ten factors were analyzed in both groups including triglyceride (TG), high-density lipoprotein, low-density lipoprotein, alkaline phosphatase, alpha fetoprotein, carbohydrate antigen-199, cancer antigen-153, apolipoproteins AI, apolipoprotein B, and cytokeratin fragment 19 (CYFRA21-1). Independent t tests, binary logistic regression, and receiver operating characteristic curves were used to assess the data.The EM group had significantly higher CYFRA21-1 and lower TG compared with the no eye metastasis group. Areas under the curve for CYFRA21-1, TG and CYFRA21-1/TG were 0.966, 0.771, and 0.976, respectively. The corresponding cut-off values were 12.12 ng/ml, 0.41 mmol/L, and 13.5. The sensitivity and specificity of CYFRA21-1/TG were 100% and 92.2%, respectively.The increased ratio of CYFRA21-1 to TG can be an accurate method to detect EM in patients with NPC.

Optimization of the CDC Protocol of Molecular Diagnosis of COVID-19 for Timely Diagnosis.

Coronavirus disease 2019 (COVID-19), the current uncontrolled outbreak of infectious disease, has caused significant challenges throughout the world. A reliable rapid diagnostic test for COVID-19 is demanded worldwide. The real-time reverse transcriptase polymerase chain was one of the most quickly established methods in the novel viral pandemic and was considered as the gold standard for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this report, we illustrate our experience of applying a protocol from the Taiwan CDC and achieving assay optimization in the immediate circumstances to meet the urgent medical and public health needs.

Evolutionary histories of coxsackievirus B5 and swine vesicular disease virus reconstructed by phylodynamic and sequence variation analyses.

Coxsackievirus (CV)-B5 is a common human enterovirus reported worldwide; swine vesicular disease virus (SVDV) is a porcine variant of CV-B5. To clarify the transmission dynamics and molecular basis of host switching between CV-B5 and SVDV, we analysed and compared the VP1 and partial 3Dpol gene regions of these two viruses. Spatiotemporal dynamics of viral transmission were estimated using a Bayesian statistical inference framework. The detected selection events were used to analyse the key molecules associated with host switching. Analyses of VP1 sequences revealed six CV-B5 genotypes (A1-A4 and B1-B2) and three SVDV genotypes (I-III). Analyses of partial 3Dpol revealed five clusters (A-E). The genotypes evolved sequentially over different periods, albeit with some overlap. The major hub of CV-B5 transmission was in China whereas the major hubs of SVDV transmission were in Italy. Network analysis based on deduced amino acid sequences showed a diverse extension of the VP1 structural protein, whereas most sequences were clustered into two haplotypes in the partial 3Dpol region. Residue 178 of VP1 showed four epistatic interactions with residues known to play essential roles in viral host tropism, cell entry, and viral decoating.

Phylodynamic Characterization of an Ocular-Tropism Coxsackievirus A24 Variant.

Recent phylodynamic studies have focused on using tree topology patterns to elucidate interactions among the epidemiological, evolutionary, and demographic characteristics of infectious agents. However, because studies of viral phylodynamics tend to focus on epidemic outbreaks, tree topology signatures of tissue-tropism pathogens might not be clearly identified. Therefore, this study used a novel Bayesian evolutionary approach to analyze the A24 variant of coxsackievirus (CV-A24v), an ocular-tropism agent of acute hemorrhagic conjunctivitis. Analyses of the 915-nucleotide VP1 and 690-nt 3Dpol regions of 21 strains isolated in Taiwan and worldwide during 1985-2010 revealed a clear chronological trend in both the VP1 and 3Dpol phylogenetic trees: the emergence of a single dominant cluster in each outbreak. The VP1 sequences included three genotypes: GI (prototype), GIII (isolated 1985-1999), and GIV (isolated after 2000); no VP1 sequences from GII strains have been deposited in GenBank. Another five genotypes identified in the 3Dpol region had support values >0.9. Geographic and demographic transitions among CV-A24v clusters were clearly identified by Bayes algorithm. The transmission route was mapped from India to China and then to Taiwan, and each prevalent viral population declined before new clusters emerged. Notably, the VP1 and 3Dpol genes had high nucleotide sequence similarities (94.1% and 95.2%, respectively). The lack of co-circulating lineages and narrow tissue tropism affected the CV-A24v gene pool.

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.

Transmission and Demographic Dynamics of Coxsackievirus B1.

The infectious activity of coxsackievirus B1 (CV-B1) in Taiwan was high from 2008 to 2010, following an alarming increase in severe neonate disease in the United States (US). To examine the relationship between CV-B1 strains isolated in Taiwan and those from other parts of the world, we performed a phylodynamic study using VP1 and partial 3Dpol (414 nt) sequences from 22 strains of CV-B1 isolated in Taiwan (1989-2010) and compared them to sequences from strains isolated worldwide. Phylogenetic trees were constructed by neighbor-joining, maximum likelihood, and Bayesian Monte Carlo Markov Chain methods. Four genotypes (GI-IV) in the VP1 region of CV-B1 and three genotypes (GA-C) in the 3Dpol region of enterovirus B were identified and had high support values. The phylogenetic analysis indicates that the GI and GIII strains in VP1 were geographically distributed in Taiwan (1993-1994) and in India (2007-2009). On the other hand, the GII and GIV strains appear to have a wider spatiotemporal distribution and ladder-like topology A stair-like phylogeny was observed in the VP1 region indicating that the phylogeny of the virus may be affected by different selection pressures in the specified regions. Further, most of the GI and GII strains in the VP1 tree were clustered together in GA in the 3D tree, while the GIV strains diverged into GB and GC. Taken together, these data provide important insights into the population dynamics of CV-B1 and indicate that incongruencies in specific gene regions may contribute to spatiotemporal patterns of epidemicity for this virus.

Molecular Epidemiology and Phylogenetic Analysis of Human Adenovirus Caused an Outbreak in Taiwan during 2011.

An outbreak of adenovirus has been surveyed in Taiwan in 2011. To better understand the evolution and epidemiology of adenovirus in Taiwan, full-length sequence of hexon and fiber coapsid protein was analyzed using series of phylogenetic and dynamic evolution tools. Six different serotypes were identified in this outbreak and the species B was predominant (HAdV-3, 71.50%; HAdV-7, 15.46%). The most frequent diagnosis was acute tonsillitis (54.59%) and bronchitis (47.83%). Phylogenetic analysis revealed that hexon protein gene sequences were highly conserved for HAdV-3 and HAdV-7 circulation in Taiwan. However, comparison of restriction fragment length polymorphism (RFLP) analysis and phylogenetic trees of fiber gene in HAdV-7 clearly indicated that the predominant genotype in Taiwan has shifted from 7b to 7d. Several positive selection sites were observed in hexon protein. The estimated nucleotide substitution rates of hexon protein of HAdV-3 and HAdV-7 were 0.234×10-3 substitutions/site/year (95% HPD: 0.387~0.095×10-3) and 1.107×10-3 (95% HPD: 0. 541~1.604) respectively; those of the fiber protein of HAdV-3 and HAdV-7 were 1.085×10-3 (95% HPD: 1.767~0.486) and 0.132×10-3 (95% HPD: 0.283~0.014) respectively. Phylodynamic analysis by Bayesian skyline plot (BSP) suggested that using individual gene to evaluate the effective population size might possibly cause miscalculation. In summary, the virus evolution is ongoing, and continuous surveillance of this virus evolution will contribute to the control of the epidemic.

The development of a beacon-based real-time PCR for cytomegalovirus and its application in immunocompromised patients.

BACKGROUND: Because of the high cost of commercially available quantitative PCR kits, we developed a beacon- based real-time PCR (B-rt-PCR) for Cytomegalovirus (CMV) viral load determination. METHODS: A total of 197 samples from 60 immunocompromised patients, who were bone marrow transplantation recipients or had hematological malignancies, were tested using B-rt-PCR, COBAS Amplicor CMV Monitor test (Amplicor CMV test), and conventional CMV PCR. The correlation results among these 3 assays were calculated. RESULTS: In these 197 samples, the CMV viral load determined by B-rt-PCR for positive specimens ranged from 19.8 to 4148.7 copies/10(5) peripheral blood mononuclear cells (PBMCs). When any positive result of B-rt-PCR, the Amplicor CMV test, or conventional PCR was considered as "CMV positive" for the 56 specimens tested by all three methods, we found that the positive and negative predictive values, respectively, were 100% and 98.6% for B-rt-PCR, 100% and 46.2% for the Amplicor CMV test, and 100% and 89.4% for conventional PCR. These three methods had good specificity (all 100%). However, the sensitivity rate of B-rt-PCR (96.3%) was higher compared to the Amplicor CMV test (46.2%) and conventional PCR (89.4%). CONCLUSIONS: The B-rt-PCR is evaluated to be a sensitive method for CMV detection in immunocompromised patients.

Rapid identification of M. abscessus and M. massiliense by MALDI-TOF mass spectrometry with a comparison to sequencing methods and antimicrobial susceptibility patterns.

AIM: Development of the matrix-assisted laser desorption ionization-TOF (MALDI-TOF) mass spectrometry method to rapidly identify Mycobacteria abscessus and Mycobacteria massiliense from M. abscessus complex in clinical microbiology laboratories. MATERIALS & METHODS: Of 128 M. abscessus complex clinical isolates, sequence analysis identified 64 as M. massiliense and 64 as M. abscessus. MALDI-TOF mass spectrometry with clustering analysis created a model to differentiate these two species. Multilocus sequence typing was used to confirm our model. RESULTS: Using a model containing five signals, 100% species recognition was achieved for 50 strains of each species. The sequence type (ST) cluster of M. abscessus was distinct from the cluster of M. massiliense. ST1 was prevalent (59%) among M. abscessus isolates, and ST117 was common (41%) among M. massiliense isolates. CONCLUSION: Molecular methods are more costly and time-consuming and may not be available in the clinical microbial laboratory. Subsequently, MALDI-TOF analysis could be a rapid identification method in the future.

Molecular epidemiology of Echovirus 30 in Taiwan, 1988-2008.

To investigate the molecular epidemiology of Taiwanese Echovirus 30 (E-30) strains, we analyzed the 876 bp sequence of the VP1 gene from 32 Taiwanese strains isolated in 1988-2008, 498 reference sequences, and one Echovirus 21 strain as the out-group. Phylogenetic analysis detected six E-30 genotypes (designated GI-GVI) that had circulated globally during the past five decades. The genotypes varied widely in geographic distribution and circulation half-life. The GI, GII, and GV were ancient genotypes in which the first strains emerged in the 1950s. The GIII was a reemerging genotype, in which strains had first appeared in Colombia in 1995 before reemerging in the New Independent States (NIS) in 2003. The GIV, an emerging genotype that recently appeared in Asia in 2003, was closely related to the ancient genotypes. The GVI was the circulating genotype, which included eight clusters (A-H) that had circulated since 1967. No GVI-A, C, D, or E strains have been identified during the past 10 years. The GVI-B first appeared in China in 1984 and later in Russia and Asia in the 2000s. The GVI-F, G, and H strains, which comprised the prevalent clusters, had been dominant in Asia Pacific area, globally, and Europe, respectively. Taiwanese strains were classified into GVI-D (1988-1989), GVI-F (1993-2004), and GVI-G (1993-2008). The quiescence period of E-30 is longer in Taiwan (5-8 years) than in other countries (3-5 years).

Coxsackievirus B4 in southern Taiwan: molecular epidemiology.

BACKGROUND: Enterovirus outbreaks caused by Coxsackievirus B4 (CB4) in Taiwan in 2004 and 2008. OBJECTIVE: To retrospectively analyze the molecular epidemiology and pathogenicity of CB4 in Taiwan. STUDY DESIGN: This study analyzed twenty-three CB4 strains isolated in Taiwan during 1993-2004. Sequence variations data were obtained using 420 bp of VP4/VP2 region and 331 bp of 3' VP1 region. Phylogenetic dendrograms were constructed with other CB4 sequences in Genebank. The clinical manifestations of CB4 infection were examined by retrospectively reviewing medical records of infected patients. RESULTS: Three CB4 genotypes were identified: genotypes II, IVb and VIII. Genotype VIII, a new and geographically distinct cluster, has been isolated in South Korea, China and Taiwan. This genotype was isolated in twelve of twenty-three CB4 patients treated in Taiwan during 1997-2004. Eight of twenty-three strains belonging to genotype II, now the major genotype worldwide, were first identified in Taiwan in 2000. Three isolates (identified 1993-1994) analyzed in this study belonged to genotype IVb. In this retrospective follow-up study of sixteen patients with CB4 infection, the median patient age at the time of infection diagnosis was 4-year-old (range, 18 days to 10-year-old), and male-female ratio was 1:1. None of the sixteen patients suffered IDDM or myocarditis after their B4 infection episodes; four had Attention Deficit Hyperactivity Disorder (ADHD) and/or tic disorders (TDs) at follow-up. CONCLUSIONS: Genotypes II and VIII of CB4 have co-circulated in Taiwan since 2000. Controlled studies are needed to evaluate a possible association between ADHD and TDs with CB4 infection.

Evolution of EV71 genogroup in Taiwan from 1998 to 2005: an emerging of subgenogroup C4 of EV71.

In Taiwan, enterovirus 71 (EV71) has played an important role in severe enterovirus-related cases every year since the devastating outbreak in 1998. Three genogroups A, B, C occur worldwide; with the B and C genogroups being subdivided into B1-B4 and C1-C4 subgenogroups respectively. To understand the mutation of the EV71 genogroup in Taiwan before and after 1998, a total of 54 worldwide strains were studied including 41 Taiwanese strains obtained in 1986 and 1998-2004. A fragment of 207 bp of the VP4 region was amplified and sequenced. Genetic analysis was performed using MEGA software (version 3.0) for the nucleotide sequence alignment and phylogenetic analysis. In Taiwan, the subgenogroup B1 was predominant before 1998 while subgenogroup C2 was the major etiologic group in 1998 outbreak. A minor etiologic group outbreak in 1998, subgenogroup B4, became predominant during the period from 1999 to 2003. In this study, subgenogroup C4 emerged and became predominant in 2004 in Taiwan. The nucleotide differences between B1 and C2, C2 and B4, B4 and C4 were 20%-26%, 19%-27%, 18%-22%, respectively. Nucleotide sequence alignment revealed 67 substitutions. Most of the substitutions (62/67) were silent mutations. This is the first report about the emergence of EV71 subgenogroup C4 in Taiwan.