A Non-Invasive Method for Monitoring Osteogenesis and Osseointegration Using Near-Infrared Fluorescent Imaging: A Model of Maxilla Implantation in Rats.

Currently, computed tomography and conventional X-ray radiography usually generate a micro-artifact around metal implants. This metal artifact frequently causes false positive or negative diagnoses of bone maturation or pathological peri-implantitis around implants. In an attempt to repair the artifacts, a highly specific nanoprobe, an osteogenic biomarker, and nano-Au-Pamidronate were designed to monitor the osteogenesis. In total, 12 Sprague Dawley rats were included in the study and could be chategorized in 3 groups: 4 rats in the X-ray and CT group, 4 rats in the NIRF group, and 4 rats in the sham group. A titanium alloy screw was implanted in the anterior hard palate. The X-ray, CT, and NIRF images were taken 28 days after implantation. The X-ray showed that the tissue surrounded the implant tightly; however, a gap of metal artifacts was noted around the interface between dental implants and palatal bone. Compared to the CT image, a fluorescence image was noted around the implant site in the NIRF group. Furthermore, the histological implant-bone tissue also exhibited a significant NIRF signal. In conclusion, this novel NIRF molecular imaging system precisely identifies the image loss caused by metal artifacts and can be applied to monitoring bone maturation around orthopedic implants. In addition, by observing the new bone formation, a new principle and timetable for an implant osseointegrated with bone can be established and a new type of implant fixture or surface treatment can be evaluated using this system.

Two new, near-infrared, fluorescent probes as potential tools for imaging bone repair.

A precise imaging technique to evaluate osteogenesis, osteodifferentiation, and osseointegration following peri-implant surgery is in high clinical demand. Herein, we report the generation of two new, near-infrared (NIR) fluorescent probes for use in the molecular imaging of bone repair. The first probe aims to monitor the in vitro differentiation of human mesenchymal stem cells (MSCs) into osteoblasts. A NIR fluorochrome was conjugated to a cyclic peptide that binds to integrin α5ß1, a factor that promotes osteogenesis in MSCs and therefore functioned as an osteoblast-specific marker. The second probe aims to monitor osteogenesis, and was generated by conjugating the drug pamidronate to a NIR fluorescent gold nanocluster. Pamidronate specifically binds to hydroxyapatite (HA), a mineral present in bone that is produced by osteoblasts, and therefore provides a functional marker for new bone formation. Our results show that both probes bind to their specific targets in vitro-differentiated osteoblasts, and not to undifferentiated MSCs, and emit NIR fluorescence for functional detection. This in vitro work demonstrates the ability of these probes to bind to active osteoblasts and their mineral deposits and highlight their potential utility as clinical tools for the imaging of the osseointegration process at the molecular level.

Molecular characterization and phylogenetic analysis of dengue viruses imported into Taiwan during 2011-2016.

A total of 1,596 laboratory-confirmed imported dengue cases were identified in Taiwan during 2011-2016. Most of the imported cases arrived from Southeast Asia as well as the Indian subcontinent, the Pacific region, Latin America, Australia and Africa. Phylogenetic analyses of the complete envelope protein gene sequences from 784 imported dengue virus (DENV) isolates were conducted, and the results suggest that the DENV-1 genotype I and DENV-2 Cosmopolitan genotype comprise the predominant serotype/genotype of DENV strains circulating in Southeast Asia. The DENV-1 genotype III, DENV-3 genotype III and DENV-4 genotype I and II strains were found to be newly emerging in several Southeast Asian countries. Our results also showed that geographical restrictions of DENV-1 genotype I, DENV-1 genotype III and DENV-2 Cosmopolitan genotype are becoming blurred, indicating the extensive introductions and continuous expansions of DENV strains between nations in Southeast Asia. In this study, we present the geographic distribution and dynamic transmission of DENV strains circulating in Southeast Asian countries. In addition, we demonstrated local dengue epidemics caused by several imported DENV strains in Taiwan during 2011-2016.

Correction: The Critical Role of Early Dengue Surveillance and Limitations of Clinical Reporting - Implications for Non-Endemic Countries.

[This corrects the article DOI: 10.1371/journal.pone.0160230.].

Imported Chikungunya Virus Strains, Taiwan, 2006-2014.

We identified 78 imported chikungunya cases in Taiwan during 2006-2014. Sixty-six (84.6%) cases were initially suspected to be dengue, which indicates the necessity for laboratory diagnostics in differentiation between dengue and chikungunya. Results also emphasize the need for active surveillance of febrile illness at points of entry.

The Critical Role of Early Dengue Surveillance and Limitations of Clinical Reporting - Implications for Non-Endemic Countries.

The increasing dengue burden and epidemic severity worldwide have highlighted the need to improve surveillance. In non-endemic areas such as Taiwan, where outbreaks start mostly with imported cases from Southeast Asia, a closer examination of surveillance dynamics to detect cases early is necessary. To evaluate problems with dengue surveillance and investigate the involvement of different factors at various epidemic stages, we investigated 632 laboratory-confirmed indigenous dengue cases in Kaohsiung City, Taiwan during 2009-2010. The estimated sensitivity of clinical surveillance was 82.4% (521/632). Initially, the modified serological surveillance (targeting only the contacts of laboratory-confirmed dengue cases) identified clinically unrecognized afebrile cases in younger patients who visited private clinics and accounted for 30.4% (35/115) of the early-stage cases. Multivariate regression indicated that hospital/medical center visits [Adjusted Odds Ratio (aOR): 11.6, 95% confidence interval (CI): 6.3-21.4], middle epidemic stage [aOR: 2.4 (1.2-4.7)], fever [aOR: 2.3 (2.3-12.9)], and musculo-articular pain [aOR: 1.9 (1.05-3.3)] were significantly associated with clinical reporting. However, cases with pruritus/rash [aOR: 0.47 (0.26-0.83)] and diarrhea [aOR: 0.47 (0.26-0.85)] were underreported. In conclusion, multiple factors contributed to dengue surveillance problems. To prevent a large-scale epidemic and minimize severe dengue cases, there is a need for integrated surveillance incorporating entomological, clinical, serological, and virological surveillance systems to detect early cases, followed by immediate prevention and control measures and continuous evaluation to ensure effectiveness. This effort will be particularly important for an arbovirus, such as Zika virus, with a high asymptomatic infection ratio. For dengue- non-endemic countries, we recommend serological surveillance be implemented in areas with high Aedes mosquito indices or many breeding sites. Syndromic surveillance, spatial analysis and monitoring changes in epidemiological characteristics using a geographical information system, as well as epidemic prediction models involving epidemiological, meteorological and environmental variables will be helpful for early risk communication to increase awareness.

Laboratory-Based Surveillance and Molecular Characterization of Dengue Viruses in Taiwan, 2014.

We present the results of a laboratory-based surveillance of dengue in Taiwan in 2014. A total of 240 imported dengue cases were identified. The patients had arrived from 16 countries, and Malaysia, Indonesia, the Philippines, and China were the most frequent importing countries. Phylogenetic analyses showed that genotype I of dengue virus type 1 (DENV-1) and the cosmopolitan genotype of DENV-2 were the predominant DENV strains circulating in southeast Asia. The 2014 dengue epidemic was the largest ever to occur in Taiwan since World War II, and there were 15,492 laboratory-confirmed indigenous dengue cases. Phylogenetic analysis showed that the explosive dengue epidemic in southern Taiwan was caused by a DENV-1 strain of genotype I imported from Indonesia. There were several possible causes of this outbreak, including delayed notification of the outbreak, limited staff and resources for control measures, abnormal weather conditions, and a serious gas pipeline explosion in the dengue hot spot areas in Kaohsiung City. However, the results of this surveillance indicated that both active and passive surveillance systems should be strengthened so appropriate public health measures can be taken promptly to prevent large-scale dengue outbreaks.

Laboratory diagnostics of dengue fever: an emphasis on the role of commercial dengue virus nonstructural protein 1 antigen rapid test.

BACKGROUND/PURPOSE(S): In 2008, the Dengue NS1 Ag STRIP (Bio-Rad Laboratories, Marnes-la-Coquette, France) was introduced to routine dengue diagnostics in Taiwan, in addition to real-time reverse-transcription polymerase chain reaction (PCR), virus isolation, and capture immunoglobulin (Ig)M/IgG enzyme-linked immunosorbent assay (ELISA). This study aimed to evaluate the benefit of this assay and factors influencing the results of these diagnostic tests. METHODS: Retrospectively, the authors enrolled laboratory-confirmed adult dengue patients from July 2008 to January 2012 in a tertiary hospital. The sensitivities of each test alone and in combination were analyzed by the duration of illness (early stage: day 0-day 3 and late stage: day 4-day 8). The factors influencing sensitivity of the Dengue NS1 Ag STRIP were examined. RESULTS: There were 392 patients enrolled. The overall sensitivity of the Dengue NS1 Ag STRIP was 68.37% and PCR was 71.94%. With the assistance of the Dengue NS1 Ag STRIP, a diagnosis was made in 10.97% of patients without the need for second convalescent samples, and 4.34% more cases were detected. Independent factors for reduced Dengue NS1 Ag STRIP sensitivity were dengue virus (DENV) IgG seropositivity and a sample taken after the fifth day of illness. At the early stage, the PCR and the Dengue NS1 Ag STRIP combination had the highest sensitivity rate than other combinations. At the late stage, a combination of the Dengue NS1 Ag STRIP and capture IgM/IgG ELISA had better sensitivity rates. PCR and capture IgM/IgG ELISA in combination had sensitivity above 90% through the course of illness. CONCLUSION: Dengue NS1 Ag STRIP is a useful tool for early dengue diagnosis. Its use can increase the diagnostic sensitivity and decrease the need of convalescent samples. Seeking treatment late (days postonset > 4) and DENV IgG seropositivity independently decrease the sensitivity of the Dengue NS1 Ag STRIP.

Molecular characterization and phylogenetic analysis of dengue viruses imported into Taiwan during 2008-2010.

We present our surveillance results on imported dengue cases in Taiwan during 2008-2010. A total of 734 imported dengue patients were identified. The travelers were arriving from 18 countries, including Southeast Asia, the Indian subcontinent, South Pacific islands, and Latin America. Gene sequences from 358 dengue virus (DENV) isolates were used to perform phylogenetic analyses, thus, providing an updated view of the geographic distribution and dynamic transmission of DENV strains circulating in these countries. Our results suggest that the DENV-1 genotype I and DENV-2 Cosmopolitan genotype comprise the predominant DENV strains circulating in Southeast Asian countries. The DENV-3 Genotype III strain was found to be newly emerging in several Southeast Asian countries, however, the Asian genotype 2 and the Asian/American genotype of DENV-2 strains appeared to be less prevalent in Southeast Asia. Furthermore, imported dengue viruses are representative of the overall patterns of serotype/genotype frequencies of dengue outbreaks that occurred in Taiwan.

Use of a single-tube nested real-time PCR assay to facilitate the early diagnosis of acute Q fever.

We have developed a single-tube nested real-time PCR (STN-RT PCR) assay using the repetitive, transposon-like element IS1111 as the DNA target to facilitate early diagnosis of acute Q fever. The use of our proposed diagnostic procedures, including IgM detection by serology and the STN-RT PCR assay, significantly increased the diagnostic sensitivity for Q fever to 78%, compared to 29% when serology alone was used for subjects providing mainly acute-phase blood samples.

Serologic assessment of the risk of developing chronic Q fever in cohorts of acutely infected individuals.

OBJECTIVE: The aim of this study was to assess the clinical significance of serological profiles suggestive of chronic Q fever after acute infection. METHODS: A prospective follow-up study consisting of two separate cohorts was conducted to monitor the serological evolution of Q fever. The first cohort comprised subjects with acute Q fever diagnosed in 2004-2007 and the second enrolled subjects whose infection occurred in 2009. The indirect immunofluorescence assay was used for serological monitoring, with serum PCR testing added for subjects whose serological profiles revealed high titers of anti-phase I IgG≥800, titers suggestive of chronic Q fever. RESULTS: In the first cohort of 92 persons, seventeen (18%) subjects had serological profiles suggestive of chronic Q fever (titers of anti-phase I IgG: 1280-5120, median: 1280) after a median follow-up period of 606.5 days. After a further follow-up (median period: 592 days) exclusively for those seventeen subjects, serological resolution with fourfold decrease of titers of anti-phase I IgG was noted in five of them. In the second cohort, only one (4%) of the twenty-eight subjects had high levels of anti-phase I IgG 180 days after acute infection. All the eighteen subjects with high levels of anti-phase I IgG were asymptomatic and had negative serum PCR testing. The different prevalence of subjects with high titers of anti-phase I IgG in the two cohorts was associated with duration of follow-up period (P < .01). CONCLUSIONS: Subjects with high titers of anti-phase I IgG≥800 was not uncommon and might not be detected until more than six months after acute Q fever infection. Asymptomatic subjects with high levels of anti-phase I IgG alone should not be treated as chronic Q fever and might not need continued serological monitoring in the absence of predisposing factors to chronic Q fever.

Characteristic of dengue disease in Taiwan: 2002-2007.

Taiwan's dengue outbreaks have a unique type of transmission: starting by import from abroad in early summer, spreading out locally, and ending in the winter. This pattern repeats every year. Most of the dengue patients are adults, with dengue fever peaking in the 50-54 year age range, and dengue hemorrhagic fever in the 60-64 year age range. Two patterns of dengue infection were found: DENV-2 in 2002 with 74% of secondary infection in contrast to non-DENV-2 (DENV-1 or DENV-3) in 2004-2007 with approximately 70% of primary infection. Secondary dengue virus infection increases disease morbidity, but not mortality in adults. The active serological surveillance shows two-thirds of the dengue-infected adults are symptomatic post infection. The Taiwanese experience of adult dengue should be valuable for countries or areas where, although dengue is not endemic, the Aedes aegypti vector exists and dengue virus can be introduced by travelers.

Molecular characterization of dengue viruses imported into Taiwan during 2003-2007: geographic distribution and genotype shift.

We presented our surveillance results on imported dengue cases in Taiwan during 2003-2007. A total of 542 imported dengue patients were identified. The travelers were infected in 17 countries in Southeast Asia, the Indian subcontinent, East African islands, South Pacific islands, and Central America. Most of these imported cases were infected in Southeast Asian countries. Phylogenetic analyses were conducted to examine 288 imported dengue virus (DENV) strains introduced from 13 countries. The results provide an updated view on the geographic distribution and dynamic transmission of epidemic DENV stains circulated in Southeast Asian countries. Although the geographic distributions of genotypes of DENV-3 isolated from Southeast Asian countries remain unchanged, the introductions and local expansions of epidemic DENV-1, DENV-2, and DENV-4 strains into new areas in Asia were observed. These findings highlight the importance to strengthen laboratory-based dengue surveillance for better understanding of transmission dynamics and molecular evolution of DENVs.

Application of the dengue virus NS1 antigen rapid test for on-site detection of imported dengue cases at airports.

We used the dengue virus NS1 antigen (Ag) rapid test for on-site detection of imported dengue cases at airports. Among 22 positive cases of dengue identified from 850 patients with a fever suspected to have dengue, 17 were NS1 Ag test positive. These findings demonstrate the usefulness of the NS1 Ag rapid test in screening imported dengue cases at airports.

Effect of serotypes on clinical manifestations of dengue fever in adults.

BACKGROUND AND PURPOSE: Dengue fever (DF) is a major public health issue. However, it is unclear whether different dengue virus serotypes (DENV) are associated with different clinical manifestations and outcomes. This study investigated the association between viral serotype and clinical manifestations of DF. METHODS: Adult patients with DENV-2 and DENV-3 who were treated at Kaohsiung Medical University Hospital and Kaohsiung Municipal Hsiao-Kang Hospital, Kaohsiung, Taiwan, from January 1998 to September 2007 were enrolled. The patients' demographic data, underlying diseases, clinical manifestations, laboratory data, and disease outcomes were retrospectively analyzed. RESULTS: 294 patients had DENV-2 and 91 had DENV-3. The median age was 50 years, and 45.7% of patients were men. Patients with DENV-3 were more likely to have a malignancy (p = 0.011), myalgia (p = 0.03), skin rash (p < 0.001), ascites (p = 0.04), and fever (p = 0.003) than patients with DENV-2. Patients with DENV-3 had their lowest levels of white blood cells and platelets, and peak plasma activated partial thromboplastin time (aPTT) 1 day later than patients with DENV-2. DENV-2 infection was associated with a higher monocyte count and more prolonged aPTT early in the clinical course. Infection by DENV-2 more commonly occurred as a secondary infection, while infection by DENV-3 was more common as a primary infection (p < 0.001). There were no differences between the groups in organ involvement, disease severity, duration of hospital stay, and medical expenditure. CONCLUSION: The symptoms, signs, and laboratory findings appear to be different for patients infected with DENV-2 and DENV-3, but there is no difference in outcomes.

Laboratory-based dengue surveillance in Taiwan, 2005: a molecular epidemiologic study.

We present the results of laboratory-based dengue surveillance in Taiwan for 2005. A phylogenetic study showed that multiple dengue epidemics were caused by three different imported dengue virus (DENV) strains. A strain of DENV-3 (genotype I) imported from the Philippines first appeared in the southern part of Kaohsiung City and later spread to Kaohsiung County from August to December, which resulted in 77 cases of dengue. Another strain of DENV-3 (genotype II) imported from Vietnam first appeared in the central part of Kaohsiung City and later spread to Kaohsiung County from September to December, which resulted in 35 cases of dengue. A strain of DENV-2 (American/Asian genotype) imported from Vietnam first appeared in Tainan City and later spread to Kaohsiung City/County from October to December, which resulted in 60 cases of dengue. This study provides molecular epidemiologic evidence that most dengue in Taiwan is caused by imported strains of the virus.

Study of IgG subclass profiles of anti-HBs in populations with different HBV infection status.

To study IgG-specific subclasses of hepatitis B virus (HBV) surface antigen (anti-HBs), in different populations in Taiwan, a comparison was made between 104 chronic carriers (60 male and 44 female) and 439 recovered individuals (247 male and 192 female). Biochemical analyses of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were also performed. Among the 104 chronic carriers, 21 patients had abnormal ALT and AST levels (> 25 IU/ml). When comparing the patients with abnormal ALT and AST levels to chronic carriers with normal ALT and AST levels, no statistical difference was observed for anti-HBs levels (p > 0.05). The IgG subclass pattern of the relative anti-HBs IgG subclass titers was IgG1 > IgG3 = IgG4 in both chronic carriers and recovered individuals (p < 0.05). IgG1 is the predominant anti-HBs antibody after HBV infection, in either chronic carriers or in HBV-cured individuals. This finding is partly inconsistent with data reported from other group who suggested in individuals naturally infected, the anti-HBs IgG consists mainly of IgG3 and IgG1. In contrast to that of our previous studies of anti-HBe and anti-HBc, the mean OD values of anti-HBs total IgG, and all IgG subclasses except for IgG2, of either males or females, were significantly higher in recovered individuals than in chronic carriers, while the mean OD values of anti-HBe and anti-HBc were significantly higher in chronic carriers than in recovered individuals (P < 0.05). The IgG subclass profile of anti-HBs in chronic carriers was not changed with liver inflammation and was independent of sex and age, except in individuals with abnormal ALT and AST for whom anti-HBs IgG1 was not significantly higher than IgG3 (p > 0.05), in spite of that whose mean O.D. value is higher.

The study of IgG subclass profiles of anti-hbc in populations with different status of HBV infection.

To study IgG subclasses for the hepatitis B virus (HBV) core antigen (anti-HBc) in different populations, a comparison was made between 104 chronic carriers (60 male and 44 female) and 434 recovered individuals (247 male and 192 female). Biochemistry analyses of AST (aspartate aminotransferase) and ALT (alanine aminotransferase) were also performed. Among the 104 chronic carriers, 21 patients were found to be ALT and AST abnormal (> 25 IU/ml). After comparing these ALT and AST abnormal patients with other ALT and AST normal chronic carriers, no statistical difference was observed in the OD values of the anti-HBe (p > 0.05). The ELISA results showed the anti-HBc IgG subclass pattern was IgG1 > IgG3 > IgG4 in chronic carriers and IgG3 > IgG1 > IgG4 in recovered individuals (p < 0.05). This result suggests the IgG1/IgG3 ratio may be related with HBV status. However, in spite of the different anti-HBc IgG1/IgG3 patterns demonstrated in different populations, both anti-HBc IgG1 and IgG3 concentrations were significantly higher in chronic carriers (p < 0.05). Therefore, both the anti-HBc IgG1/IgG3 ratio and their amounts differed. They may play a significant role in chronic carriers and recovered individuals. The anti-HBc IgG subclass profiles of chronic carriers were not changed regardless of liver inflammation, and were independent of sex and age.