Clinical Implementation of ß-Tubulin Gene-Based Aspergillus Polymerase Chain Reaction for Enhanced Aspergillus Diagnosis in Patients with Hematologic Diseases: A Prospective Observational Study.

The ß-tubulin (benA) gene is a promising target for the identification of Aspergillus species. Assessment of the clinical implementation and performance of benA gene-based Aspergillus polymerase chain reaction (PCR) remains warranted. In this study, we assessed the analytical performance of the BenA probe PCR in comparison with the Aspergenius kit. We prospectively collected bronchoalveolar lavage (BAL) fluid via diagnostic bronchoscopy from adult patients with hematologic diseases. BenA gene-based multiplex real-time PCR and sequential melting temperature analysis were performed to detect the azole resistance of Aspergillus fumigatus. In total, 76 BAL fluids in 75 patients suspicious of invasive pulmonary aspergillosis (IPA) were collected. Before the application of PCR, the prevalence of proven and probable IPA was 32.9%. However, after implementing the benA gene-based PCR, 15.8% (12 out of 76) of potential IPA cases were reclassified as probable IPA. The analytical performance of the BenA probe PCR in BAL samples was comparable to that of the Aspergenius kit. The diagnostic performance was as follows: sensitivity, 52.0%; specificity, 64.7%; positive predictive value, 41.9%; negative predictive value, 73.3%; positive likelihood ratio, 1.473; and negative likelihood ratio, 0.741. Moreover, benA gene-based Aspergillus PCR discriminated all major sections of Aspergillus, including cryptic species such as Aspergillus tubingensis. Sequential melting temperature analysis successfully detected 2 isolates (15.4%) of A. fumigatus carrying resistant mutations. BenA gene-based Aspergillus PCR with melting temperature analysis enhances diagnostic accuracy and detects not only cryptic species but also resistant mutations of A. fumigatus. It shows promise for clinical applications in the diagnosis of IPA.

Development of multiplex real-time PCR for rapid identification and quantitative analysis of Aspergillus species.

The identification of Aspergillus species and azole resistance is highly important for the treatment of invasive aspergillosis (IA), which requires improvements in current fungal diagnostic methods. We aimed to develop multiplex real-time PCR to identify major Aspergillus section and azole resistance. BenA and cyp51A genes were used to design primers, probes, and control DNA for multiplex PCR. Qualitative and quantitative analysis was conducted for 71 Aspergillus and 47 non-Aspergillus isolates. Further, the limit of detection (LOD) and limit of quantitation (LOQ) from hyphae or conidia were determined according to the culture time. Newly developed real-time PCR showed 100% specificity to each Aspergillus section (Fumigati, Nigri, Flavi, and Terrei), without cross-reaction between different sections. In quantitative analysis of sensitivity measurements, LOD and LOQ were 40 fg and 400 fg, respectively. Melting temperature analysis of the cyp51A promoter to identify azole resistance showed temperatures of 83.0 ± 0.3°C and 85.6 ± 0.6°C for susceptible A. fumigatus and resistant isolates with TR34 mutation, respectively. The minimum culture time and fungal colony size required for successful detection were 24 h and 0.4 cm in diameter, respectively. The developed multiplex real-time PCR can identify common Aspergillus sections quantitatively and detect presence of the TR34 mutation. Further, this method shows high sensitivity and specificity, allowing successful detection of early-stage fungal colonies within a day of incubation. These results can provide a template for rapid and accurate diagnosis of IA.

A Simple Method for the Design and Development of Flavivirus NS1 Recombinant Proteins Using an In Silico Approach.

Even in countries that are currently not facing a flavivirus epidemic, the spread of mosquito-borne flaviviruses presents an increasing public threat, owing to climate change, international travel, and other factors. Many of these countries lack the resources (viral strains, clinical specimens, etc.) needed for the research that could help cope with the threat imposed by flaviviruses, and therefore, an alternative approach is needed. Using an in silico approach to global databases, we aimed to design and develop flavivirus NS1 recombinant proteins with due consideration towards antigenic variation. NS1 genes analyzed in this study included a total of 6,823 sequences, from Dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), Zika virus (ZIKV), and Yellow fever virus (YKV). We extracted and analyzed 316 DENV NS1 sequence types (STs), 59 JEV STs, 75 WNV STs, 30 YFV STs, and 43 ZIKV STs using a simple algorithm based on phylogenetic analysis. STs were reclassified according to the variation of the major epitope by MHC II binding. 78 DENV epitope type (EpT), 29 JEV EpTs, 29 WNV EpTs, 12 YFV EpTs, and 5 ZIKV EpTs were extracted according to their major epitopes. Also, frequency results showed that there were dominant EpTs in all flavivirus. Fifteen STs were selected and purified for the expression of recombinant antigen in Escherichia coli by sodium dodecyl sulfate extraction. Our study details a novel in silico approach for the development of flavivirus diagnostics, including a simple way to screen the important peptide regions.

Epidemiology and Antifungal Susceptibility Profile of Aspergillus Species: Comparison between Environmental and Clinical Isolates from Patients with Hematologic Malignancies.

Global data on the epidemiology and susceptibility of Aspergillus are crucial in the management of invasive aspergillosis. Here, we aimed to determine the characteristics of clinical and environmental Aspergillus isolates, focusing mainly on hematologic malignancy patients. We prospectively collected all consecutive cases and clinical isolates of culture-positive proven/probable invasive aspergillosis patients from January 2016 to April 2018 and sampled the air inside and outside the hospital. Cryptic species-level identification of Aspergillus, antifungal susceptibilities, and cyp51 gene sequencing were performed, and clinical data were analyzed. This study was conducted as part of the Catholic Hematology Hospital Fungi Epidemiology (CAFÉ) study. A total of 207 proven/probable invasive aspergillosis and 102 clinical and 129 environmental Aspergillus isolates were included in this analysis. The incidence of proven/probable invasive aspergillosis was 1.3 cases/1,000 patient-days during the study period. Cryptic Aspergillus species accounted for 33.8%, with no differences in proportions between the clinical and environmental isolates. Section Nigri presented a high proportion (70.5%) of cryptic species, mainly from A. tubingensis and A. awamori: the former being dominant in environmental samples, and the latter being more common in clinical isolates (P < 0.001). Of 91 A. fumigatus isolates, azole-resistant A. fumigatus was found in 5.3% of all A. fumigatus isolates. Three isolates presented the TR34/L98H mutation of the cyp51A gene. Patients with invasive aspergillosis caused by azole-resistant A. fumigatus showed 100% all-cause mortality at 100 days. This study demonstrates the significant portion of cryptic Aspergillus species and clinical implications of azole resistance and underscores the comparison between clinical and environmental isolates.

Profiles of Environmental Mold: Indoor and Outdoor Air Sampling in a Hematology Hospital in Seoul, South Korea.

Inhalation of fungal spores can cause various spectrums of fungal diseases in immunocompromised hosts. The aim of this study was to evaluate the concentrations and profiles of fungal species in air collected at different locations in hematology wards and outside of the hospital in Seoul St. Mary's Hospital over the course of a year. Air sampling was performed at four locations-outside the hospital (O), in the general ward (GW), in the lounge in the cleanroom (CRL), and in the patients' room in the cleanroom (CRR)-by using Andersen single-stage air sampler at every two weeks between May 2017 and May 2018. The results showed higher mean fungal density in summer, and the concentrations of fungi decreased as follows: O (954.8 colony-forming units, CFU/m³) > GW (4.2 CFU/m³) > CRL (0.7 CFU/m³) > CRR (0 CFU/m³). Aspergillus was most prevalent both inside (47%) and outside (62%) of the hospital. However, the outdoor fungal profile was more diverse than the indoor profile. Within the hospital, Penicillium was the second most dominant species. In conclusion, the outdoor fungal profile is diverse even in Seoul, a highly urbanized area in Korea. The distribution of indoor air fungi is significantly different from outdoor due to air quality systems. Heating, ventilation, and air conditioning (HVAC), as well as high-efficiency particulate air (HEPA)-filtered systems should be established to effectively reduce levels of indoor fungi.

The effects of forward and backward walking according to treadmill inclination in children with cerebral palsy.

[Purpose] This study investigated the effects of forward and backward walking using different treadmill incline positions on lower muscle activity in children with cerebral palsy, to provide baseline data for gait training intensity. [Subjects and Methods] Nineteen subjects with cerebral palsy walked forward and backward at a self-selected pace on a treadmill with inclines of 0%, 5%, 10%, and 15%. Activation of the rectus femoris, biceps femoris, tibialisanterior, and lateral gastrocnemius was measured using surface electromyography during the stance phase. [Results] As treadmill incline increased during forward walking, muscle activation of the paralyzed lower limbs did not significantly change. However, as treadmill incline increased during backward walking, rectus femoris activation significantly increased and a significant difference was found between treadmill inclines of 0% and 10%. A comparison of backward and forward walking showed a significant difference in rectus femoris activation at treadmill inclines of 0%, 5%, and 10%. Activation of the tibialis anterior was only significantly higher for backward walking at the 10% gradient. [Conclusion] Backward walking may strengthen the rectus femoris and tibialis anterior in walking training for cerebral palsy. Gradient adjustment of the treadmill can be used to select the intensity of walking training.

Effects of balance training by knee joint motions on muscle activity in adult men with functional ankle instability.

[Purpose] This study examined the effects of balance training by applying knee joint movements on muscle activity in male adults with functional ankle instability. [Subjects and Methods] 28 adults with functional ankle instability, divided randomly into an experimental group, which performed balance training by applying knee joint movements for 20 minutes and ankle joint exercises for 10 minutes, and a control group, which performed ankle joint exercise for 30 minutes. Exercises were completed three times a week for 8 weeks. Electromyographic values of the tibialis anterior, peroneus longus, peroneus brevis, and the lateral gastrocnemius muscles were obtained to compare and analyze muscle activity before and after the experiments in each group. [Results] The experimental group had significant increases in muscle activity in the tibialis anterior, peroneus longus, and lateral gastrocnemius muscles, while muscle activity in the peroneus brevis increased without significance. The control group had significant increases in muscle activity in the tibialis anterior and peroneus longus, while muscle activity in the peroneus brevis and lateral gastrocnemius muscles increased without significance. [Conclusion] In conclusion, balance training by applying knee joint movements can be recommended as a treatment method for patients with functional ankle instability.

Comparison of plantar pressure between flat and normal feet when crossing an obstacle at different heights.

OBJECTIVE: The purpose of this study is to compare the plantar foot pressure and the center of pressure (COP) pathway of normal and flat feet while crossing an obstacle at different heights. METHODS: Nineteen subjects (10 normal feet, 9 flat feet) participated in this study. The plantar foot surface was divided into the following seven regions for pressure measurement: two toe regions, three forefoot regions, one midfoot region, and one heel region. A one-way ANOVA with repeated measurements was used to compare the plantar foot pressure of normal and flat feet according to the obstacle height. RESULTS: The trend analysis showed a quadratic trend during level walking for the normal foot group, but a linear trend appeared as the obstacle height increased. In the flat foot group, the trend analysis showed a linear trend regardless of the obstacle height. In the 2nd-3rd metatarsal head region, the plantar pressure of the flat foot group increased more than the normal foot group as the obstacle height increased; however, in the 4th-5th metatarsal head region, the plantar pressure in flat feet was lower than in normal feet. In the heel region, the plantar pressure in both groups generally increased as the obstacle height increased, but the plantar pressure in the flat foot group was lower than in the normal foot group. CONCLUSION: We believe that, due to a loss of longitudinal arch, the COP path and plantar pressure of flat feet may be different from normal feet when crossing obstacles of different heights.

The effect of exercise using a sliding rehabilitation machine on the gait function of children with cerebral palsy.

[Purpose] The purpose of the present study was to examine the effect of strength training using a sliding rehabilitation machine (SRM) on the gait function of cerebral palsy children. [Subjects and Methods] Thirteen children aged 6-18 years participated in the SRM training for 8 weeks (30 min/day, 2 times/week). The SRM is designed for the performance of a closed-kinetic chain exercise in which a tilt table is moved up and down using wheels on the table. Participants began in a position of flexion of the 3 lower joints (hips, knees, and ankles) on the SRM. In each exercise session, they extended and flexed the 3 joints. The level of exercise was set by changing the inclination of the tilt table. Functional gait ability was measured with the 6-minute walk test (6MWT), 10-m walk test (10MWT), and timed up-and-go test (TUG) before and after the training. Muscle strength was also measured using an isokinetic dynamometer. [Results] Nine of the thirteen children completed the entire study. The peak torques of the knee extensor and flexor group muscles significantly improved after training with the SRM. The total distance of the 6 MWT significantly increased after training. The times of the 10 MWT and the TUG significantly improved after training. The changes in muscle tone were also investigated using the MAS (Modified Ashworth Scale) and Tardieu scale, but no significant changes were found in muscle tone between the pre- and post-test measurements. [Conclusion] The findings demonstrate the effect of the SRM intervention which resulted in improved muscle strength and functional gait.