Multiplex PCR assay for the identification of eight Anopheles species belonging to the Hyrcanus, Barbirostris and Lindesayi groups.

BACKGROUND: Genus Anopheles mosquitoes are the primary vectors of human malaria, which is a serious threat to public health worldwide. To reduce the spread of malaria and identify the malaria infection rates in mosquitoes, accurate species identification is needed. Malaria re-emerged in 1993 in the Republic of Korea (ROK), with numbers peaking in 2004 before decreasing to current levels. Eight Anopheles species (Anopheles sinensis, Anopheles pullus, Anopheles belenrae, Anopheles lesteri, Anopheles kleini, Anopheles sineroides, Anopheles koreicus, Anopheles lindesayi) are distributed throughout Korea. Members of the Anopheles Hyrcanus group currently cannot be identified morphologically. The other species of Anopheles can be identified morphologically, except when specimens are damaged in traps. The purpose of this study was to develop a rapid and accurate method for simultaneous molecular identification of the eight Anopheles species present in the ROK. METHODS: Anopheles spp. used in this study were collected near/in the demilitarized zone in ROK, where most malaria cases are reported. DNA from 165 of the Anopheles specimens was used to develop a multiplex PCR assay. The internal transcribed spacer 2 (ITS2) region of each species was sequenced and analysed for molecular identification. RESULTS: DNA from a total of 165 Anopheles specimens was identified to species using a multiplex diagnostic system. These included: 20 An. sinensis, 21 An. koreicus, 17 An. lindesayi, 25 An. kleini, 11 An. lesteri, 22 An. sineroides, 23 An. belenrae, and 26 An. pullus. Each species was clearly distinguished by electrophoresis as follows: 1,112 bp for An. sinensis; 925 bp for An. koreicus; 650 bp for An. lindesayi; 527 bp for An. kleini; 436 bp for An. lesteri; 315 bp for An. sineroides; 260 bp for An. belenrae; and, 157 bp for An. pullus. CONCLUSION: A multiplex PCR assay was developed to identify Anopheles spp. distributed in ROK. This method can be used to accurately identify Anopheles species that are difficult to identify morphologically to determine species distributions and malaria infection rates.

Seasonal Abundance of Culicoides at Yongsan US Army Garrison (USAG) and Camp Humphreys USAG, Republic of Korea, 2010-2013 and 2014-2017.

Biting midges (Culicoides: Ceratopogonidae) were collected using New Jersey light traps at Yongsan US Army Garrison (USAG;urban), Seoul Metropolitan city and Camp Humphreys USAG (rural), Pyeongtaek, Gyeonggi-do (province), Republic of Korea , from May-October 2010-2013 and 2015-2017, to determine species composition and seasonal distribution patterns in urban and rural habitats. A total of 9,958 female (53.85%) and 8,533 male (46.15%) Culicoides comprising 16 species were collected. Overall, the most commonly collected species was Culicoides arakawae (74.3%), followed by C. circumscriptus (16.2%), C. kibunensis (2.5%), C. nasuensis (2.2%), C. clavipalpis (1.4%), and C. pallidulus (1.3%), while the remaining 10 species accounted for <2.1% of all Culicoides spp. collected. The 2 predominant species collected were C. circumscriptus (47.4%) and C. arakawae (33.4%) at Yongsan, and C. arakawae (90.4%) and C. circumscriptus (3.9%) at Camp Humphreys. The seasonal abundance of these 2 species varied between years and between sites but on average peaked in August-September for C. arakawae and June-July for C. circumscriptus. Annual variations in abundance were observed for most species collected during this study. Unusually high proportions of male specimens were observed for most species at both sites which may be due to the use of the New Jersey trap.

Seasonal Abundance of Deer and Horse Flies (Diptera: Tabanidae) in the Northern Part of Gyeonggi-do, Republic of Korea.

The seasonal abundance of horse and deer flies (family Tabanidae) was analyzed using Mosquito Magnet(®) traps at 5 sites located near/in the demilitarized zone, northern Gyeonggi-do, Republic of Korea from late April to early October for 4 consecutive years (2010-2013). A total of 2,999 horse and deer flies (tabanids) belonging to 5 genera and 20 species were collected. Chrysops mlokosiewiczi (90.9%) was the most frequently collected, followed by Haematopota koryoensis (4.8%) and C. suavis (1.0%). The remaining 17 species comprised only of 3.3% of all species collected. C. mlokosiewiczi demonstrated bimodal peak populations during mid-June and early August, while H. koryoensis demonstrated a unimodal peak during mid-July. Overall numbers of tabanids collected were influenced by the previous year's winter temperatures and precipitation. Population abundance was influenced by habitat with most of tabanids collected from habitats near forested areas, followed by rice paddies, and a beef farm.

Species Diversity and Seasonal Distribution of Culicoides spp. (Diptera: Ceratopogonidae) in Jeju-do, Republic of Korea.

Biting midges belonging to the genus Culicoides (Diptera: Ceratopogonidae) were collected by Mosquito Magnet(®) and black light traps at 5 sites on Jeju-do, Republic of Korea (Korea), from May-November 2013 to determine species diversity and seasonal distribution. A total of 4,267 specimens were collected, of which 99.9% were female. The most common species was Culicoides tainanus (91.8%), followed by C. lungchiensis (7.2%) and C. punctatus (0.6%), while the remaining 4 species accounted for <0.5% of all Culicoides spp. that were collected. High numbers of C. tainanus were collected in May, followed by decreasing numbers through August, and then increasing numbers through November when surveillance was terminated. Peak numbers of C. lungchiensis were collected during September, with low numbers collected from May-August and October-November. The presence of C. lungchiensis in Korea was confirmed by morphological and molecular analyses.

Distribution and mosquito hosts of Chaoyang virus, a newly reported flavivirus from the Republic of Korea, 2008-2011.

In total, 183,602 female culicine mosquitoes were captured by Mosquito Magnet, black light, and New Jersey light traps, and manual aspiration of resting blood-fed mosquitoes, in the Republic of Korea from 2008 to 2011. Culicine mosquitoes were identified to species, placed in pools of up to 30 mosquitoes each, and screened for flavivirus RNA by using an SYBR green I-based reverse transcription-polymerase chain reaction assay. Thirty-two of the 8,199 pools assayed were positive by quantitative polymerase chain reaction for Chaoyang virus (CHAOV), an insect-specific virus [26 Aedes vexans nipponii Theobald, 3 Culex pipiens L., 1 Aedes albopictus (Skuse), 1 Aedes bekkui Mogi, and 1 Armigeres subalbatus (Coquillett)]. The maximum likelihood estimations (estimated number of virus-positive mosquitoes/1,000 mosquitoes) for Ae. bekkui, Ae. albopictus, Ar. subalbatus, Ae. vexans nipponii, and Cx. pipiens positive for CHAOV were 5.37, 3.29, 0.77, 0.27, and 0.26, respectively. CHAOV is an insect-specific virus, and there is currently no evidence to suggest a role in animal or human disease.

First collection records of Culiseta (Culicella) nipponica in the Republic of Korea.

The 1st confirmed record of Culiseta nipponica for the Republic of Korea was made at Osan Air Base and Tongilchon, Gyeonggi Province, and at the Incheon International Airport, Incheon metropolitan area, using carbon dioxide-baited light and Mosquito Magnet traps. Larval surveys near adult collection sites during June and August 2013 were negative for Culiseta nipponica.

Seasonal abundance of Culicoides (Diptera: Ceratopogonidae) collected by mosquito Magnet® in Northern Gyeonggi-do (Province), Korea.

Biting midges (Culicoides: Ceratopogonidae) were collected by Mosquito Magnet® traps at the Neutral Nations Supervisory Commission (NNSC) camp and Daeseongdong village inside the demilitarized zone (DMZ) and near the military demarcation line (MDL) separating North and South Korea and at Warrior Base (US Army training site) and Tongilchon 3 km south of the DMZ in northern Gyeonggi Province, Republic of Korea (ROK), from May-October 2010-2012, to determine their seasonal distributions. A total of 18,647 Culicoides females (18,399; 98.7%) and males (248; 1.3%) comprising 16 species were collected. Overall, the most commonly collected species was Culicoides nipponensis (42.9%), followed by C. erairai (29.2%), C. punctatus (20.3%), C. arakawae (3.3%), C. pallidulus (1.8%), and C. circumscriptus (1.4%), while the remaining 10 species accounted for only 1.1% of all Culicoides spp. collected. The seasonal distribution of C. nipponensis was bimodal, with high numbers collected during May-June and again during September. C. erairai was more frequently collected during June-July, followed by sharply decreased populations from August-October. C. punctatus was collected in low numbers from May-September with high numbers collected during October. C. erairai was predominantly collected from the NNSC camp (85.1% of all C. erairai collected) located adjacent to the MDL at Panmunjeom in the northernmost part of Gyeonggi-do (Province), while other sites yielded low numbers of specimens.

Three species of Culicoides Latreille (Diptera: Ceratopogonidae) newly recorded from the Republic of Korea.

Light trap surveys of adult Culicoides Latreille in the Republic of Korea (ROK) resulted in the capture of three previously unreported species, C. nasuensis Kitaoka, C. pallidulus Yu and C. jacobsoni Macfie. These new records are supported by supplementary morphological descriptions and DNA barcodes (mitochondrial cytochrome oxidase I or COI). An updated checklist of species reported from the ROK is provided.

Seasonal abundance of biting midges, Culicoides spp. (Diptera: Ceratopogonidae), collected at cowsheds in the southern part of the Republic of Korea.

Black light traps were used to measure the seasonal and geographical distribution of Culicoides spp. (biting midges or no-see-ums) at 9 cowsheds in the southern half of the Republic of Korea (ROK) from June through October 2010. A total of 25,242 Culicoides females (24,852; 98.5%) and males (390; 1.5%) comprising of 9 species were collected. The most commonly collected species was Culicoides punctatus (73.0%) followed by C. arakawae (25.7%), while the remaining 7 species accounted for <1.0% of all Culicoides spp. collected. The mean number of Culicoides spp. collected per trap night (Trap Index [TI]) was highest for C. punctatus (409.3), followed by C. arakawae (144.2), C. tainanus (4.1), C. oxystoma (1.2), C. circumscriptus (0.7), C. homotomus (0.6), C. erairai (0.4), C. kibunensis (0.3), and C. nipponensis (0.04). Peak TIs were observed for C. punctatus (1,188.7) and C. arakawae (539.0) during July and August, respectively. C. punctatus and C. arakawae have been implicated in the transmission of arboviruses and other pathogens of veterinary importance that adversely impact on animal and bird husbandry.

Species Diversity of Anopheles Mosquitoes and Plasmodium vivax Infection Rates, Gyeonggi Province, Republic of Korea During 2020.

There are currently >300 malaria cases reported annually in the Republic of Korea (ROK), with most cases attributed to exposure in northern Gangwon and Gyeonggi provinces near the demilitarized zone (DMZ). The species diversity and malaria infection rate were determined for a sample of Anopheles mosquitoes collected from May to early November 2020 for six sites in a malaria high-risk area in/near the DMZ and two malaria low-risk areas in southern Gyeonggi province using Mosquito Magnet traps in the ROK. A total of 1864 Anopheles spp. were identified to species by PCR. Overall, An. kleini (31.4%, 510/1622) was the most frequently species assayed, followed by An. pullus (25.5%, 413/1622), An. sineroides (23.9%, 387/1622), and An. sinensis (10.2%, 165/1622), while the other four species only accunted for 9.1% (147/1622) collected in/near the DMZ. Only three species, An. pullus, An. sinensis, and An. sineroides were collected at Humphreys US Army Garrison (USAG) (235 individuals), while only An. sinensis was collected at Yongsan USAG (7 individuals). A total of 36 Anopheles specimens belonging to five species collected in/near the DMZ were positive for Plasmodium vivax by PCR. Anopheles kleini (9) was the most frequent species positive for P. vivax, followed by An. belenrae (8), An. pullus (8), An. sinensis (5), An. sineroides (5), and a member of the Anopheles Lindesayi Complex in the ROK (1). This is the first report of P. vivax in a member of the An. Lindesayi Complex in the ROK. These findings can assist in guiding future malaria vector management in the ROK.

Japanese encephalitis virus in culicine mosquitoes (Diptera: Culicidae) collected at Daeseongdong, a village in the demilitarized zone of the Republic of Korea.

In total, 22,846 (17,793 culicines and 5,053 Anopheles spp.) female mosquitoes were captured by a Mosquito Magnet trap at Daeseongdong, a small village adjacent to the military demarcation line (center of the demilitarized zone) in northern Gyeonggi Province, Republic of Korea (ROK). Culicine mosquitoes were identified to species, placed in pools of up to 30 mosquitoes each, and screened for flavivirus using a SYBR Green I-based real-time polymerase chain reaction. In total, 51/660 pools positive for flaviviruses and confirmed by DNA sequencing of the NS5 region, were positive for Japanese encephalitis virus (family Flaviviridae, genus Flavivirus, JEV) (50 Culex tritaeniorhynchus Giles and one Culex bitaeniorhynchus Giles). The JEV maximum likelihood estimations (MLEs) (estimated number of viral RNA-positive mosquitoes per 1,000) for Cx. tritaeniorhynchus and Cx. bitaeniorhynchus were 9.7 and 0.9, respectively. This is the first report of a Cx. bitaeniorhynchus positive for JEV in the ROK. JEV is a local civilian and military health threat and a significant concern for nonimmune (unvaccinated) U.S. soldiers, civilians, and family members deployed to the ROK.

Mosquito species composition and Plasmodium vivax infection rates on Baengnyeong-do (island), Republic of Korea.

Vivax malaria is a significant military and civilian health threat in the north of the Republic of Korea (ROK). The island of Baengnyeong-do is the westernmost point of the ROK and is located close to the southwestern coast of the Democratic People's Republic of Korea (DPRK). Mosquitoes were collected using a black light trap on Baengnyeong-do, and Anopheles spp. were assayed by PCR, to identify the species, and screened for sporozoites of Plasmodium vivax. Of a subsample of 257 mosquitoes, Anopheles lesteri was the most frequently collected (49.8%), followed by Anopheles sinensis (22.6%), Anopheles pullus (18.7%), Anopheles kleini (7.8%), and Anopheles belenrae (1.2%). The overall sporozoite rate was 3.1%, with the highest rates observed in An. kleini (15.0%), An. sinensis (5.2%), and An. lesteri (1.6%). No sporozoite positive An. pullus or An. belenrae were observed. The results extend our knowledge of the distribution and potential role in malaria transmission of An. kleini, An. lesteri, and An. sinensis, for an area previously considered to be at a low risk for contracting vivax malaria.

Accuracy of the detection of infratentorial stroke lesions using perfusion CT: an experimenter-blinded study.

INTRODUCTION: Although perfusion CT (PCT) for the detection of supratentorial stroke is well established, there is a dearth of evidence of its effectiveness in the detection of infratentorial stroke. Hence, this study compared sensitivity, specificity, and accuracy of PCT maps between infratentorial and supratentorial stroke lesions. METHODS: One hundred patients with acute stroke who had successfully undergone near whole-brain PCT with the toggling table technique and follow-up MRI were included. Wilcoxon Mann-Whitney test was performed at P<0.01. RESULTS: There was no significant statistical difference in the accuracy (91.79% vs. 93.23% in regional cerebral blood volume; 92.26% vs. 95.31% in regional cerebral blood flow; 89.17% vs. 92.71% in mean transit time; 89.76% vs. 92.19% in time to peak; P>0.01 in all PCT maps) between supratentorial and infratentorial stroke. Also, there was no remarkable difference in both sensitivity and specificity of PCT maps. CONCLUSION: This was the first study to investigate the accuracy of PCT with the toggling table technique in detection of infratentorial stroke lesions. Clinically, PCT is highly reliable and accurate in detecting infratentorial stroke lesions.

Does Nasal Septal Deviation and Concha Bullosa Have Effect on Maxillary Sinus Volume and Maxillary Sinusitis?: A Retrospective Study.

Purpose: This study aimed to determine whether nasal septal deviation and concha bullosa affect the maxillary sinus volume, and whether this effect is associated with the prevalence of chronic sinusitis. Materials and Methods: This study retrospectively reviewed 209 paranasal sinus CT (PNS CT) images of patients with sinonasal symptoms from January 2017 to December 2018. The maxillary sinus volume was measured twice by a radiologist, and statistical analysis was performed using SAS 9.4. Results: Intersex comparison of the maxillary sinus volume (on left and right sides) revealed that the volume was significantly larger on both the sides (p < 0.0001) in men compared with that in women. Concha bullosa was found to occur mainly in the concave cavity of the septal deviation (p < 0.0001). No significant association was found between nasal septal deviation and maxillary sinusitis (p = 0.8756) as well as between concha bullosa and maxillary sinusitis prevalence (p = 0.3401) or maxillary sinus volume (both: p = 0.6289, Rt.: p = 0.9522, Lt.: p = 0.9201). Conclusion: Although nasal septal deviation and the location of concha bullosa may affect each other, maxillary sinus volume and maxillary sinusitis were neither associated with nasal septal deviation nor concha bullosa.

Wing size and parity as markers of cohort demography for potential Anopheles (Culicidae: Diptera) malaria vectors in the Republic of Korea.

Wing lengths of parous (P) and nulliparous (NP) PCR-identified female Anopheles belenrae, An. kleini, An. pullus, and An. sinensis were determined from weekly trap collections at Camp Humphreys (CH), Ganghwa Island (GH), and Warrior Base (WB), Republic of Korea (ROK) during Jun-Oct, 2009. Wing length was greatest at the beginning and end of the study period. Wing length of NPs tended to be less than that of Ps before the period of maximum greening (Jul-Aug) but greater thereafter. Larger specimens tended to be Ps, and weekly wing length of Ps appeared less variable than NPs, possibly due to selection. A bimodal wing length frequency distribution of An. sinensis suggested two forms comprising small- (≤4.5 mm, SW) and large-winged females (>4.5 mm, LW). LW comprised the majority of peaks in abundance, however %SW, while still a minority, often increased during these times suggesting a density-dependent effect. At WB and GH, a two to three-week periodicity in %SW was obvious for An. sinensis and An. kleini. Analyses of weather station and satellite data showed that smaller-winged An. sinensis were associated with warmer, more humid, and greener times of the year. SW and LW specimens possibly result from agricultural practices that are common across large areas; regular synchronous peaks of SW and LW were observed from different sites. Peaks in SW Ps followed peaks in NPs in a 'ripple effect' one to two weeks apart, suggesting that wing length combined with parity could be used to follow the emergence and survival of mosquito cohorts.

Temporal Trend of Aedes albopictus in Local Urban Parks of the Republic of Korea.

Aedes albopictus (Skuse) was first reported in the Republic of Korea in 1941 (Suwon, Gyeonggi Province). Recently, a mosquito-monitoring program was established in response to the potential development of autochthonous infections of dengue, chikungunya, and Zika viruses, to identify the regional characteristics of Ae. albopictus in Suwon. Daily collections of adult mosquitoes were conducted using Blackhole UV-LED traps and BG-Sentinel traps baited with octenol, carbon dioxide or both at five sites in Suwon (Sungkyunkwan University, Manseok, Seoho, Olympic, and Jungang parks) from 1 July to 1 November 2016. Based on the ecological cycle of mosquitoes, an estimated association between the daily mean temperatures [2 wk prior to the collection date (lag 2 wk)] and relative population abundance were developed using a generalized linear model. The overall proportion of female mosquitoes collected from all traps was 88.3%. Higher female trap indices were observed for mosquitoes collected at BG-Sentinel traps. Additionally, the BG-sentinel trap was more effective collecting Ae. albopictus. Culex pipiens (Coquillett) was the most frequently collected mosquito using UV-LED traps, followed by Ae. vexans nipponii (Theobald), Ochlerotatus koreicus (Edwards), Cx. inatomi (Kamimura and Wada), and members of the Anopheles Hyrcanus Group. Within a certain temperature range, daily mean temperatures 2 wk prior to the collection period was significantly associated with a maximum abundance of Ae. albopictus (P < 0.05) at 26.2°C (95% confidence interval: 24.3-29.4°C). The BG-Sentinel trap is an effective trap for monitoring relative population abundance of Ae. albopictus, and trap catches are significantly correlated with daily mean temperatures.

Focal nodular hyperplasia-like nodules in alcoholic liver cirrhosis: radiologic-pathologic correlation.

OBJECTIVE: The purpose of this study was to describe our experience with three patients who had pathologically proven focal nodular hyperplasia (FNH)-like nodules that radiologically mimicked hepatocellular carcinoma (HCC). CONCLUSION: FNH-like nodules may radiologically mimic HCC, appearing as hypervascular masses on contrast-enhanced CT images and as high-signal-intensity masses on superparamagnetic iron oxide-enhanced MR images. Pathologically, there is the presence of a high number of unpaired arteries and sinusoidal capillarization, which may mimic HCC. Thus, it is important to differentiate FNH-like nodules radiologically, pathologically, and clinically from HCC.

Actinomycosis of the gallbladder mimicking carcinoma: a case report with US and CT findings.

We describe a case of actinomycosis of the gallbladder mimicking carcinoma. Sonography showed a hypoechoic mass replacing gallbladder lumen and engulfing a stone; contrast-enhanced computed tomography showed a heterogeneously enhanced thickened gallbladder wall with subtle, disrupted luminal surface enhancement, which formed a mass. As a result of the clinical and radiologic presentation, our impression was of gallbladder carcinoma. Actinomycosis should be included in the differential diagnosis when sonography and computed tomography findings show a mass engulfing the stone in the gallbladder and extensive pericholecystic infiltration with extension to neighboring abdominal wall muscle.

Comparison of Adult Mosquito Black-Light and Light-Emitting Diode Traps at Three Cowsheds Located in Malaria-Endemic Areas of the Republic of Korea.

Adult mosquito surveillance and field trials evaluated selected commercially available ultraviolet black-light (BL) and light-emitting diode (LED) traps at three sites where vivax malaria is endemic from May to October 2015 in northwestern Republic of Korea. Collections totaled 283,929 adult mosquitoes (280,355 [98.74%] females and 3,574 [1.26%] males) comprising 17 species (including six members of the Anopheles Hyrcanus Group) belonging to six genera. The four most predominant female species collected were Aedes vexans nipponii (Theobald) (83.84%), followed by Anopheles Hyrcanus Group (13.66%), Culex pipiens Group (1.67%), and Culex tritaeniorhynchus Giles (0.54%). Overall, LED traps (188,125) collected significantly more female mosquitoes compared with BL traps (92,230; P = 0.0001, P < 0.05). Results from these field evaluations significantly enhance vector and disease surveillance efforts, especially for the primary vectors of malaria (Anopheles Hyrcanus Group) and Japanese encephalitis (Cx. tritaeniorhynchus).

Analysis of Genes with Alternatively Spliced Transcripts in the Leaf, Root, Panicle and Seed of Rice Using a Long Oligomer Microarray and RNA-Seq.

Pre-mRNA splicing further increases protein diversity acquired through evolution. The underlying driving forces for this phenomenon are unknown, especially in terms of gene expression. A rice alternatively spliced transcript detection microarray (ASDM) and RNA sequencing (RNA-Seq) were applied to differentiate the transcriptome of 4 representative organs of Oryza sativa L. cv. Ilmi: leaves, roots, 1-cm-stage panicles and young seeds at 21 days after pollination. Comparison of data obtained by microarray and RNA-Seq showed a bell-shaped distribution and a co-lineation for highly expressed genes. Transcripts were classified according to the degree of organ enrichment using a coefficient value (CV, the ratio of the standard deviation to the mean values): highly variable (CVI), variable (CVII), and constitutive (CVIII) groups. A higher index of the portion of loci with alternatively spliced transcripts in a group (IAST) value was observed for the constitutive group. Genes of the highly variable group showed the characteristics of the examined organs, and alternatively spliced transcripts tended to exhibit the same organ specificity or less organ preferences, with avoidance of 'organ distinctness'. In addition, within a locus, a tendency of higher expression was found for transcripts with a longer coding sequence (CDS), and a spliced intron was the most commonly found type of alternative splicing for an extended CDS. Thus, pre-mRNA splicing might have evolved to retain maximum functionality in terms of organ preference and multiplicity.